A Compilation of Culture Media - Color (Danh Luc MT Nuoi Cay VSV)

SOCIETY OF AMERICAN BACTERIOLOGISTS
MONOGRAPHS ON SYSTEMATIC
BACTERIOLOGY
Volume I. General Systematic Bacteriology.

R. E. Buchanan, Iowa State College.
597 pages. $6.00, net postpaid.
Volume II. A Compilation of Culture Mediafor

the Cultivation of Microorgan-
isms. Max Levine and H. W.
Schoenlein, Iowa State College, xvi
+ 969 pages. $15.00

A COMPILATION OF CULTURE MEDIA FOR THE
CULTIVATION OF MICROORGANISMS
MONOGRAPHS ON SYSTEMATIC BACTERIOLOGY
A Compilation of Culture
Media
FOR THE CULTIVATION OF
MICROORGANISMS
BY
MAX LEVINE, Ph.D.
Professor of Sanitaryand Technical Bacteriology, Bacteriologist,
Iowa Engineering Experiment Station
AND
H. W. SCHOENLEIN, M.S.
Formerly Fellow in the Department of Bacteriology,
Iowa State College, Ames, Iowa
PREPARED AT THE REQUEST OF

THE SOCIETY OF AMERICAN BACTERIOLOGISTS
AND FINANCED BY A GRANT FROM THE
DIGESTIVE FERMENTS COMPANY, DETROIT, MICHIGAN
BALTIMORE
THE WILLIAMS & WILKINS COMPANY
1930
Copyright 1930
THE WILLIAMS & WILKINS COMPANY
Made in the United States of America
Published June, 1930
All rights reserved
Composed and Printed at the

WAVERLY PRESS, INC.
FOR
Thk Williams & Wilkins Company
Baltimore, Md., U. S. A.
CONTENTS
I. FOREWORD xi
II. PREFACE xiii
III. INTRODUCTION xv
IV. KEY TO THE MAIN GROUPS OF CULTURE MEDIA 1
V. KEY TO THE SUB-GROUPS OF LIQUID MEDIA.
Group I (Med. 1-1394) 1
A. Sub-group lA. Water only as a basal medium 1
B. Sub-group IB. Key to sections of liquid media in which ail constituents
are inorganic. (Med. 2-113) 3
1. Nitrogen present as free N2 only.

a. Incomplete "basal" solutions requiring addition of other nutri-
ents. Section 1 (Med. 2-25) 3
b. Complete nutrient solutions.
Section 2 (Med. 26-29) 10
2. Nitrogen supplied as ammonium salts.
a. Incomplete or "basal" solutions.

b. Complete media primarily for organisms oxidizing NH3 to NO2.
c. Complete media primarily for organisms not oxidizing NH3 to
NO2. Section 5 (Med. 73-85) 25
3. Nitrogen supplied as nitrites. Section 6 (Med. 86-95) 28

4. Nitrogen supplied as nitrates.
a. Incomplete or "basal" solutions.
Section 7 (Med. 96-106) 31

b. Complete nutrient solutions.
Section 8 (Med. 107-113) 34
C. Sub-group IC. Key to sections of liquidmedia in which one or more con-
stituents are organic. (Med. 114-1394) 36
1. Chemical composition of all constituents known.

a. Nitrogen present only as free N2.
Section 1 (Med. 114H90) 37
b. Nitrogen present as ammonium salts.
Section 2 (Med. 191-323) 59
c. Nitrogen present as nitrites.
Section 3 (Med. 324-327) 91

d. Nitrogen present as nitrates.
Section 4 (Med. 328-365) 92
e. Nitrogen present as amino acids.
Section 5 (Med. 366-479) 102

f. Organic nitrogen other than amino acids present.
Section 6 (Med. 480-515) 135
2. Chemical composition of one or more constituents not known.
a. Media containing commercial digests.
(1) Additional constituents inorganic.

Section 7 (Med. 516-565) 144
-i 35b i) 1
Viii CONTENTS
(2) Incomplete or "basal" solutions with additional organic

non-nitrogenous materials of known composition.
Section 8 (Med. 566-588) 157
(3) Complete media as in (2).
Section 9 (Med. 589-647) 167
(4) Additional constituents nitrogenous and of known chemi-
cal composition. Section 10 (Med. 648-683) 182
(5) Additional materials of plant origin. Chemical composi-

tion not known. Section 11 (Med. 684-712) 191
(6) Additional material of animal origin of unknown chemical

composition (other than extracts or infusions).
Section 12 (Med. 713-747) 198
(7) Media containing animal extracts or infusions in addition
to commercial digests.
Section 13 (Med. 748-977) 210
b. Media containing non-commercial digests.
Section 14 (Med. 978-1140) 299
c. Media not containing digests.
(1) Containing plant derivatives of unknown chemical com-
position. Section 15 (Med. 1141-1228) 329
(2) Containing animal derivatives.

Section 16 (Med. 1229-1376) 355
(3) Containing extracts of soil, ashes, etc.
Section 17 (Med. 1377-1394) 402
VI. KEY TO THE SUB-GROUPS OF REVERSIBLY SOLID AGAR MEDIA.
Group II (Med. 1395-2198) 408
A. Sub-group IIA. Key to media containing water and agar only.
(Media 1395-1401) 408
B. Sub-group IIB. Key to sections of liquefiable agar media, with all con-
stituents (other than agar) inorganic. (Media 1402-1430) 411
1. Nitrogen present only as free N2. Section 1 (Med. 1402-1410) 411
2. Nitrogen supplied as ammonium salts.

Section 2 (Med. 1411-1424) 413
3. Nitrogen supplied as nitrites and nitrates.
Section 3 (Med. 1425-1430) 417
C. Sub-group lie. Key to sections of liquefiable agar media containing or-
ganic materials (in addition to agar).
(Med. 1431-2198) 419
1. Composition of all added constituents known.
a. Inorganic nitrogen supplied. Section 1 (Med. 1431-1495) 419
b. Organic nitrogen supplied. Section 2 (Med. 1496-1519) 436
2. Composition of some of the added constituents not known.

a. Agar media containing commercial digests; other materials
of
known composition. Section 3 (Med. 1530-1596) 443
b. Agar media containing commercial digests together with ma-

terials of plant or soil origin.
Section 4 (Med. 1597-1627) 461
^^
CONTENTS
materials of ani-
c. Agar media containing commercial digests and
mal origin other than extracts or infusions.
Section 5 (Med. 1628-1660) ....... 469
media containing commercial digests and extracts or in-
d Agar
1661-2028) 481
fusions of animal origin. Section 6 (Med.
digests.
e. Agar media containing non-commercial
Section 7 (Med. 2029-2078) 650 .
plant origin but

f Agar media containing unknown constituents of
Section 8 (Med. 2079-2141) 674
no digests.
animal origin
e Aear media containing unknown constituents of
Section 9 (Med. 2142-2192) 690
but no digests.
of ash or soil origin

h Agar media containing unknown constituents
Section 10 (Med. 2193-2198) 708
but no digests.
MEDIA.
VII KEY TO THE SUB-GROUPS OF REVERSIBLY SOLID GELATIN
2199-2371) 711
Group III (Med.
A Sub-group IIIA. Key to gelatin media containing no other organic
(Med. 2199-2204) 711
materials.
liquefiable gelatin media containing

B. Sub-group IIIB. Key to sections of
(Med. 2205-2371) 713
additional organic materials.
1 Gelatin media with other constituents of known composition.

all
Section 1 (Med. 2205-2234) 713
materials of unknown
2 Gelatin media containing digests and other
Section 2 (Med. 2235-2323) 718
composition.
composition other
3 Gelatin media containing materials of unknown
Section 3 (Med. 2324-2371) 752
than digests.
KEY TO REVERSIBLY SOLID MEDIA OTHER THAN AGAR OR

GELATIN.
VTII
Group IV (Med. 2372-2382) 765
MEDIA; INITIALLY
IX KEY TO SUB-GROUPS OF IRREVERSIBLE SOLID
LIQUID; SOLIDIFYING AGENT ORGANIC.
Group V (Med. 2383-2466) 768
of plant
A Sub-group VA. Irreversibly solid media, solidified by materials
(Med. 2383-2397) 768
origin.
deriva-
B Sub-group. VB. Irreversibly solid media, solidified by blood or its
(Med. 2398-2430) 772
thres.
solidified by materials of animal

C. Sub-group VC. Irreversibly solid media
origin other than blood (eggs, etc.).
(Med. 2431-2466) 788
AGENT INOR-
X KEY TO IRREVERSIBLE SOLID MEDIA; SOLIDIFYING2467-2485)
Group VI (Med. 803
GANIC.
X CONTENTS
XI. KEY TO SUB-GROUP OF MEDIA INITIALLY SOLID.

Group VII (Med. 2486-2543) 813
A. Sub-group VIIA. Initially solid organic substrates.
(Med. 2486-2516) 813
B. Sub-group VIIB. Initially solid inorganic substrates.
(Med. 2517-2543) 825
XII. MEDIUM NAME INDEX 833
XIII. CONSTITUENTS INDEX 869
XIV. USE INDEX 891
XV. AUTHOR INDEX 899
XVI. REFERENCES 915
FOREWORD
and media used for the
The contributions relating to the various substrates
cultivation of bacteria, yeasts, and molds, are
widely scattered in the hterature.
For some years it has been
Very frequently they are not adequately indexed.
felt that a key to the various media
and substrates proposed for the growth of
were not
microorganisms would prove helpful. The fact that these formulae
of dupHcation m
the hterature. A
readily available has led to a great amount
undertaken and the formulae tor
search of the hterature has therefore been
that have been proposed brought together.
The
about 7000 various media
task of securing this material was performed
most acceptably by Mr. H. W.
of the keys The task
Schoenlein, who was also associated in the development
of planning the presentation, and of final editmg
of organizing the compilation,
to curtail
was undertaken by Dr. Max Levine. It was found possible greatly
the number of separate formulae by use of the subheading Variants m many
in the following pages was made possible

by a
The compilation presented
Society of American Bacteriologists by the Digestive Ferments
grant to the
of American
Company of Detroit, Michigan. At the request of the Society
at the Iowa State College
Bacteriologists, the Department of Bacteriology
helpfulness, and support of the
accepted the task of compilation. The interest,
progress of the work cannot be
Digestive Ferments Company throughout the
too greatly appreciated.
of classification of the media, together with
the arrangement of
The system
keys, indices, etc., should make possible a ready review of the media which
have been proposed growing different types of organisms and containing
for
different ingredients. The primary subdivisions into Groops on the basis of
agent is the logical one
physical characteristics and nature of the sohdifying
of usefulness. The
both from the standpoint of history and the standpoint
of the constitu-
secondary subdivisions based upon the chemical characteristics
ents of the media are in fine with modern trend
toward emphasis upon physiol-
ogy and metabolism of microorganisms.
It is hoped that this compendium of
information will prove very generally
useful to all bacteriologists and in all bacteriological laboratories.
R. E. Buchanan.
PREFACE
The rapid growth of the science of Bacteriology has led to the development of
numerous combinations of materials for the propogation of microorganisms.
It was felt that the collection and orderly arrangement of such culture media
would be of assistance in the future development of the science of Bacteriology,
and the authors were gratified with the opportunity to perform this service.
In spite of the efforts to thoroughly cover the literature, this compilation is
probably incomplete, particularly with respect to formulae published in bulletins
and monographs which were not available or consulted. It is hoped that this
compilation will help acquaint the bacteriologists with the numerous types and
combinations of materials which have been employed for cultivating micro-
organisms, and if it serves to check the needless publication of new formulae,
which are really old, the authors will feel well repaid for their time and efforts.
To Mr. H. G. Dunham, of the Digestive Ferments Company, we are greatly
indebted for helpful criticisms, and suggestions.
To Mrs. C. H. Werkman, Miss Ruth Confare and Miss Lois Kratoska, the
authors extend cordial thanks for assistance in typewriting and correcting the
manuscript.
To Dr. R. E. Buchanan the authors wish to particularly express their indebt-
edness and gratitude for the fatherly advice, encouragement, and assistance, so
graciously rendered at all times.
Max Levine,
h. w. schoenlein.
Iowa State College,
Ames, Iowa,
July 1, 1929
INTRODUCTION
At the request of the Society of American Bacteriologists, the Hterature was
surveyed for formulae of culture media reported useful for the growth of bacteria
and other microorganisms. The standard periodicals such as the various Cen-
tralblatts fiir Bakteriology, ZeitschriftHygiene, Annals of the Pasteur
ftir
Institute, Journal of Hygiene, Journal of Pathology and Bacteriology, and all

of the American bacteriological journals were perused from their inception to
the year 1926. A number of other periodicals, as well as text books, monographs
and many Experiment Station bulletins were included in the survey.
Formulae of about seven thousand culture media were gleaned from the
literature. These were arranged and classified into approximately twenty five
hundred quite distinct media. A number and a distinctive name was assigned
to each medium which is described under the following headings:
Constituents,
Preparation, Sterilization, Use, Variants, so as to facilitate comparison of differ-
ent formulae.
A dichotomous key, several indices and complete references to all articles
from which formulae were taken are also included in this compilation. In the
key, and all indices, the numbers refer to medium numbers and not pages.
The Medium Name Index includes an alphabetical Ust of all media described
herein.
In the Constituents Index an attempt was made to list every medium in
which a particular substance was employed.
The Author Index is self explanatory.
The Use Index is necessarily incomplete. It includes only those media for
which a specific use was indicated in the original article reviewed.
The various media were subdivided into seven "groups" on the basis of their
physical states. Thus, all the liquid media are considered in a group by them-
selves and the solid media are further subdivided, on the nature of the solidifying
agent and physical properties of these agents, as, for example, whether initially
hquid, reversibly liquid and solid, or permanently solid.
Each group, if sufficiently complicated to warrant further classification, is
then subdivided into ''sub-groups" on the basis of the presence or absence of
additional organic constituents.
The sub-groups are further subdivided into sections on the basis of the nature
of the nitrogen, carbon, and inorganic constituents.
For each section there is prepared a key, generally dichotomous, by means of
which each medium therein may be located. The various keys are distributed
throughout the book immediately preceding their respective media.
The problem of a suitable means of identification of individual media was a
particularly difficult one. The scheme finally adopted was to assign a number in
/
XVI INTRODUCTION /
conjunction with a specific name, the latter including the chief nitrogen and
carbon source, where possible, and the name of the author who first listed the
medium. Thus "273 Ayers' Glucose Ammonium Phosphate Solution," indi-
cates that Ayers first described the medium, the nitrogen source was ammonia,
the carbon source glucose, and that the medium is No. 273 in this collection.
In addition to the carbon and nitrogen source the nature of the solidifying
agent is also employed in naming culture media, as, for example, "1553 Greig
Smith's Sucrose Peptone Agar."

Where medium was not available the fact is
the original article containing a
indicated by placing the author from whose publication the medium was ob-
tained in parentheses after the name of the medium. Thus "2156 Beck's
Glycerol Serum Agar (Klimmer)" indicates that the medium was obtained from
Klimmer who referred to Beck as the author.
If the same medium is described by different authors, only the earliest avail-
able author's name appears in the medium name, but the other authors are given
under the heading "references" at the end of the description of the medium.
Where several media differ only with respect to the relative quantities of
constituents employed, or in the method of preparation, the earliest described
medium takes precedence, and all others are described as variants.
One of the vexing questions which arose was that of the disposition of media
in which it was specified that a number of substances might be substituted for
each other. For example, if the availability of various carbon sources is being
studied, shall each new combination, as the employment of glucose, sucrose,
glycerol, etc., be considered a distinct medium? To do so would have markedly
extended the number of media to be considered.
The term "basal" was introduced to take care of this situation. This was ap-
plied to the formula exclusive of the substituted constituents which then became
added nutrients. The term "basal" occurring in the name of a medium there-
fore designates that the formula is not in iteslf a complete medium, but that it
serves as a base to which other constituents are added.
A serious difficulty in naming media arose in connection with the use of beef
extracts and meat infusions. This was particularly troublesome when authors
did not specify whether extract or infusions were employed. Where the use of
extracts was specified in the articles reviewed, these terms appear in the names
of the respective media, as e.g., "779 Dunham's Infusion Broth" or "1695
Heinemann's Meat Extract Agar." Where the author did not specify the
nature of the material employed (i.e., whether extract or infusion) the term
"bouillon" is employed, thus "936 Kendall, Day and Walker's Mannitol
Bouillon."
COMPOSITION OF CULTURAL MEDIA AND SUBSTRATES USED
IN BACTERIOLOGY
Any classification of media and substrates used in bacteriology must of
necessity be artificial and arbitrary in form. In the classification here adopted
an effort made to bring closely related media together, although it is
has been
some cases even the primary divisions tend to separate media
realized that in
having much the same essential composition and used for much the same
purposes.
The 7 primary divisions which have been adopted are as follows:
PRIMARY CLASSIFICATION OF MEDIA

Ai. Liquid media Group I (Med. 1-1394)
A2. Solid media.
Bi. Initially liquid.
Ci. Reversible. Reversibly liquid and solid. Liquefiable by heat.
Di. Solidified by the addition of agar-agar. . Group II (Med. 1395-2198)
D2. Solidified by the addition of gelatin. . Group III (Med. 2199-2371)
D3. Solidified by the addition of other materials
Group IV (Med. 2372-2382)
C2. Irreversible. Not reversibly liquid and solid.
Di. Solidifying agent organic Group V (Med. 2383-2466)
D2. Solidifying agent inorganic Group VI (Med. 2467-2485)
B.. Initially solid Group VII (Med. 2486-2543)
GROUP I. LIQUID MEDIA

The term "Liquid Medium" is here de-
fined to include all media containing suffi-
SUBGROUP I-A
cient water and so lacking
* in viscosity as to
,

, The water used in xu
rrû j.
the i
u x i
i
bacteriological
be readily poured from one vessel to another.
laboratory as a medium or in the prepara-
A liquid medium is considered one which tion of media may be either distilled or a
, ,, ,, ,. ,.^ , , ,
does not set or solidify when cooled to
^^tural water. In theory distilled water is
""^ ^'''*'
^ '" preferred, although distilled water
o? wateT''^*"''
mi. ,' -n I-1- -1 1-
J.- J X
unless carefully prepared may contain gas-
eous or
1
i he classihcation 01 liquid media adopted n- -i- i- u -n j.
, ., . ^ , , , !
j.
metallic impurities which will inter-
is based primarily upon whether the nitro-
j-^j.^ ^^^-^ j^^ ^^^
gen supplied is inorganic or organic.
Distilled water' for the bacteriological
Subgroups of liquid media laboratory.
Ai. Water only Subgroup I-A Surface and ground water for media.
A2. Water with other constituents
Bi. All constituents of medium inorganic 1, Molisch's Basal Nutrient Solution
Subgroup I-B (Med. 2 to 113)
B2. One or more constituents organic Constituents:
Subgroup I-C (Med. 114 to 1394) 1. Sea water 1000.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Preparation magnesium citrate 0.05%

(1) Dissolve one of the added nutrients potassium citrate 0.05%
in amount indicated in 1. calcium butyrate 0.05%
(2) Tube in tall tubes. Fill within a sodium acetate 0.05%
centimeter of the plug. iron ammonium 0.05%
citrate
Sterilization: Method not given. iron magnesium 0.05%
citrate
Use: To determine the constituents essen- iron ammonium citrate 0.05% + gyp-
tial for the growth of purpurbacteria, sum
Rhodobacterium capsulatum and Rhodo- infused hay
hacillus palustris (Molisch). The cul- infused hay + iron ammonium citrate
tures were incubated in diffused light. 0.05%
The author reported generally no growth iron potassium citrate 0.05%
using sucrose, dextrin, glycerol, ammo- iron ammonium tartrate 0.05%
nium tartrate or asparagin alone, but gen- iron ammonium oxalate 0.05%
erally growth with the other materials iron glycero phosphate 0.05%
and combinations listed. iron lactate 0.05%
Added nutrients and variants: The author iron magnesium lactate 0.05%
suggested the following: manganese peptone 0.025%
(a) Add one of the following materials: manganese peptone 0.025% +
sucrose 1.0% K2HPO4 0.1%
dextrin 1.0% manganese peptone 0.025% +
inulin 1.0% K2HPO4 0.1% + KNO3 0.1% +
asparagin 1.0% MgS04 0.1%
peptone 1.0% manganese peptone 0.025% +
glycerol 1.0% K2HPO4 0.1% + KNO3 0.1%
peptone 1.0% + sucrose 0.5% manganese peptone 0.025% +
peptone 1.0% + glycerol 0.5% MgS04 0.1%
peptone 1.0% + dextrin 0.5% manganese peptone 0.025% +
peptone 1.0% + asparagin 0.5% KNO3 0.1%
peptone 1.0% + inulin 0.5% manganese peptone 0.025% +
asparagin 0.5% + dextrin 0.5% gypsum 0.1%
asparagin 0.5% + inulin 0.5% manganese peptone 0.025% +
ammonium tartrate 1.0% K4Fe (CN)6 0.1%
(b) Used Moldau river water added one manganese peptone 0.025% +
of the following materials: citrate iron peptone
sucrose 1.0% or 0.5% peptone 0.5%
dextrin 1.0% or 0.5% peptone + MnCOs in excess
inulin 1.0% or 0.5% peptone + FeCOa in excess
asparagin 1.0% or 0.5% iron albuminate 0.05%
glycerol 1.0% or 0.5% iron peptonate 0.05%
peptone 1.0% manganese lactate 0.05%
peptone 1..% + sucrose 1.0% or 0.5% manganese phospholactate 0.05%
peptone 1.0% + dextrin 1.0% or 0.5% manganese salicylicate 0.05%
peptone 1.0%, + inulin 1.0% or 0.5% manganese fluorate 0.05% + peptone
peptone 1.0% + asparagin 1.0% or 0.5%
0.5% manganese citrate 0.05%
peptone 1.0% + glycerol 1.0% or manganese oxalate 0.05%
0.5% manganese glycerinate 0.05%
asparagin 0.5% +
dextrin 0.5%
iron sulphide peptone 0.05%
asparagin 0.5% + glycerol 0.5 g. manganese acetate 0.05%
dextrin 0.5% +
inulin 0.5% (c) Sea water without any addition.
ammonium tartrate 1.0% (d) Moldau river water without any
sodium citrate (neutral) 0.05% addition.
ammonium citrate 0.05% Reference: Molisch (1907, p. 68).
SUBGROUP I-B Ci. Sodium salts present.

Di. Calcium salts present.
Liquid Media in which All Constituents Ashby's Basal Solution. (Hoffmann and
are Inorganic Hammer) 4
von Wahl's Basal Salt Solution 5
Liquid media in which all constituents Locke's Solution 6
are inorganic may be classified most con- Winogradsky's Basal Inorganic Salt
veniently on the basis of the type of nitro- Solution (Heinemann) 7
gen provided. Charrin and Dissard's Basal Salt Solu-
tion 8
Key to the sections of Subgroup I-B
Gerlach and Vogel's Basal Solu-
A I. Nitrogen present as free or elementary tion 9
nitrogen only. Rettger, Berman and Sturges' Basal
Bi. Incomplete media, "basal"
so-called Solution 10
solutions, requiring the addition of other D2. Calcium salts not present.
nutrients Section 1 (Med. 2-25) Omeliansky's Basal Salt Solution 11
B^. Complete solutions, used as media with- Percival's Basal Salt Solution 12
out additions Section 2 (Med. 26-29) Nawiasky's Basal Salt Solution 13
A^. Nitrogen supplied as ammonium salts. C2. Sodium salts not present.
Bi. Incomplete media, or basal solutions. Stoklasa's Basal Solution 14
Section 3 (Med. 30-56) Heinze's Basal Salt Solution 15
B2. Complete media. Sohngen's Basal Solution 16
Ci. Media primarily for organisms oxidiz- van Delden's Basal Gypsum Solu-
ing ammonia to nitrites for growth tion 17
energy Section 4 (Med. 57-72) Czapek's Basal Solution 18
C2. Media not primarily for organisms oxi- Buchanan's Basal Salt Solution 19
dizing ammonia to nitrites for growth B2. Salts of mono, di, and trivalent cations
energy Section 5 (Med. 73-85) present.
A3. Nitrogen supplied as nitrites. Ci. Sodium Salts present.
Media primarily organisms oxidizing
for
Fuhrmann's Basal Solution 20
nitrites to nitrates for growth energy. Munter's Basal Salt Solution 21
Section 6 (Med. 86-95) Meyer's Basal Salt Solution. (Per-
A4. Nitrogen supplied as nitrates. otti) 22
Bi. Incomplete or basal solutions requiring C2. Sodium Salts not present.
the addition of other nutrients. Dox's Inorganic Salt Solution for
Section 7 (Med. 96-106) Fungi (Tanner) 23
B2, Complete nutrient solutions.
Gage's Basal Salt Solution 24
Section 8 (Med. 107-113) Bijerinck and van Delden's Basal Salt
Solution 25
SUBGROUP I-B. SECTION 1
Inorganic basal solutions of known 2. Physiological Salt Solution (Normal

chemical composition, nitrogen present saline)
only as atmospheric nitrogen; incomplete
solutions requiring the addition of other Constituents
nutrients. 1. Water 1000.0 cc.
Ai. Containing salts of monovalent cations 2. NaCl (c.p.) 8.5 g.
only. Preparation: (1) Dissolve 2 in 1.
Physiological Salt Solution. (Normal Sterilization: As desired.

saline) 2 Uses: Diluent and serological work in the
Molar Salt Solution 3 laboratory.
A2. Containing salts of monovalent and Variants: Frost used 6.0 g. NaCl per liter
other cations. References: Original author not found.
Bi. Salts of mono and divalent cations This formula was taken from Giltner
present. (1921, p. 399). Frost (1903, p. 18).
3. Molar Salt Solution 4. MgS04 0.3 g.

5. NaCl 0.1 g.
Constituents :
Preparation
1. Water 1000.0 cc.
(1) Dissolve and 5 in 1.
2, 3, 4
2. NaCI (best commercial
(2) Add one added nutrients, or
of the
grade) 60.0 g.
combinations of added nutrients.
Preparation: (1) Dissolve 2 in 1.
Sterilization : Not specified.
Sterilization: As desired.
Use: Cultivation of carrot bacillus (Bacil-
Uses: Diluent.
lus daucarum) and other organisms caus-
References: Giltner (1921, p. 399).
ing vegetable spoilage.
4. Ashby's Basal Solution (Hoffmann

Added nutrients The author added one of
:
the following materials or combinations

and Hammer)
of materials:
Constituents (a) peptone 10.0 g.
1. Distilled Water 1000.0 cc. fsucrose 30.0 g.
2. KH2PO4 0.2 g. (b) \ asparagin 10.0 g.
3. MgS04 0.2 g. [glycerol 10.0 g.
4. NaCl 0.2 g.
. , fsucrose 30.0 g.
5. CaS04 0.1 g.
\asparagin 1-0 g.
6. CaCOs 5.0 g.
[glycerol 30.0 g.
Preparation : ,^)
\asparagin 1.0 or 10.0 g.
(1) Dissolve the KH2PO4 in a small
amount of water, and add tenth nor- , . /lactose 30.0 g.
mal NaOH to neutral point to phenol- \asparagin 10.0 g.
phthalein. (sucrose 5.0 g.

(2) Dissolve 3, 4, 5 and 6 in remainder of 1 glycerol 10.0 g.
(3) Mix (1) and (2). ammonium
(4) Add suitable carbon source. tartrate 10.0 g.
(5) Distribute in 20.0 cc. lots in 150 cc. (sucrose 5.0 g.
Erlenmeyer flasks. glycerol 10.0 g.
Sterilization: Method not specified. KNO3 10.0 g.
Uses: For study of the nitrogen fixing
(h)(KNO. 0.5g.
bacteria from soil. ^ ^ [soda 0.5 g.
Added Nutrients: Hoffmann and Hammer [sucrose 5.0 g.
suggest the following list of carbon
(i) jglucose 5.0 g.
sources:
[glycerol 5.0 g.
Mannitol 20 g.
-., fglucose 30.0 g.
Maltose 20 g.
[asparagin 10.0 g.
Sucrose 20 g.
Galactose 20 g. . . /(NH4)2S04 0.0025 g.
^^ \Na2CO3 5.0 g.
Inulin 20 g.
Dextrose 20 g. Reference: von Wahl (1906, p. 496).
Levulose 20 g.
Dextrin 20 g. 6. Locke's Solution
RafRnose 20 g. Constituents
Lactose 20 g. 1. Water 1000.0 cc.
References: Hoffmann and Hammer (1910 2. NaCl (0.9 to 1.0%)... 9.0 to 10.0 g.
p. 128). 3. KCl
(0.01%) 0.1 g.
4. CaCl2 (0.02%) 0.2 g.
5. von Wahl's Basal Salt Solution Preparation: (1) Dissolve 2, 3 and 4 in 1.
Constituents Sterilization: As desired.

1. Distilled water 1000.0 cc. Uses: Commonly used as a diluent. Tis-
2. Potassium phosphate 1.0 g. sue neither swells nor shrinks in this solu-
3. CaCh 0.1 g. tion. It is also used as a basis for culture
media, various nitrogen or carbon sources 3. CaCOs 0.5 g.
being added. 4. NaCl 0.5 g.
Reference: Park, Williams and Krumwiede 5. FeS04 some

(1924, p. 122). Preparation
(1) Dissolve 2, 3, 4, 5 and one of the car-
7, Winogradsky's Basal Inorganic Salt bon sources listed under added nu-
Solution (Heinemann) trients in 1.
(2) Distribute in flat bottomed flasks.

Constituents
Sterilization : Steam for 30 minutes on each
1. Distilled Water 1000.0 cc.
of 3 successive days.
2. MgS04 0.5 g.
Use: Study of nitrogen assimilation by
3. CaCl. 0.01 g.
azotobacter and other bacteria from the
4. NaCl 2.0 g.
Preparation: (1) Dissolve 2, 3 and 4 in 1 soil. More nitrogen was assimilated

using dextrose as a source of carbon than
calcium propionate. Little nitrogen was
Uses: Used in soil bacteriological work as
assimilated if the flasks were sealed after
a base to which other materials are added.
inoculation.
Reference: Heinemann (1922, p. 37).
Added nutrients
8. Charrin and Dissard's Basal Salt (a) The authors suggested the use of the
Solution following carbon sources:
Glucose 2.0 g.
Constituents:
Water 1000.0 cc. Calcium propionate 1.0 g.
1.
(b) Percival used the same basic solution
2. KH2PO4 0.1 g.
3. Na2HP04 0.1 g.
and added 2.0 g. glucose.
Variants: Bonazzi added 10.0 g. of glucose,
4. CaCh 0.05 g.
designated the use of deep well water
5. MgS04 0.05 g.
KHCO3 0.134 g. added 1.0 g. CaCOs, specified the addition
6.
of 0.02 to 0.5 g. FeS04-7H20, and also
Preparation
and 6 in stated that 0.23 g. KNO3 and 1.264 g,
(1) Dissolve 2, 3, 4, 5 1.
Adjustment of reaction not given.

Ca(N03)2 might be added if desired.
(2)
Distribute in 50.0 cc. lots in flasks
References: Gerlach and Vogel (1902, p.
(3)
671), Percival (1920, p. 181), Bonazzi
and add 1.0 or 0.5 g. of one of the
added nutrients to each flask. (1921, p. 339).
Sterilization: Autoclave at 120C. Time

10. Rettger, Berman and Sturges' Basal
not specified.
Solution
Use: To study pigment production by
Bacillus pyocyaneus. The authors re- Constituents
ported that asparagin gave the best pig- 1. Water 1000.0 cc.
ment; peptone gave a little pigment; no 2. NaCl 5.0 g.
growth using urea. 3. Na2S04 2.0 g.

Added nutrients: The authors added 0.5 or 4. CaCl2 1.0 g.
1.0 g. of one of the following organic 5. KH2PO4 1.0 g.
materials to each 50.0 cc. lot of medium: Preparation

peptone glycogen (1) Dissolve 2, 3, 4 and 5 in 1.
asparagin acetic acid (2) Add one of the added nutrients listed
urea lactic acid below.
glucose Sterilization: Generally by filtering
Reference: Charrin and Dissard (1893, p. through a Berkefeld filter, in some cases
182). by heat.
Use To study proteolysis by Proteus vul-
:
9. Gerlach and Vogel's Basal Solution garis, B. prodigiosus and B. subtilis.

Constituents: Added nutrients
1. Water 1000.0 cc. (a) Various chemically pure proteins
2. K2HPO4 0.5g. added alone or in combination.
6 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(b) Egg albumin prepared by method of 12. Percival's Basal Solution

Hopkins and Pinkus (1899); amount
Constituents
not specified.
1. Distilled water 1000.0 cc.
Peptone 10.0 g.
(c)
2. K2HPO4 5.0 g.
Reference: Rettger, Berman and Sturges
3. MgS04 2.5 g.
(1916, pp. 15-33).
4. NaCl 2.5 g.
Preparation
11. Omeliansky's Basal Solution
(1) Dissolve 2, 3 and 4 in 1.
Constituents: (2) Add 10.0 g. of one of the added nu-

1. Distilled water 1000.0 cc. trients.
2. Potassium phosphate 0.5 g. Sterilization : Not specified.
3. MgS04 0.03 g. Use: To demonstrate ammonia production
4. NaCl 0.5 g. from protein.
5. Na.CO, 1.0 g. Added nutrients
Preparation (a) Percival added 1.0% of fibrin or
(1) Dissolve 2, 3, 4 and 5 in 1. gelatin.
(2) Add one of the added nutrients as (b) Harvey added 10.0 g. of one of the
indicated under added nutrients. following materials: peptone, fibrin,
Sterilization: The various media under gelatin.
'added nutrients' are sterilized differently. (c) Omeliansky used 1.0 g. potassium
The method for each medium will be given phosphate, 0.5 g. MgSO., 0.1 g. NaCl
with the nutrient. in the basic solution and added one
Use: To study nitrate and nitrite produc- of the listed proteins. He studied
tion from organic nitrogen. Author re- the production of aroma by Bad.
ported that in the concentrations used ester oar omaticum. Growth with
all tests for NH3, nitrites or nitrates were aroma production took place with all
negative. Nessler's reagent was used to materials except casein and keratin.
determine ammonia amyl iodine reaction One of the following materials was
for nitrite and diphenylamine for nitrate. added:
Added nutrients: The author added one of egg albumin (Kahlbaum) 10.0 g.
the following materials to the basic solu- blood albumin (Kahlbaum) 10.0 g. . . .
tion: peptone (Okuney) 10.0 g.

(a) Asparagin 1.0%. Filter through a casein (Hammorsten) (Merck). 10.0 g.
Chamberland filter to sterilize. keratin (Zyzkia, Merck) 10.0 g.
(b) Urea 1.0%. Filter through a Cham- References: Percival (1920, p. 112) Harvey
berland filter to sterilize. (1921-22, p. 102), Omeliansky (1923, pp.
(c) Bouillon 50.0 cc. Both basic solution 409, 411).
and bouillon to be sterile. Method
of sterilization not given.
13. Nawiasky's Basal Salt Solution
(d) Egg white 1.0%. Sterilize in the Constituents
steamer. 1. Water 1000.0 cc.
(e) Fresh urine 50.0 cc. Filter through a 2. NaCl 5.0 g.
Chamberland filter to sterilize. The 3. K2HPO4 2.0 g.
urine gave a slight ammonia test with 4. MgS04 0.5 g.
Nessler's reagent. To remove all Preparation
ammonia add 1.0% soda to the fresh (1) Dissolve 2, 3 and 4 in 1.
and store the filtrate for
urine, filter (2) Neutralize and make slightly alkaline
2daysat25C. overHsSOi. Then fil- (3) Add one of the organic materials
ter through a Chamberland filter as listed in added nutrients to (2).
above to sterilize. Sterilization: Method not given.
Reference: Omeliansky (1899 p. 481). Use: Cultivation of Proteus vulgaris.
Added nutrients: 16. Sohngen's Basal Solution

asparagin 5.0 g
Constituents :
glycocoll 4.0 g
1. Water 75.0 cc.
alanin 6.25 g
isobutylaldehyde 1.6
2. K2HPO4 0.05 g.
g
leucine 2.0
3. CaCOa
g
Preparation :
aspartic acid 4.5 g
(1) Dissolve 2 in 1 and add 3.
Reference: Nawiasky (1908, p. 215).
(2) Place in a liter Erlenmeyer flask.
14. Stoklasa's Basal Solution (3) Add suitable carbon source.
Constituents :
Sterilization : Method not specified.
1. Water (River, Tap) 1000.0 cc. Uses: To study the effect of filter paper
2. Dipotassium phosphate 0.5 g. (colloid) on bacterial processes, particu-
3. CaCOs 1.0 g. larly fixation of atmospheric nitrogen.
Preparation :
Added Nutrients: The author employed
(1) Dissolve 2 and 3 in 1.
the following:
(2) Add suitable carbon source. (a) 1.0 g. glucose + 0.1 g. CaCOs
(3) Distribute in large Erlenmeyer flasks (b) 1.0 g. glucose + 0.1 g. CaCOs + 10.0 g.
(40 cm. X 15 cm.) paper cut in squares of 1 sq. cm.

filter
Sterilization: Sterilize in streaming steam (c) 1.0 g. mannitol + 0.1 g. CaCOa
or in the autoclave. (d) 1.0 g. mannitol + 0.1 g. CaCOj +
Uses: To study nitrogen assimilation by 10.0 g. filter paper
Azotobacter and Radiobacter as in- Reference: Sohngen (1913, p. 628).

fluenced by various carbohydrates.
Added nutrients: The following carbohy- 17. van Delden's Basal Gypsum Solution
drates were suggested by the author to
serve as carbon sources: Constituents :
l-arabinose saccharose 1. Water 1000.0 cc.

1-xylose rhamnose 2. Gypsum 0.4 g.
d-glucose lactose 3. K2HP04 0.5 g.
d-galactose maltose Preparation :
d-fructose (1) Dissolve 2 and 3 in 1.

Reference: Stoklasa (1908 p. 491). (2) Additional nutrients are added as in-
dicated below.
15. Heinze's Basal Solution Sterilization: Not specified.
Constituents : Use: To study sulphate reduction by Mi-
1. Distilled water 1000.0 cc. crospira desulfuricans.
2. CaCh 0.2 g. Added nutrients and variants The author :
3. MgS04 0.4 g. used one of the following combinations:

4. KH2PO4 2.0 g. (a) Added 0.5 g. glucose and 0.5 g. pep-
Preparation : tone.
(1) Dissolve 2, 3 and 4 in 1. (b) Added 1.0 g. glucose, 1.0 g. CaCO,,
(2) Add 1.0% of one of the listed added specified the use of tap water and used
nutrients 1.2 g. gypsum.
(3) Adjustment of reaction not specified. (c) Specified the use of tap water, added
Sterilization : Not specified. 0.5 g.NH4CI, added 0.5 g. glucose and
Use : To study oxalic acid formation. Oxa- used 1.2 g. gypsum.
lic acid was formed in quite small amounts. (d) Specified the use of tap water, added
Heavy formation of HNO3 and ammonia sodium acetate, 0.5
0.5 g. g. asparagin
occurred. and used 1.2 g. gypsum.
Added nutrients: The author employed (e) Specified the use of tap water, added
1.0% of one of the following: asparagin, used 1.2 g. gypsum
1.0 g.
peptone and added 1.25 g. of sodium succinate
hemialbumose or potassium succinate or calcium
gelatin citrate or potassium tartrate or po-
Reference: Heinze (1905, p. 18). tassium malate.
(f) Specified the use of tap water, added Maltose 2.0% + sodium succinate 1.0%
1.0 g. asparagin and used 1.2 g. gyp- Maltose 2.0% + sodium asparaginate
sum. 1.0%
Reference: van Delden (1903-04, p. 83). Maltose 2.0% + ammonium citrate 1.0%
18. Czapek's Basal Solution (Waksman) Maltose 2.0% + peptone 1.0%
Constituents
Maltose 2.0% + asparagin 1.0%
Raffinose 2.0%
1. Distilled water 1000.0 ce.
Melitose 2.0%
2. K2HPO4 1.0 g.
Inulin 2.0%
3. KCl 0.5 g.
Glycerol 1.0, 3.0 or 5.0%
4. MgS04 0.5 g.
Glycerol 1.0, 3.0 or 5.0% + ammonium
5. FeS04 0.01 g.
phosphate 0.5%
Preparation
Inositol 2.0%
2, 3, 4 and 5 in 1.
(1) Dissolve
Mannitol 2.0%
As desired.
Sterilization:
Uses: Used by many investigators
Ammonium formate 1.0%, 2.0% or 5.0%
as a
basic mineral or salt solution for com-
Ammonium Tartrate 1.0%
parison of availability of various nitrogen
Ammonium citrate 1.0%
Sodium, potassium or calcium butyrate
and carbon nutrients.
0.5%, 1.0% or 2.0%
Reference: Waksman (1918, p. 479).
Sodium butyrate (0.5%) 5.0 g. + ammo-
19. Buchanan's Basal Solution nium phosphate 0.5%
Constituents: Calcium butyrate (1.0%) 1.0 g. + am-
1. Water 1000.0 cc. monium phosphate 0.5%
2. KH.PO4 2.0 g. Potassium valerianate 0.5%, 1.0% or
3. MgS04 (0.01%) 0.1 g. 2.0%
Preparation Sodium succinate 0.5%, 1.0% or 2.0%
(1) Dissolve 2, 3 and one of the added Sodium succinate 1.0% + ammonium
nutrients in 1. phosphate 0.5%
(2) Cool on ice. Calcium lactate 0.5%, 1.0% or 2.0%
(3) Filter to remove insoluble precipi- Calcium lactate 1.0% + ammonium phos-
tates. phate 0.5%
(4) Tube. Sodium citrate 1.0%
Sterilization: Intermittently in flowing Sodium citrate 1.0% + ammonium phos-
steam on each of 3 successive days. phate 0.5%
Use To study growth of Bacillus radicicola
: Asparagin 1.0%
bacteroids, and to study gum production Asparagin 1.0, 2.0, 3.0 or 5.0% + am-
by Bacillus radicicola. Author reported monium phosphate 0.5%
that generally carbohydrates and gluco- Asparagin 1.0% + sodium asparaginate
sides favored, while peptones inhibited 1.0%
the growth of B. radicicola; mannitol Sodium asparaginate 1.0, 3.0 or 5.0% +
especially favored growth. ammonium phosphate 0.5%
Added nutrients: One of the following Sodium asparaginate 0.5, 1.0,3.0 or 5.0%
materials or combinations was added: Nutrose 0.1, 0.5, 1.0, 2.0 or 5.0%
Arabinose 2.0% Nutrose 1.0% + ammonium tartrate
Rhamnose (Isodulcitol) 2.0% 0.5%
Glucose 2.0% Nutrose 1.0% + ammonium phosphate
2.0% + peptone 1.0% 0.5%
Mannose 2.0% Nutrose 1.0% + asparagin 1.0%
Galactose 2.0% Peptone (Witte) 1.0%
Levulose 2.0% Peptone (Witte) 1.0% + Ammonium
Sucrose 0.1, 10.0, 20.0, 30.0, or 50.0% phosphate 0.5%
Sucrose 2.0% + peptone 1.0% Peptone (Witte) 1.0% + sodium aspar-
Sucrose 2.0% + KNO3 0.05 to 0.5% aginate 1.0%
Sucrose 2.0% + nutrose 1.0% Peptone (Witte) 1.0% + asparagin 1.0%
Peptone (Witte) 1.0% + KNO3 0.1 or (3) Distribute in 100.0 cc. lots in Erlen-
1.0% meyer flasks.
Amygdalin 2.0% (4) Add 0.5 g. of one of the added nu-
Amygdalin 2.0% + ammonium phos- trients to each flask.
phate 0.5% Sterilization: Not specified.
Salicin 2.0% + ammonium phosphate Use: To study the availability of nitrogen
0.5% for Actinomycetes odorifer, Act. chromo-
Salicin 2.0% genes, Act. albus I and II, Act. S. a, b,
Ammonium phosphate and c. Good growth was obtained with

References: Buchanan (1909, pp. 382, 391, albumin, hemi albumin, casein, asparagin,
62), Tanner (1909, p. 57) and alanine; little growth obtained with
tyrosin, while generally no growth with
20. Fuhrmann's Basal Solution
urea, sulphocarbamide, or dicyandiamide.
Constituents Added nutrients: The author employed 0.5
1. Water 1000.0 cc. g. of one of the following nitrogen sources
2. KH2PO4 1.0 g. for each 100.0 cc. of medium:
3. CaCla 0.1 g. albumin urea
4. MgS04-7H20 0.3 g. hemi albumin sulphocarbamide
5. NaCl 0.1 g. casein alanine
6. FesCle 0.01 g. asparagin tyrosine
Preparation dicyandiamide
(1) Dissolve 2, 3, 4, 5 and 6 in 1. Reference: Munter (1913 p. 373).
(2) Distribute in 100.0 cc. lots.
Sterilization: Method not specified. 22. Meyer's Basal Salt Solution (Perotti)
TJses: To study the availability of various Constituents
nitrogen and carbon sources for Pseudo- 1. Distilled water 1000.0 cc.
monas cerevisiae. 2. K2HPO4 10 g.
Added Nutrients Make : the following addi- 3. CaCh 0.1 g.
tions to each 100.0 cc: 4. MgS04 0.1 g.
(a) Asparagin 1.0 g. 5. xNaCl 0.1 g.
(b) Asparagin 1.0 g. + glucose 1.5 g. 6. Iron chloride 0.01 g.
(c) Asparagin 1.0 g. + sucrose 0.75 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6
(d) Potassium nitrate 1.0 g. + glycerol in 1.
1.0 g. Sterilization: Not specified.
(e) Potassium nitrate 1.0 g. + sucrose Uses: Used by Perotti as a basis for the
0.75 g. preparation of various solutions for the
(f) Potassium nitrate 1.0 g. + glucose study of the dicyandiamid bacteria of
1-5 g. the soil.
(g) Ammonium chloride 1.0 g. + sucrose Variants
0.75 g. (a) Buchanan used a basal solution con-
(h) Ammonium chloride 1.0 g. + glucose taining the following materials:
1.5 g.
1. Water 1000.0 cc.
Reference: Fuhrmann (1906, p. 319). 2. KH0PO4 0.1 g.
3. CaCl. 0.1 g.
21. Miinter's Basal Salt Solution
4. MgS04-7H20 0.3 g.
Constituents 5. NaCl 0.1 g.
1. Water 1000.0 cc. 6. Fe2Cla 0.01 g.
2. MgSOi 0.5 g. Buchanan added one of the following
3. NaCl 0.5 g. materials, and used the media to deter-
4. CaCl2 0.1 g. mine the constituents essential for the
5. K2HPO4 1.0 g. growth of Bacillus thermophilus. Growth
6. FeCls trace was obtained with all materials except as-
Preparation paragin alone.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. asparagin 1.0%
(2) Neutralize with CaCOs and filter. asparagin 1.0% + glycerol 1.0%
asparagin 1.0% + filter paper 1.0% Dulcitol Pentosan from dulse

urea 1.0% + peptone 1.0% Sorbitol Sinistrin
peptone 1.0% + glucose Sucrose Lichenin
peptone 1.0% + lactose 1.0% Galactose
peptone 1.0% + sucrose 1.0% Maltose
(b) Klaeser added 2.0g KNO3 and lO.Og. Mannan from Salep
of glucose. He used the solution to Reference: Gage (1910 p. 21).
study nitrate reduction.
25. Beijerinck and van Delden's Basal
(c) Stapp used l.Og HK2PO4 instead
Solution
of KH2PO4. He dissolved 3.0g
Na2HP04 and 0.5 g uric acid in 45.0 Constituents
cc.water and added 50.0 cc. of the basal 1. Water 1000.0 cc.
solution. The medium was used for 2. MgS04.7H20 8.0 mg.
the isolation of uric acid splitting 3. MnS04.4H20 0.5 mg.
bacteria from the feces and soil. The 4. FeCl3.3H20 0.5 mg.
organisms studied were Bad. co- Preparation: (1) Dissolve 2, 3 and 4 in 1.
bay ae, Bad. capri, Bad. guano, Bad. Sterilization: Not specified.
muscili, Bad. hoUandicus. Uses: Used by Beijerinck and van Del-
Reference : Buchanan (1906 p. 73). Perotti den as a mineral mixture to be added in
(1908 p. 220), Klaeser (1914 p. 38a) Stapp small quantities to a considerable variety
(1920 p. 3). of liquid media.
Reference: Beijerinck and van Delden
23. Box's Inorganic Salt Solution For Fungi (1903 p. 41).
(Tanner)
Constituents
Inorganic liquid media of known composi-
1. Distilled Water 3000.0 cc.
tion, nitrogen as free nitrogen only, com-
2. MgS04 1.5 g.
plete solutions, used as media without
3. K2HPO4 3.0 g.
addition of other nutrients.
4. KCl 1.5 g.
5. FeS04 "Complete" inorganic non-nitrogenous
0.03 g.
media have been described for the cultiva-
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
tion of certain iron bacteria and for certain
algae. In each case it will be noted that
Uses: Cultivation of fungi.
either "well water" or a heavy inoculum of
Reference: Tanner (1919 p. 65).
soil or soil extract is used. It is probable
therefore that ammoniacal or nitrate nitro-
24. Gage's Basal Salt Solution
gen is usually present, and may in some
Constituents cases be essential for the growth of the mi-
1. Water 1000.0 cc. croorganisms.
2. KH2PO4 0.5 g. The important media of this section may
3. MgS04 0.2 g. be differentiated as follows:
4. CaCl2 0.02 g. Ai. Medium employed primarily for iron
5. Fe2(S04)3 1 drop of 10% solution. bacteria.
Preparation Ellis' Ferric Hydroxide Solution 26
(1) Dissolve 2, 3, 4 and 5 in 1. A2. Media for algae. (Cyanophyceae or
(2) Add 1.5 g. of suitable carbon source. blue-green).
Sterilization: Method not specified. Beijerinck's Basal Phosphate Solution. 27
Uses Study of nitrogen fixation and of the
:
Heinze's Basal Solution A 28
morphology of the nitrogen fixing bacteria. Heinze's Basal Solution B 29
Added nutrients: The author suggests the
addition of 1.5 g. of one of the following 26. Ellis' Ferric Hydroxide Solution
carbon sources: Constituents:
Mannitol Galacton from slip- 1. Well water.
Glucose pery elm. 2. Ferric hydroxide.

CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 11
Preparation: (1) Add freshly precipitated 29. Heinze's Basal Solution B

ferric hydroxide (amount not specified)
Constituents
to sterile well water in sterile flask.
1. Water 1000.0 cc.
Uses: Inoculate with Spirophyllum fer-
2. Potassium phosphate neu-
rugineum (one of the iron bacteria) and
tral 0.2 g.
expose to sunlight.
Reference: Ellis (1907 p. 511).
3. MgS04 0.2 g.
4. K2SO4 0.2 g.
27. Beijerinck's Basal Phosphate Solution

5. CaCOa 0.1 g.
6. Iron chloride trace
Constituents Preparation: (1) Dissolve 2, 3, 4, 5 and 6
1. Water 100.0 cc. in 1.
2. K2HPO4 0.02 g. Sterilization: Not specified.

Preparation Uses: Inoculate with a suspension of soil.
(1) Dissolve 2 in 1. Soil algae, such as Nostoc punctiforme
(2) Place in a 3 liter flask and plug with will develop. If soil bacteria are present
cotton. Nostoc punctiforme will grow and nitro-
Sterilization : Not specified. For purposes gen will be assimilated.
used, probably generally unnecessary. Reference: Heinze (1906 p. 653).
Uses: Add an inoculum of 1.0 or 2.0 g.
garden earth and incubate at 16-20C. SUBGROUP I-B. SECTION 3
(In the light?) Oligonitrophilic organ- Inorganic liquid solutions of known com-
isms of the Cyanophyceae (blue-green position; nitrogen supplied as ammonium
algae) will develop, the medium becom- salts; incomplete or basal solutions re-
ing bluish green. quiring the addition of other nutrients.
Variants: van Delden specified the use of The several solutions falling in this sec-
Grabenwasser." tion may be differentiated as follows:
Reference: Beijerinck (1901 p. 562), van Ai.* Containing ammonia as ammonium
Delden (1903-04 p. 85). Lohnis (1913 chloride.
p. 115). Boas' Basal Ammonium Chloride Solu-
tion 30
28. Heinze's Basal Solution A Wherry's Basal Ammonium Chloride
Constituents Solution B 31
1. Distilled water 1000.0 cc. Kendall, Walker and Day's Basal Am-
2. KHsPOi 2.0 g. monium Chloride Solution 32
3. CaCla 0.2 g. Lohnis' Basal Ammonium Chloride
4. FeCls in 10% solution... 20 drops. Salt Solution 33
Preparation A2. Containing ammonia as ammonium
(1) Dissolve 2, 3 and 4 in 1. If K3PO4 is sulphate.
used, the solution may be acidified Bi. Not containing additional salts.
with sulphuric acid and neutralized Buchanan's Basal Ammonium Sul-

with soda. phate Solution 34
(2) Distribute in 100 cc. lots. Cathelineau's Basal Ammonium Sul-
Sterilization: Not specified. phate Solution 35
Uses : Inoculate with 1.0 cc. of a suspension B2. Containing additional salts.
of 1.0 g. of soil in 100.0 g. sterile distilled Pere's Basal Ammonium Sulphate
water. Incubate (in the light?). The Solution 36
soilCyanophyceae (blue green algae) will Henneberg's Basal Ammonium Sul-
develop. Nitrogen may possibly be phate Solution 37
assimilated (fixed) in small amounts. Omeliansky's Basal Ammonium Sul-
Variants: The author substituted K2HPO4 phate Solution 38
or K3PO4 for KH2PO4.
Reference: Heinze (1906 p. 703). See A2, A3, A4 and A5
Kita's Basal Ammonium Sulphate (3) Distribute in 40.0 cc. lots in 100.0 cc,
Solution 39 Erlenmeyer jena glass flasks.
Grimm's Basal Ammonium Sulphate Sterilization: Not specified.
Solution 40 Uses Used to study the formation of starch
:
Waksman and Joffe's Basal Sulphur by molds, Aspergillus orijzae. If starch

Ammonium Sulphate Solution 41 isformed, a blue color appears when iodine
Bokorny's Basal Ammonium Sulphate is added to the culture.
Solution, (Vierling) 42 Added nutrients: The author used 5.0%
Verkade and Sohngen's Basal Ammo- of one of the following carbon sources:
nium Sulphate Solution 43 sucrose glucose
Starkey's Basal Ammonium Sulphate levulose galactose
Solution 44 maltose
A3. Containing ammonia as salts of phos- Reference: Boas (1922 p. 8).
phoric acid.
Ayers and Rupp's Basal Sodium Am- 31. Wherry's Basal Ammonium Chloride
monium Phosphate Solution 45 Solution B
Committee S. A. B. Basal Ammonium
Phosphate Solution 46 Constituents:
Kendall, Day and Walker's Basal Am- 1. Redistilled water 1000.0 cc.
monium Phosphate Solution 47 2. NajCOa 1.0 g.
Laurent's Basal Ammoniima Phosphate 3. KCl 1.0 g.
Solution 48 4. CaCl2 1.0 g.
Fermi's Basal Ammonium Phosphate 5. MgS04 1.0 g.
Solution 49 6. NH4CI 2.0 g.

Palladin's Basal Ammonium Phos- 7. KH2PO4 2.0 g.
phate Solution 50 Preparation
Schukow's Basal Ammonium Phos- (1) Dissolve 2, 3, and 4 in 1. This is
phate Solution 51 called solution "B".

Koser's Basal Ammonium Phosphate (2) Dissolve 6 and 7 in (1).
Solution 52 (3) Distribute in 50 cc. lots and add one
A4 Containing ammonia as ammonium of the test materials to each lot.
nitrate. Sterilization: Not specified.
Munter's Basal Ammonium Nitrate Use: To study acid fastness of tubercle
Solution 53 bacilli. Growth started first on methyl
von Bronsart's Basal Ammonium Ni- alcohol. After 20 days the order of vigor
trate Solution 54 of growth was as follows: Propyl, butyl,
Adolf Mayer's Basal Ammonium Ni- ethyl and methyl alcohol. Culture be-
trate Solution (Smith) 55 came strongly acid fast only in propyl
As. Containing ammonia as Ammonium alcohol.
Carbonate. Added nutrients: The following carbon
Prazmowski's Basal Ammonium Car- sources were used:
bonate Solution (Smith) 56 ethyl alcohol 2.0%
propyl alcohol 2.0%
30. Boas' Basal Ammonitun Chloride
amyl alcohol 2.0%
Solution
butyl alcohol 2.0%
methyl alcohol 2.0%
Constituents leucine 0.1 g.
1. Water 1000.0 cc. tyrosine 0.1 g.
2. NH4CI (0.25%) 2.5 g. alanine 0. 1 g.
3. KH2PO4 (0.25%) 2.5 g. glycocoU 0.1 g.

4. MgS04 (0.15%) 1.5 g. aspartic acid 0.1 g.
Preparation glutamic acid 0.1 g.
(1) Dissolve 2, 3 and 4 in 1. (Salts to be Kahlbaum C. P.)
(2) Add 5.0% of one of the carbohydrates. Reference: Wherry (1913 p. 116).
32. Kendall, Walker and Day's Basal Am- (2) Then add 0.5% ammonium sulphate
monium Chloride Solution to (1).
Sterilization: Sterilize on each of 3 succes-
Constituents:
sive days for 20 minutes in flowing steam.
1. Redistilled water 1000.0 cc.
Use : To study gum production of Bacillus
2. NH4CI 4.0 g.
radicicola.
3. NaCl 5.0 g.
Added nutrients: The following carbon
4. Na2HP04 sources (2.0%) were used by the author:
Preparation
arabinose levulose lactose
rhamnose melitose raffinose
(2) Add one of the nutrients listed below.
glucose inositol mannitol
(3) Distribute in 100.0 cc. quantities in
mannose sucrose inulin
flasks.
galactose maltose starch
Sterilization: Method not given.
Reference: Buchanan (1909 p. 388).
Use: To study lipase production by tuber-
cle bacilli. Different esters were added 35. Cathelineau's Basal Ammonium Sul-
to clear bacteria free culture broth and phate Solution
incubated for 24 hours. Amount of acid
Constituents
produced measured in terms of N/50
1. Water 100.0 cc.
NaOH, determined lipase production.
2. (NH4)2S04 1.0 g.
Added nutrients: The authors used ethyl
Preparation
alcohol, glycerol or mannitol as carbon
(1) Dissolve 2 in 1.
sources.
(2) Add one of the following to (1).
Reference: Kendall, Walker and Day (1914
(a) Glucose 5.0 g.
p. 455).
(b) Sodium succinate 5.0 g.
(c) Sodium phosphate 5.0 g.
33. Lohnis' Basal Ammonium Chloride
(d) Sodium succinate 5.0 g.
Solution
Glucose 5.0 g.
Constituents: (e) Glucose 5.0 g.
1. Water (tap) 1000.0 cc. Sodium phosphate 5.0 g.
2. NH4CI 0.5 g. (f) Sodium succinate 5.0 g.
3. K2HPO4 0.5 g. Sodium phosphate 5.0 g.
Preparation Sterilization: Method not given.
(1) Dissolve 2 and 3 in 1. Use: To study pigment production and
(2) Add 1.0% of one of the listed nutrients fluorescence by Bacillus viridis (Lesage).
Sterilization: Not specified. Pigment production was independent of
Use: Methane production by cellulose de- the amount of phosphates present.
composers. Added nutrients: The author added the
Added nutrients: The author added 1.0% following carbon sources and salts:
of one of the following materials: (a) Glucose 5.0 g.
acetate starch (b) Sodium succinate 5.0 g.
butyrate sugar (c) Glucose 5.0 g.
lactate peptone Sodium succinate 5.0 g.
gum wool (d) Glucose 5.0 g.
Reference: Lohnis (1913 p. 93). Sodium phosphate 5.0 g.
(e) Sodium succinate 5.0 g.
34. Buchanan's Basal Ammonium Sulphate Sodium phosphate 5.0 g.
Solution Reference: Cathelineau (1896 p. 235).
Constituents:
1. Water 1000.0 cc.
36. Pere's Basal Ammonium Sulphate
Solution
2. (NH4)2S04 (0.5%) 5.0 g.
Preparation Constituents
(1) Add 2.0% of one of the carbon sources 1. Water 100.0 cc.
to water and dissolve. 2. Ammonium phosphate 1.0 g.
3. (NH4)2S04 0.5 g. 5. NaCl Trace

4. Potassium phosphate 0.25 g. 6. Chalk
Preparation Preparation
(1) Dissolve 2, 3 and 4 in 1. (1) Dissolve 2, 3, 4 and 5 in 1.
(2) Add one of the test materials in (2) Prepare a 1.0% solution of one of the
amounts given to (1). test materials.
(3) The reaction must be neutral. If (3) Distribute in test tubes.
acid after sterilization, add a few- (4) Add chalk to each tube.
drops of ammonia. Sterilization: Not specified.
Sterilization: Method not given. Use: To study the fermentation ability of
Use: To study the decomposition of car- Bacterium for micicum under either anaero-
bonaceous bodies (polyatomic alcohols, bic or aerobic conditions.
starch, etc.)by Tyrothrix tenuis, Bacilhis Added nutrients : The author used 10 grams
mesentericus vulgatus and Bacillus sub- of the following carbon sources:
tilis. Glucose Dextrin
Added nutrients: The author used the Sucrose Amylum
following substances: Galactose Inulin
Mannitol 10.0 g. Lactose Arabinose
Glycerol 5.0 g. Mannitol Gum arable
Glycerose, amount not given Dulcitol Ethylene glycol
Potato starch 2.0 g. Maltose Glycerol
Sucrose 5.0 g. Erythritol
Glucose 5.0% = 5.0 g Reference: Omeliansky (1903-4 p. 258).
Reference: Per6 (1896 p. 421).
37. Henneberg's Basal Ammonium Sul-

39. Kita's Basal Ammonium Sulphate
Solution
phate Solution
Constituents
Constituents:
Water 1. Water 1000.0 cc.
1. 1000.0 cc.
2. KH2PO4 3.0 g.
2. MgS04 0.25 g.
3. (NH4)2S04 3.0 g.
3. KH2PO4 5.0 g.
4. FeCla solution drops
4. MgS04 2.0 g.
5. Carbon source. 5. (NH4)2S04 5.0 g.
Preparation
Preparation
Dissolve 2, 3 and 4 in 1. (1) Dissolve 2, 3 and 5 in 1.
(1)
(2) Do not adjust the reaction. (2) Add several drops of a ferric chloride
solution to (1).
(3) Add any desired carbon source.
Sterilization: Not specified.
(3) Add 50.0 g. of any desired carbohy-
drate to (2).
Use : To study
a suitable carbon source for
acetic acid organisms, B. pasteurianum,
B. aceti, B. oxydans and B. Kutzingianum.

Use: To study utilization of various carbon
sources by molds, Aspergillus okazaki,
Glucose was found to be a suitable carbon
Aspergillus candidus, Aspergillus albus,
source, but methyl, ethyl or propyl alco-
Aspergillis lamarii, Pseudorhizopus, As-
hol, acetic acid or ammonium tartrate
pergillus glaucus.
were not suitable sources of carbon.
Reference: Kita (1913 p. 434).
Reference: Henneberg (1898 p. 18).
38. Omeliansky's Basal Ammonium Sul- 40. Grimm's Basal Ammonium Sulphate
phate Solution Solution
Constituents: Constituents
1. Distilled water 1000.0 cc. 1. Distilled water 1000.0 cc.
2. Potassium phosphate 0.1 g. 2. K2HPO4 0.5 g.
3. (NH4)2S04 0.1 g. 3. MgS04 0.5 g.
4. MgS04 0.05 g. 4. (NH4)2S04 5.0 g.
Preparation : 41. Waksman and Joffe's Basal Sulphur

(1) Dissolve 2, 3 and 4 in 1. Ammonium Sulphate Solution
(2) Distribute in 30.0 cc. lots in 200.0 cc.
Constituents:
Erlenmeyer flasks.
(3) After sterilization add one of the test
2. (NH4)2S04 2.0 g.
materials to (2). (Amount not speci-
3. K2HP0< 1.0 g.
fied).
4. MgS04 0.5 g.
5. KCl 0.5 g.
Use: To study the utilization of various
6. FeS04 0.01 g.
carbon sources by Oospora lactis and
7. Sulfur 10.0 g.
Aspergillus repens. Growth with ethyl
8. Ca3(P04)2, 2.5 or 10.0 g.
alcohol, isobutyl alcohol, acetic acid,
Preparation :
ethyl ether, propyl, isopropyl and amyl
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and one of
formic acid, methyl, ethyl, propyl, iso-
the added nutrients in 1.
propyl, isobutyl and amyl acetic acid.
(2) Distribute in 100.0 cc. portions in
Other materials give very little or no
250.0 cc. flasks.
growth.
Added nutrients: The author used the Sterilization : Sterilize in flowing steam for
following carbon sources: 30 minutes on 3 consecutive days.
Pentan Use: Enrichment and isolation of sulphur

bacteria, Thiobacillus thiooxidans. If
Heptan
Hexan glucose be omitted fungi do not grow.
Octan Medium supports the growth of all sul-
Methyl alcohol phur oxidizers.
Ethyl alcohol Added nutrients:
Propyl alcohol (a) The authors added 10.0 glucose or any
Isopropyl alcohol other organic or inorganic stimulator.

Butyl alcohol (b) Used the basic solution without any
Isobutyl alcohol additions.
Amyl alcohol Reference: Waksman and Joffe (1922
Formic acid p. 239).
Acetic acid
Propionic acid Ammonium
42. Bokomy's (Vierling) Basal
Butyric acid normal
Sulphate Solution
Isobutyric acid
Valeric acid Constituents:
Ethyl ether 1. Water 1000.0 cc.
Propyl ether 2. (NH4)2S04 1-0 g.
Formaldehyde 3. KH2PO4 0.2 g.

Acetaldehyde 4. MgS04 0.1 g.
Propionic aldehyde Preparation :
Methyl formic acid (1) Dissolve 2, 3 and 4 in 1.

Ethyl formic acid (2) Add 2.0 g. of one of the carbon sources
Propyl formic acid
Isobutyl formic acid
Use: Author used the solution to study
Methyl acetic acid
urease production.
Ethyl acetic acid
Propyl acetic acid Added nutrients: The author added the
Isobutyl acetic acid following carbon sources:
Methyl iodine Glycerol Arabinose
Ethyl iodine Ethyl alcohol Sorbose
Ethyl bromide Lactose Xylose
Propyl bromide Galactose Mannose
Chloroform Rhamnose
Reference: Grimm (1914 p. 648). Reference: Vierling (1920 p. 31).
43. Verkade and Sohngen's Basal Ammo- Preparation

nium Sulphate Solution (1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Add one of the added nutrients.
Constituents
Sterilization : Heat in flowing steam for 30
1. Water 1000.0 cc.
minutes on each of three consecutive
2. K2HPO4 (0.05%) 0.5 g
days.
3. MgS04 (0.05%) 0.5 g
Added nutrients : The author added one of
4. FeCU (0.01%) 0.1g the following:
5. MnS04 (0.01%) 0.1 g
(a) Sulphur 1.0%. The sulphur was
6. (NH4) 2SO4 (0.05%) 0.5 g
weighed out and placed in individual
7. CaCOa
flasks, the basic solution added and
Preparation
sterilized.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(b) Sodium thiosulphate 0.5%. The
(2) Add 0.25% of one of the test materials
thiosulphate was dissolved in water,
and an excess of CaCOs.
sterilized and added to the sterile
Sterilization: Autoclave at 110C. for 10
basic solution in the necessary
minutes.
amounts.
Use: To study the availability of organic
Use: Cultivation of Thiobacillus thioxidans
acids as a carbon source for Aspergillus
and to study oxidation of sulphur.
niger and Pcnicillium glaucum. Fumaric,
Reference: Starkey (1925 p. 138).
citric aconitic, oleic and erucic acids
give good growth. 45. Ayers and Rupp's Basal Sodium Ammo-
Added nutrients: nium Phosphate Solution
maleic acid
Constituents:
fumaric acid
citric acid
2. Sodium ammonium phosphate. . .1.5 g.
itaconic acid
Preparation
phenylitaconic acid
allocinnamic acid
(2) Add one of the combinations given
cinnamic acid
under added nutrients.
aconite acid
(3) Adjust to pH = 7.0
isocrotonic acid
crotonic acid
Use: To study acid and alkali production.
BB-dimethylacrylic acid
Added nutrients and modifications: The
angelic acid
author added one of the following com-
tiglic acid
binations:
oleic acid
(a) Glucose 5.0 g.
elaidic acid
citric acid (neutralized with
erucic acid
NaOH) 2.5 g.
brassidic acid
KCl 0.2 g.
Variants: The authors substituted 0.05%
(b) glucose 5.0 g.
KNO3 for (NH 4)280 4.
KCl 0.2 g.
Reference: Verkade and Sohngen (1920
(c) citric acid (neutralized with
p. 82).
NaOH) 2.5 g.
KCl 0.2 g.
44. Starkey's Basal Ammonium Sulphate
(d) Used sodium ammonium phos-
3.6 g.
Solution
phate in the basic solution and added
Constituents KH2PO4 1.2 g. and glucose 5.0 g. To
1. Distilled water 1000.0 cc. this solution was added 1.2 g. of one
2. (NH4)2S04 0.2 to 0.4 g. of the following acids neutralized
3. KH2PO4 3.0 to 4.0 g. with NaOH: formic acid, acetic acid,
4. CaCls 0.25 g. lactic acid, succinic acid.
5. MgS04 0.5 g. (e) Same as (d) without the addition of
6. FeSOi 0.01 g. acids.
Reference: Ayers and Rupp (1918 pp. 199, 2. (xNH4)2HP04 4.0 g.

209). 3. NaCl 5.0 g.
Preparation
46. Committee S. A. B. Basal Ammonium
Phosphate Solution
(2) Add one of the added nutrients listed
Constituents below.
1. Water 1000.0 cc. (3) Distribute in 100.0 cc. lots.
2. (NH4)H2P04 1.0 g. Sterilization: Not specified.
3. KCl 0.2 g. Use: To study metabolism and lipase pro-
4. Brom Cresol Purple (Satu- duction by tubercle bacilli. Authors
rated aqueous solution) 2.0 cc. used alizarin, neutral red and phenol-
Preparation phthalein to study reaction changes.
(1) Dissolve 2, 3 and 4 in 1. Ziehl-Nielsen stain for staining. Differ-
(2) Adjust to pH = 7 by addition of ent esters were added to clear bacteria
NaOH. free culture broth, incubated for 24 hours
(3) Add fermentable substances. and the amount of acid produced, (meas-
(4) Tube. ured in terms of N/50 NaOH) determined
Sterilization: Method not specified. lipase production.
Uses To test the ability of microorganisms
: Added nutrients: The authors added one
to produce acid in a solution having of the following nutrients:
ammonia as the nitrogen source and a mannitol 10.0 g.
suitable fermentable carbon compound. glycerol 30.0 g.
Added nutrients: Suitable amounts of any sodium acetate
of the carbohydrates or of the polyatomic Reference: Kendall, Day and Walker (1914
alcohols, etc., may
be used. p. 434), Kendall, Walker and Day (1914
Variant. Authors also prepared the me- p. 455).
dium as follows:
(1) Dissolve 1.0g. (NH4)H2P04, 0.2 g.
48. Laurent's Basal Ammonium Sulphate
NaCl, 0.2g.MgSO4 and 10.0 g. of one
Solution
of the added nutrients in 1000.0 cc.
water. Constituents:
(2) Adjust to pH = 7.0 by the addition of 1. Water 1000.0 cc.
NaOH. 2. Ammonium phosphate (neu-
Indicator may be added: tral) 2.5 g.
(3)
Litmus 3. Potassium phosphate (neu-
Phenol red, 2.0 cc. saturated aqueous tral) 2.5 g.
solution per liter 4. MgS04 1.0 g.
Brom cresol purple, 2.0 cc. saturated Preparation: (1) Dissolve 2, 3 and 4 in 1.
aqueous solution per liter Sterilization: As desired; not specified.

Brom cresol purple, 1.0 cc. saturated Uses Used with the addition of 1.0% of any
:
aqueous solution per liter one of a considerable variety of nitroge-

Cresol red, 1.0 cc. saturated aqueous nous and carbon compounds.
solution per liter Added nutrients
Brom cresol green 2.0 cc. of a 2.0% (a) The following were employed:
alcoholic solution per liter saccharose potassium lactate
Brom-chlor phenol blue 5.0 cc. of lactose potassium citrate
saturated aqueous solution per liter glucose potassium tartrate
Reference Committee Society of American
: mannite ammonium bimalate
Bacteriologists (1922 p. 523), (1923 p. 11). glycerine sodium butyrate
peptone sodium hippurate
47. Kendall, Day and Walker's Basal
asparagin sodium formate
Ammonium Phosphate Solution
potassium sue- potassium acetate
Constituents: cinate
1. Redistilled water 1000.0 cc. These were used by Laurent in his
studies of Bacillus fluorescens (3) Distribute in thin layers in Erlen-

putidus. meyer flasks.
(b) Laurent cultivated Sclerotinia Liber- Sterilization : Method not specified.
tiana using the basic solution and Use: Cultivation of Chlorothecium sac-
adding 1.0% peptone, 2.5% lactose, charophilnm.
2.5% glycerol and 2.5% potassium Variants: The author suggests the follow-
tartrate. ing carbon sources:
Variants : Smith cultivated plant parasites saccharose glucose
on a medium composed of 2.0 g K2HPO4, mannitol raffinose
0.1 g ammonium phosphate, 0.19 MgS04, Reference: Palladia (1903 p. 146).
and used 5.09 sodium formate or acetate
as an added nutrient. 51. Schukow's Basal Ammonium Phosphate
Reference: Laurent (1899 p. 43, 81). Smith Solution
(1905 p. 50). Constituents
1. Water 1000.0 cc.
49. Fermi's Basal Ammonium Phosphate 2. Ammonium phosphate 5.0 g.
Solution 3. Potassium phosphate 1-0 g.
Constituents 4. MgS04 0.5 g.
1. Water 1000.0 cc. Preparation
2. Ammonium Phosphate (1) Dissolve 2, 3 and 4 in 1.
(0.5 to
1.0%) 5.0 to 10.0 g. (2) Add 9.0 to 10.0% of one of the organic
3. KH2PO4 (0.5%) 5.0 g. acids to (1).
4. MgS04 (0.5%) 5.0 g. (3) Adjustment of reaction not specified.
5. K3PO4 (0.05%) 0.5 g. (4) Distribute into fermentation flasks
Preparation (sealed with H2SO4).
(1) Dissolve 2, 3, and 5 in 1.
4 (5) After inoculation plug the flask with
(2) Add one of the added nutrients. a cork and seal with paraffin.
Sterilization: Not specified. Sterilization: Sterilize in a steamer
Use: Cultivation of schizomycetes. (method not given).
Added nutrients: The author added 5.0% Use: To study the utilization of acids by
glycerol or 5.0% sucrose. yeast. The author found 0.1 g. tartaric,
Variants: Tanner gave the following as 0.15 g. malic and 0.26 g. citric acid was
Fermi's Solution: used in 65 days by the yeast. Acid de-
1. Distilled water 1000.0 cc. terminations were made by titration with
2. MgS04-7H20 0.2 g. normal NaOH, using litmus as an indi-
3. K2HPO4 1.0 g. cator.
4. (XH4).HP04 10.0 g. Added nutrients and variants
5. Glycerol 45.0 g. (a) The author added 9.0 to 10.0% of one
Reference: Fermi (1892 p. 26), Tanner of the following organic acids:
(1919 p. 68). tartaric malic citric
(b) Henneberg specified the use of 0.3%
50. Palladin's Basal Ammonium Phosphate KH,P04 and used 0.1% MgS04 in-
Solution stead of 0.05%. He added 5.0% glu-
Constituents cose and used it for the cultivation of
1. Water 1000.0 cc. lactic acid bacteria.
2. Ammonium phosphate 4.7 g. Reference: Schukow (1896 p. 607), Henne-
3. Potassium phosphate 3.0 g. berg (1903 p. 7).
4. MgS04 1.0 g.
5. CaClj 1.0 g. 52. Koser's Basal Ammonium Phosphate
6. FeCl2 Trace Solution
(1) Dissolve 2, 3, 4, 5 and 6 in 1. 1. Distilled water 1000.0 cc.
(2) Add sufficient carbohydrate to make 2. NaCl 5.0 g.
solution one fourth molar. 3. MgS04-7H20 0.2 g.
CULTURE MEDIA FOR CULTIVATIOX OF MICRO ORGAXISMS 19
4. (NH4)H2P04 1.0 g. Sterilization: Not specified.

5. K,HP04 1.0 g. Uses: To study the availability of various
Preparation carbon sources for Actinomyces odorifer,
(1) Dissolve 2, 3, 4, 5 and one of the test Act. chromogenes, Act. albus and others.
materials in 1. Solution is clear at Glycerin laevulose, dextrose, galactose,
pH = 6.7 to 6.8. mannite and starch were good carbon
(2) Add one of the test materials. Add sources for all the actinomycetes studied.
the different organic acids to (1) in The remaining carbohydrates serve as
such amounts so that when brought to suitable carbon sources for some of the
pH reading of 6.8 by addition of actinomyces but not all. Oxalic acid,
NaOH the resulting concentration of tartaric acid, and hippuric acid are not
the Na salt will be approximately suitable carbon sources for the actinomy-
0.2%. cetes studied. The remaining acids are
(3) Tube in 8 to 10 cc. lots. generally fair carbon sources for most of
Sterilization: Autoclave at 15 pounds pres- the actinomycetes studied.
sure for 15 minutes. Added nutrients: The author used the
Use: To study the utilization of organic following carbon sources:
salts and acids by the colon-aerogenes sucrose glucose
group. Glucose and lactose were added mannitol arabinose
as controls to determine if the solution galactose laevulose
would support the growth of the organism. lactose glycerol
Added nutrients: The author added ap- inulin starch
proximately 0.2% of the sodium salt of Variants: The author omitted the CaCU
the following acids. In case of citric and CaCOs and used one of the following
acid 0.1 to0.5% was used, carbon sources. The acid was neutral-
glucose 0.2% malic ized and made slightly alkaline by the
lactose 0.2% lactic addition of Na.COs or CaCOs- 0.025 g.
sodmm salt of one of glyceric nitrogen in the form of NH4NO3 and 0.25
the organic acids citric g. of the test material was added to each
acetic tartaric 50.0 cc. of medium.
propionic mucic oxalic acid zitric acid
n-valeric malonic acetic acid hippuric acid
n-butyric oxalic succinic acid uric acid
iso-valeric benzoic malic acid humus acid
n-caproic salicylic tartaric acid aspartic acid
succinic o-phthalic Reference: Miinter (1913 pp. 368, 371).
Reference: Koser (1923 p. 497).
53. Miinter's Basal Ammonium Nitrate 54. von Bronsart's Basal Ammonium Nitrate
Solution Solution
1. Water 1000.0 cc. 1. Water 1000.00 cc.
2. MgS04 0.5 g. 2. iMgS04 (0.25%) 2.5 g.
3. NaCl 0.5 g. 3. KH2PO4 (0.25%) 2.5 g.
4. CaCl. 0.2 g. 4. NH4NO3 (1.0%) 10.0 g.
5. K2HPO4 1.0 g. Preparation
6. NH4NO3 (1) Dissolve 2, 3 and 4 in 1.
7. CaCOa (2) Add one of the test materials to (1).
Preparation Sterilization: Not given.
(1) Dissolve 2, 3, 4 and 5 in 1. Use: To study utilization of various car-
(2) Neutralize by the addition of CaCOs. bon sources by Xylaria, Xylaria arhua-
(3) To each 100.0 cc. of (2) add 0.025 g. cula, Xylaria polymorpha, Xylaria hy-
of nitrogen in the form of XH4NO3. poxylon. Growth best using starch as a
(4) Add 1.0 g. of one of the test materials carbon source with Xylaria arbuscula,
to (3). and levulose produces best growth with
Xylaria polytnorpha, Xylaria hypoxylon Ai.* Ammonia present as ammonium chlo-

growth very poor in any of the solutions. ride.
Added nutrients The author used the
: fol- Hewlett's Ammonium Chloride Solution
lowing carbon sources: (Johnson) 57
Starch levulose Beijerinck and Minkman's Ammonium
inulin glucose Chloride Solution 58
dextrin mannitol Aj. Ammonia present as ammonium car-
Reference: von Bronsart (1919 p. 57). bonate.
Prazmowski's Ammonium Carbonate
55. Adolf Mayers' Basal Ammonium Nitrate Solution. (Tanner) 59
Solution (Smith) A3. Ammonia present as ammonium sul-
Constituents: phate.
1. Distilled water 1000.0 cc. Bi. Without additional salts.
2. MgS04 10.0 g. Wimmer's Ammonium Sulphate Solu-
3. NH4NO3 15.0 g. tion 60
4. Ca3(P04)2 0.1 g. B2. With other salts.
5. Potassium phosphate 10.0 g. Ci. Magnesium carbonate added.
Preparation Di. Phosphates not added.
(1) Dissolve 2, 3, 4, 5 and one of the added Winogradsky's Ammonium Sulphate
nutrients in 1. Solution (Harvey) 61
Sterilization: Not specified. D2. Phosphates added.
Use: Study of phosphorescent and acid El. All additional salts of mono valent
forming bacteria. cations.
Variants: Add 3.0% NaCl if luminous bac- Buhlert and Fickenday's Ammonium
teria are to be cultivated, and an excess Sulphate Solution 62
of pure CaCOs if acid forming bacteria E,i. Additional salts of mono and di valent
are to be grown. cations.

Added nutrients Smith added any desired
: Fi. Calcium salts present.
carbohydrate, alcohol, etc. Winogradsky's Ammonium Sulphate
Reference: Smith (1905 p. 197). Solution 63
Giltner's Modified Winogradsky's Am-
56. Prazmowski's Basal Ammonium Car- monium Sulphate Solution 64
bonate Solution (Smith) F2. Calcium salts not present.
Constituents Winogradsky and Omeliansky's Am-
1. Distilled water 1000.0 cc. monium Sulphate Solution 65
2. K2HPO4 5.0 g E3. Containing Additional salts of tri-
3. MgS04 5.0 g. valent cations.

4. (NH4)2C03 5.0 g. Gibbs' Ammonium Sulphate Solution.
5. CaCl, 0.5 g. (modified by Gowda) 68
Preparation C2. Magnesium carbonate not added.
(1) Dissolve 2, 3, 4, 5 and one of the added Di. Calcium carbonate, chalk or lime
nutrients in 1. added.
Sterilization: Not specified. El. Magnesium sulphate added.
Use: Study of phosphorescent and acid Omeliansky's Ammonium Sulphate
forming bacteria. (Harvey)
Solution. 67
Added nutrients Smith added any desired
: E2. Magnesium sulphate not added.
carbohydrate, alcohol, etc. Christensen's Ammonium Sulphate
Reference: Smith (1905 p. 197). Solution 68
D2. Calcium carbonate chalk or lime not
SUBGROUP I-B. SECTION 4 added.
Inorganic liquid media of known composi- Gage's Ammonium Sulphate Solu-
tion; nitrogen supplied as ammonium salts; tion 69
complete nutrient solutions primarily for

study of organisms oxidizing ammonia to
nitrites. See A2 A3 and A4.
Omeliansky's Ammonium Sulphate 60. Wimmer's Ammonium Sulphate

Solution. (Lohnis-Arnd) 70 Solution
Lolinis' Ammonium Sulphate Solu-
Constituents:
tion 71
A4. Ammonia present as a salt of phos-
2. (NH4)2S04 2.0 or 4.0 g.
phoric acid.
Preparation: (1) Dissolve 2 in 1.
Stutzer's Ammonium Magnesium
Phosphate Solution 72
Use: For study of nitrification (production
57. Hewlett's (Johnson) Ammonium Chlo- of nitrites) by soil bacteria.
ride Solution. Reference: Wimmer (1904 p. 140).
Constituents
61. Winogradsky Ammonium Sulphate
Solution (Harvey)
2. NH4CI 0.5 g.
3. Potassium phosphate 0.1 g. Constituents
4. MgS04 0.02 g. 1. Distilled water 1000.0 cc.
5. CaCl. 0.01 g. 2. (NH4)2S04 1.0 g.
6. CaCOs 5.0 g. 3. K2SO4 1.0 g.
Preparation 4. MgS04 7.5 g.
(1) Dissolve 2, 3, 4 and 5 in 1. Preparation: (1) Dissolve 2, 3 and 4 in 1.
(2) Add 6 and distribute into flasks. Sterilization : Not specified.
Sterilization: Not specified. Use: Study of nitrifying organisms.
Use: For the study of nitrite formation. Reference: Harvey (1921 p. 106).
Reference: Johnson (1912 p. 219).
58. Beijerinck and Minkman Ammonium 62. Buhlert and Fickendey Ammonium
Chloride Solution Sulphate Solution
Constituents: Constituents:
1. Water 100.0 cc. 1. Water 1000.0 cc.
2. K2HPO4 0.02 g. 2. (NH4)2S04 4.0 g.
3. NH4CI 0.02 g. 3. Potassium phosphate 2.0 g.
4. NaHCOs 0.1 g. 4. Basic magnesium carbonate
Preparation (4MgC03-Mg(OH)2-5H20).. 40.0 g.
(1) Dissolve 2, 3 and 4 in 1. Preparation: (1) Dissolve 2, 3 and 4 in 1.
(2) Distribute into Sohngen's culture Sterilization: Not specified.

apparatus. Use: Study of nitrifying bacteria of the
Sterilization: Not specified. soil.
Use : Inoculate with soil for study of forma- Variants
tion of nitrites. Cultivation of Bacillus (a) Fremlin's (described by Gage) solu-
sausseri. tion is simifar; the potassium phos-
Reference: Beijerinck and Minkman phate is specified as KH2PO4, and the
(1910 p. 60). proportions as follows:
1. Water (nitrite free) 1100.0 g.
59. Prazmowski's Ammonium Carbonate
2. KH2PO4 1.0 g.
Solution (Tanner)
3. (NH4)2S04 1.0 g.
Constituents 4. MgC03 1.0 g.
1. Distilled water 1000.0 cc. 2 and 3 dissolved in 1000.0 cc. water
2. K2HPO4 5.0 g. and 4 in 100.0 cc. water. These solu-
3. MgS04-7H20 5.0 g. tions are sterilized separately and
4. (NH4)2C03 5.0 g. mixed aseptically.
5. CaCla 0.5 g. (b) Percival distributed the solution in
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 25.0 cc. lots, and added 20.0 cc. of a
SterilizationNot specified.
: mixture of equal parts soil and sterile
Reference: Tanner (1919 p. 63). tat) water to each lot.
(c) Abbott gave the following solution: (3) Add several tenths grams of MgCOj
1. Water 1000.0 cc. to each lot.
2. (NH4)2S04 1.0 g. (4) Sterilize intermittently.
3. Potassium phosphate... 1.0 g. (5) Add 1.0 or 2.0 cc. of a sterile 2.0%
4. MgCOs (Basic) (NH4)2S04 solution to each lot under
(1) Dissolve 2 and 3 in 1. aseptic conditions.
(2) Flask in 100.0 cc. quantities. (c) Harvey modified Burri and Stutzer's
(3) Add 0.5 to 1.0 g. of basic MgCOs sus- solution in that he used 1.0 g.
pended in a little sterile water to K2HPO4 instead of KH2PO4 and used
each flask. 2.0 g. NaCl instead of 0.5 g.
References: Buhlert and Fickendey (1906 References: Winogradsky (1891 p. 577),
p. 404), Barthel (1910 p. 112), Percival Burri and Stutzer (1896 p. 106), Harvey
(1920 p. 208), Gage (1910 p. 14), Abbott (1921-22 p. 105) Heinemann (1905 p. 129).
(1921 p. 603). Modified Winogradsky's
64. Giltner's
Ammonium Sulphate Solution
63. Winogradsky's Ammonium Sulphate Constituents:
Solution 1. Distilled water 1000.0 cc.
2. (NH4)2S04 0.4 g.
Constituents:
3. MgS04 0.05 g.
4. K2HPO4 0.1 g.
2. Potassium phos-
5. NaoCOs 0.6 g.
phate 1 g.
CaClo trace
6.
3. MgS04 0.5 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6
4. CaCla trace
in 1.
5. MgCOs amount not specified
6. (NH4)2S04 2.0 to 2.5 g.
Use: Study of nitrate formation.
Preparation
Reference: Giltner (1921 p. 369).
(2) Distribute in flasks.
65. Winogradsky and Omeliansky's
(3) After sterilization add MgCOs Ammonium Sulphate Solution
(amount not specified) and from 2 to Constituents:
2.5 parts per thousand of (NH4)2S04. 1. Distilled water 1000.0 cc.
Sterilization: Method not specified. 2. (NH4)2S04 2.0 g.
Use: To show nitrification by organisms 3. Potassium phosphate 1-0 g.
found in the soil. Inoculate with soil. 4. MgS04 0.5 g.
Variants 5. NaCl 2.0 g.
(a) Burri and Stutzer 6. FeS04 0.4 g.
1. Water 1000.0 cc. 7. MgCOs in excess
2. NaCi 0.5 g. Preparation
3. KH2PO4 '
1.0 g. (1) Dissolve 3, 4, 5 and 6 in 1.
4. MgS04 0.5 g. (2) Distribute in 50.0 cc. lots.

5. CaCU trace (3) Add MgCOs in excess to each flask.
6. (NH4)2S04 2.0 g. (4) After sterilization add 1.0 cc. of
7. MgCOs a sterile 1.0% solution of pure
(b) Heinemann (NH4)2S04 under aseptic conditions.
1. Water 1000.0 cc. Sterilization Method not specified.
:
2. Potassium phosphate 1.0 g. Use To study nitrification by soil bacteria.

:
3. MgS04 0.5 g. Variants: The appended table gives com-

4. CaCla 0.01 g. binations used by various authors. The
5. NaCl 2.0 g. preparation of these variants is practi-
6. MgCOs cally the same as for the original. Omeli-
7. (NH4)2S04 2.0 g. ansky found that repeated inoculation
(1) Dissolve 2, 3, 4 and 5 in 1. from this medium into the same medium
(2) Distribute in 20.0 cc. lots. usually yielded a pure culture of nitrify-
ing organisms when initially inoculated (3) Distribute (1) in 5.0 cc. lots.
with soil. Giltner and Ldhnis used (4) Add 0.5 cc. of (2) to each 5.0 cc. lot
Harvey's solution (1.0 g. (NH4)2S04) and of (3).
suggested the use of either CaCOa or Sterilization: Not specified.
MgCOs. He reported that both nitrite Use: To isolate nitrite formers from the
and nitrate bacteria are stimulated if soil and to study nitrification. Tromms-
CaCOs is used instead of MgCOs. Tan- darf's reagents were used to test for the
ner used KH2PO4 in Harvey's (a) solution presence of nitrites.
instead of K2HPO4. Reference: Gowda (1924 p. 252).
OMELTANSKY
INVESTIGATOR 1899
Distilled water
(NH4)2S04
NaCl
Potassium phosphate
MgS04
FeS04
MgCO,
K2HPO4
KH2PO4
KCl
5. NaCl (0.01%) 0.1 g. 300.0 cc. Erienmeyer flasks, each con-

6. FeClj trace taining 0.5 g. pure K2CO3.
7. CaCO, Sterilization: (1) is sterilized for one hour
Reference: Harvey (1921-22 p. 103), Nik- in streaming steam. The flasks contain-
lewski (1910 p. 410). ing theK2CO3 are sterilized for 20 minutes
under a pressure of 2 atmospheres in the
68. Christensen's Ammonium Sulphate autoclave.
Solution Use: Inoculate with 5.0 g. of soil to study
nitrification.
Constituents
Reference: Arnd (1916 p. 561).
1. Water 1000.0 cc.
2. (NH4)2S04 1.6 g.
3. K0HPO4 2.0 g. 71. Lohnis' Ammonium Sulphate Solution
Preparation
Constituents:
(2) Distribute in 50 cc. lots in 500.0 cc.
2. (NH4)2S04 1.0 g.
tuberculin flasks.
3. K2HPO4 1.0 g.
(3) Add 1.0 g. of lime to each flask.
Sterilization: 20 minutes at 120C. in auto-
4. MgS04 0.5 g.
clave.
5. NaCl 2.0 g.
6. FeS04 0.4 g.
Use : Inoculate with soil to study nitrifying
Preparation
bacteria.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Reference : Christensen (1913 p. 418).
(2) During incubation at 20 to 25C.
69. Gage's Ammonium Sulphate Solution allow some ordinary gas to enter the
incubator.
Constituents
1. Water 1000.0 cc.
2. (NH4)2S04 4.0 g.
Use: To study the assimilation of Ca2 by
B. oUgocarbophilus.
3. KH2PO4 2.0 g.
4. MgS04 1.0 g.
Reference: Lohnis (1913 p. 106).
5. FeS04 0.8 g.
6. NaCI 4.0 g. 72. Stutzer's Ammonium Magnesium
Preparation Phosphate Solution
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Constituents:
(2) Distribute in Erlenraeyer flasks.
Sterilization: Method not specified.
1. Water 2000.0 cc.
Use: Study of nitrification by soil organ- 2. K2HPO4 2.0 g.
isms. 3. NaCi 0.5 g.
Reference: Gage (1910 p. 31).

4. Dry ammonium magnesium
phosphate 20.0 g.
5. FeS04 0.5 g.
70. Omeliansky 's Ammonium Sulphate Solu-
6. MgC03 20.0 g.
tion (Lohnis-Arnd)
Preparation
Constituents (1) Dissolve 2, 3 and 4 in 1000.0 cc. water.
1. Distilled water 1000.0 cc. (2) Dissolve 5 and 6 in 1000.0 cc. water.
2. (NH4)2S04 1.0 g. (3) After sterilization mix (1) and (2)
3. K2HPO4 1.0 g. in equal amounts and distribute in
4. MgS04 + 7H20 0.5 g. sterile flasks by means of sterile
5. NaCl 2.0 g. pipettes.

6. FeS04 + 7H20 0.4 g. Sterilization: Method not specified.
7. K2CO, Use: Inoculate with a loop of soil suspen-
Preparation sion. Transfer from flask to flask, using
(1) Dissolve 2, 3, 4, 5 and 6 in 1. as an enrichment medium.
(2) After sterilization distribute under Variants: Stutzer used the constituents in
aseptic precautions, in 50.0 cc. lots in the following proportions:
1. Water 1000.0 cc. Lieske's Ammonium Sulphate Solu-

2. K2HPO4 10 g. tion 80
3. NaCl 0.25 g. Gottheil's Ammonium Sulphate Solu-
4. FeS04 0.025 g. tion. (No. XI) 8.
5. Ammonium magnesium Gosling's Ammonium Sulphate Solu-

phosphate (drj-) 20.0 g. tion 82
6. MgCOs 20.0 g. Beijerinck and van Deiden's Ammo-
The medium was prepared by mixing 5 nium Sulphate Solution 83
and 6 in a mortar and adding a teaspoon- B4. Ammonia supplied as a salt of phos-
f ul or more of the mixture to flat bottomed phoric acid.
flasks. Then add 50.0 to 100.0 cc. of a Beijerinck and van Deiden's Manga-
solution of 2, 3 and 4 in 1 to each flask. nese Ammonium Phosphate Solu-
Sterilize the flasks. Method not given. tion 84
Reference: Stutzer (1901 p. 173). Beijerinck and van Deiden's Ammo-
nium Phosphate Solution 85

73. Waksman's Ammonia and Sulphur
Inorganic liquid media of known com- Solution No. 2
position, nitrogen supplied as ammonium
Constituents
salts, complete nutrient solutions, but not
1. Distilled water 1000.0 cc,
used primarily for organisms oxidizing
ammonia to nitrites.
2. (NH4)2S04 0.2 g.
A few media have been described contain- 3. iMgS04-7H20 0.5 g.
ing ammonium salts but no organic carbon

4. KH2PO4 1.0 g.
5. Ca3(P04)2 (re-precipitated) 2.5 g.
for the cultivation of microorganisms which
6. Sulfur 10.0 g.
do not secure their growth energy by oxida-
tion of ammonia. These media are of two
7. H3PO4
Preparation
general classes, those used with sulphur or
compounds for the cultivation of the (1) Dissolve 2, 3 and 4 in 1.
its
bacteria oxidizing sulphur, and those for (2) Weigh sulfur and Ca3(P04)2 sepa-
rately into the individual flasks into
other types of organisms, including the
algae. The various media may be differen-
which the medium is distributed
(100.0 cc. portions are usuall}' placed
tiated as follows:
in 250 cc. flasks).
Ai. Primarily for organisms oxidizing sul-
phur or its compounds. (3) Adjust to pH = 3.0 by the addition of
Waksman's Ammonia and Sulphur

M/1 H3PO4.
Solution no. 2
Sterilization : Sterilize in flowing steam for
73
30 minutes on 3 successive days.
Waksman's Ammonia and Sulphur
Solution 74
Use: Isolation and cultivation of Thio-
bacillus thiooxidans.
Waksman's Sulphide Solution 75
Beijerinck's Thiosulphate Solution.... 76
Reference : Waksman (1922 p. 606).
A2. Not primarily for organisms oxidizing

sulphur.
74. Waksman's Ammonia and Sulphur
Solution
Bi. Ammonia supplied as ammonium
chloride. Constituents:
Kaserer's Hydrogen Ammonium Chlo- 1. Distilled water 1000.0 cc.
ride Solution 77 2. (NH4)2S04 0.2 g.
Beijerinck and van Deiden's Ammo- 3. MgS04-7H20 0.5 g.
nium Chloride Solution 78 4. KH2PO4 3.0 g.
B2. Ammonia supplied as ammonium 5. CaCla 0.25 g.
nitrate. 6. Sulphur (powdered) 10.0 g.
Richter's Ammonium Nitrate Solu- Preparation
tion 79 (1) Dissolve 2, 3, 4 and 5 in 1.
B3. Ammonia supplied as ammonium sul- (2) Weigh out the sulphur separately in
phate. the individual flasks into which the
(1) is distributed (100 cc. in 250 cc. 4. K2HPO4 0.02 g.

flasks usually). 5. NH4CL 0.01 g.
Sterilization : Sterilize in flowing steam for 6. MgCl2 0.01 g.
30 minutes on 3 consecutive days. Preparation:
Use: Isolation and cultivation of Thio- (1) Dissolve 2, 3, 4, 5 and 6 in 1.
bacillus thiooxidans. The organisms can (2) Adjustment of reaction not specified.
grow at a pH = 1.0 with a maximum at (3) Inoculate with a little ditch or canal
pH = 3.0 to 4.0. water or ditch mud. Incubate at
Variants: The author added a trace of 28 to 30 C.
FeS04 and the following combinations of Sterilization: None required.
potassium salts of phosphoric acid to the Use: Enrichment and isolation of organ-
above solution containing no potassium isms utilizing carbonic acid, Thiobacillus
phosphate, thioparus. Two or three days after
(a) KH2PO4 3.0 g. pH = 4.2 inoculation the surface is covered with a
'KH2PO4 1.5 g.\ ^TT _ 5 4
layer of free sulphur filled with bacteria.
Variants
(c) K2HPO4 3.0 g. pH = 6.0 (a) When Beijerinck used an inoculum
. ,. /K2HPO4 3.0 g.l ^ _..
P^ - ^-^
from sea water, 3.0% NaCl was added
^^)
icaCOa 10.0 g./ to the solution.
At the various pH values the author (b) Waksman used the above solution
found starting with acid medium some with either 0.02 g. Na2HP04 or K2-
Th. thiooxidans give final pH = 1.2. Soil HPO4 and added one of the following
bacteria produce no acid at pH = 4.2 combinations:
but change pH = 8 to pH = 7.6. (1) KH2PO4 3.0 g. pH = 5.4
Reference: Waksman (1922 pp. 606, 609 (2) NaHCOa 1.0 g. pH = 8.8
to 613). fNaHCOalLOg. ^3 =
^'^'
\CaCO3 P^ ^-^
jlO.Og.
9 4
75. Waksman's Sulphide Solution
He found that Thiobacillus thio-
Constituents oxidans produced a pH = 1.4 in
1. Water 1000.0 cc. variant (b) (1).
2. K2S 5.0 g. (c) Trautwein substituted Na2HP04 for
3. NH4CI 0.1 g. K2HPO4 in the original solution and
4. MgClj 0.1 g. used the solution to grow Thionic acid
5. Na2HP04 or K2HPO4 0.2 g. bacteria (Omeliansky).
6. NaHCOs 1.0 g. Reference: Beijerinck (1903 p. 595), Waks-
7. CaC03 10.0 g. man (1922 p. 609), Trautwein (1921 p.
or CaClj 0.25 g. 518).
Preparation
77. Kaserer's Hydrogen Ammonium Chlor-
(1) Dissolve 2, 3, 4 and 5 in part of 1.
ide Solution
(2) Dissolve 6 and 7 in rest of 1.
(3) Add (1) to (2) by means of sterile Constituents:

pipettes after sterilization. 1. Water 1000.0 cc.
(4) pH = 7.5. 2. K2HPO4 (0.05%) 0.5 g.
Sterilization: Autoclave at 15 pounds pres- 3. MgS04 (0.02%) 0.2 g.
sure for 15 minutes. 4. NH4CI (0.1%) 1.0 g.
Use: To show oxidation of K2S by Thio- 5. NaHCOs (0.05%) 0.5 g.
bacillus thiooxidans 6. Iron chloride trace
Reference: Waksman (1922 p. 609-15). 7. Hydrogen
Preparation
76. Beijerinck's Thiosulphate Solution
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Constituents (2) Flask.
1. Water 100.0 cc. (3) Following sterilization, if the solu-
2. Na2S203-5H20 0.5 g. tion is sterilized, inoculate the solu-
3. NaHCOa 0.1 g. tion with soil.
(4) Prepare hydrogen gas from c.p. 3. NaHC03 (0.001%) 0.01 g.

H2SO4 and zinc. 4. (NH4)2S04 (0.001%) 0.01 g.
(5) Pass hydrogen and carbonic acid into 5. Potassium phosphate trace
the flask. 6. MgS04
Sterilization: Not specified. Preparation
Use: To show oxidation by soil forms, (1) Prepare Magnesium bicarbonate by
Bacillus oligocarbophilus and Bacillus adding pure carbonic acid to a sus-
methylicus After 5 days the amount of
. pension of Magnesium carbonate.
hydrogen was less in the flask inoculated (2) Filter.
with soil than in the control flask. (3) Dilute the filtrate 1 to 10 with water.
Variants Lantzsch using the same solution
: (4) Dissolve 3, 4, 5 and 6 in (3).
found that Actinomyces (Bacillus oligo- Sterilization: Not specified.
carbophilus oxidized hydrogen in a non- Use: Cultivation oi Leptothrix ochracea.
sterilized medium, but failed in a steril- Reference: Lieske (1919 p. 422).
ized medium.
Reference: Kascrer (1906 p. 686), Lantzsch 81. Gottheil's Ammonium Sulphate Solution
(1922 p. 316). (No. XI)
Constituents:
78. Beijerinck and van Delden's Ammonium 1. Distilled water 1000.0 cc.
Chloride Solution 2. Potassium phosphate 1.0 g.
Constituents 3. CaCla 0.1 g.
1. Distilled water 1000.0 cc. 4. MgS04 0.3 g.

2. NH4CI 0.1 to 1.0 g. 5. NaCl 0.1 g.
3. Potassium phosphate 0.2 g. 6. Iron trace

4. Beijerinck and van Delden's basic salt 7. (NH4)2S04 0.25 mg.
solution (see p. 12) 10 drops. 8. NaoCOa 0.5 g.
Preparation Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7

and 8 in 1.
(2) Add 1.0 drop of Beijerinck and van

Delden's basic salt solution (see page Use Cultivation of organisms found in the
:
12) to each 100.0 cc. of (1). soil on roots and rhizomes.
Sterilization: Not specified. Reference: Gottheil (1901 p. 432).

Use: Cultivation of Bacillus oligocarbo-
philus. 82. Gosling's Ammonium Sulphate Solution
Reference: Beijerinck and van Delden Constituents:
(1903 p. 36). 1. Distilled water 500.0 cc
2. NazCOs (dehydrated) 2.3 g
79. Richter's Ammonium Nitrate Solution
CaCOs
3. 0.25 g
Constituents: 4. K.,C03 0.08 g
1. Water 1000.0 cc. 5. FeS04 0.03 g
2. NH4NO3 3.0 g. 6. (NH4)2S04 0.005 g.
3. MgS04 1.0 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6
4. KH2PO4, FeS04 and in 1.
ZnS04 1.0 g. Sterilization: Not specified.

Preparation: (1) Dissolve 2, 3 and 4 in 1. Use: To determine sulphate reduction.
Sterilization: Not specified. Reference: Gosling (1904 p. 391).
Use: Growth oi Aspergillus niger.
Reference: Richter (1910 p. 619). 83. Beijerinck and van Delden's Ammonium
Sulphate Solution
80. Lieske's Ammonium Sulphate Solution
Constituents:
Constituents: 1. Distilled water 1000.0 cc.
1. Water 1000.0 cc. 2. (NH4)2S04 1.0 g.
2. Magnesium bicarbonate 3. Potassium phosphate 0.2 g.
4. Beijerinck and van Del- oxidizing nitrites may be differentiated as

den's mineral solution (see follows:
p. 12). A I. Employed primarily for organisms
Preparation oxidizing nitrites to nitrates.
(1) Dissolve 2 and 3 in 1. Bi. Calcium carbonate added.
(2) Add 1.0 drop of 4 to each 100.0 cc. Hewlett's Nitrite Solution (Johnson). 86
of (1). Cunningham's Nitrite Solution 87
Sterilization: Not specified. B2. Calcium carbonate not added.
Use: Growth of Bacillus oligocarbophilus. Ci. Magnesium carbonate added.
Reference: Beijerinck and van Delden Omeliansky's Nitrite Solution 88
(1903 p. 36). C2. Magnesium carbonate not added.
Di. Magnesium sulphate added.
84. Beijerinck and van Delden's Manganese Winogradsky and Omeliansky's So-
Ammonium Phosphate Solution dium Nitrite Solution 89
Constituents
Winogradsky 's Basal Sodium Nitrite
Distilled water
Solution 90
1. 1000.0 cc.
D2. Magnesium sulphate not added.
2. (NH4)2HP04 0.2 g.
Gartner's Sodium Nitrite Solution ... 91
3. NaaCOa 1.0 g.
4. Beijerinck and van Del-
Wimmer's Sodium Nitrite Solution.. 92 .
Bijerinck and van Delden's Basal

den's solution (see p. 12). . 20 drops.
Nitrite Solution 93
Preparation
Dissolve 2 and 3 in 1.
A2. Not employed primarily for organisms
(1)
oxidizing nitrites to nitrates.
(2) Add 2.0 drops of 4 to each 100.0 cc.
Gottheil's Nitrite Solution No. VIII 94 .
of (1).
Bijerinck and van Delden's Potassium
Nitrite Solution A 95
Use: Bacillus oligocarbophilus grows as a
snow white scum on the surface of the
86. Hewlett's Nitrite Solution (Johnson)
medium.
Reference: Beijerinck and van Delden Constituents
(1903 p. 41). 1. Distilled water 1000.0 cc.
2. KNO2 0.3 g.
85. Beijerinck and van Delden's Ammo- Potassium phosphate
3. 0.1 g.
nium Phosphate Solution
4. MgS04 0.05 g.
Constituents 5. CaCOs 5.0 g.
1. Distilled water 100.0 cc. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
3. (NH4)2HP04 0.02 g. Use: Study of nitrate production. Bacil-
4. NazCOa 0.1 g. lus megatherium produced no nitrates
Preparation in this solution.
(1) Dissolve 2, 3 and 4 in 1. Reference: Johnson (1912 p. 219).
(2) Reaction to be slightly alkaline.
Sterilization: Not specified. 87. Cunningham's Nitrite Solution
Use: Bacillus oligocarbophilus grows as a
thin white scum on the medium. Constituents
(1903 p. 41).
2. NaNOo 1.0 g.
3. K2HPO4 0.5 g.

4. MgS04 0.3 g.
5. NaCl 0.5 g.
Inorganic liquid media or basal solutions 6. Na.COa 0.2 g.
of known composition; nitrogen supplied Preparation
as nitrites. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
The several liquid media which have (2) Distribute in 10.0 cc. quantities in
been published for the growth of organisms 50.0 cc. Erlenmeyer flasks.
(3) Add a small quantity of sterile addition of 0.1% Na2C03 gave the best
CaCOs to each tube of sterile (2). results. The addition of FeS04 had no
Sterilization: Sterilize (2) and the CaCOa effect on nitrate production.
into autoclave. Added nutrients The authors added one of

:
Use: To study oxidation of nitrites and the following materials to determine the
nitrates. effect of organic material on nitrification.
Reference: Cunningham (1924 p. 151). glycerolO.l, 0.2, 0.4, 0.8, 1.0, 1.4 or 2.0%
glucose 0.05, 0.1, 0.2, 0.4, 0.8, 1.0 or 2.0%
88. Omelianski's Nitrite Solution urea 0.1, 1.0 or 1.6%
Constituents: asparagin 0.1, 0.6, 0.8, 1.0 or 2.0%
1. Distilled water 1000.0 cc. hay infusion 4.0, 8.0, 16.0 or 32.0%
2. NaNOs (Merck) 1.0 g. leaf infusion 4.0, 8.0, 16.0 or 32.0%
3. NaaCOs (ustum) 1.0 g. dirt infusion 4.0, 8.0, 16.0 or 32.0%
4. Potassium phosphate 0.5 g. meat infusion 2.0, 4.0, 8.0 or 10.0%
5. NaCl 0.5 g. urine 2.0, 4.0, 10.0 or 20.0%

6. FeS04 0-4 g. (NH4)2S04 (1.0% solution) 0.2, 1.0, 1.6,
7. MgS04 0.3 g. 2.0, 3.0, 4.0 or 6.0%
8. MgCOa sodium acetate 1.0, 2.0, 3.0, 4.0 or 6.0%

Preparation: sodium butyrate 0.1, 1.0, 2.0, 3.0 or 4.0%
(1) Dissolve 2, 3, 4, 5,6 and 7 in 1. Generally organic material slows up
(2) Flask in flat bottomed flasks in 50.0 oxidation unless it be present in very small
cc. lots. quantities. The more organic materials
(3) Add 0.5 g. MgCOa to each flask. present the slower the oxidation.
Sterilization: Not specified. Variants
Use: Isolation of nitrate forming organ- (a) Wimmer used the above solution
isms. The author found that repeated specifyingO.5 g. KH2PO4 and suggest-
cultivation on this medium yielded a pure ing the addition of 0.28 KCl. g.
culture. (b) Harvey used employ-

this solution
Variants: Boullanger and Massol used the ing 0.5 g. K2HPO4 and 0.3 g. anhy-
above solution with the addition of slag. drous Na2C03 instead of 1.0 g.
Reference: Omeliansky (1899 p. 548), Boul- (c) Gibbs (Gowda) used 0.5 g. K2HPO4
langer and Massol (1903 p. 494). and a trace of Fe2(S04)3 instead of
0.4 g. FeS04.
89. Winogradsky and Omeliansky's Sodium Reference: Winogradsky and Omeliansky
Nitrite Solution Wimmer (1904
(1899 p. 333), p. 140),
Constituents Smith (1905 p. 199), Harvey (1921-22 p.
1. Distilled water 1000.0 cc. 106), Gowda (1924 p. 258), Percival (1920
2. NaNOa 1-0 g- p. 149).
3. Potassium phosphate 0.5 g.

90. Winogradsky's Basal Sodium Nitrite
4. MgS04 0-3 g.
Solution
5. NaCl 0.5 g.
6. Na.COa 1-0 g. Constituents:
7. FeS04 0.4 g. 1. Distilled water 1000.0 cc.
Preparation 2. NaN02 (Merck) 1.0 g
(1) Distill water with permanganate. 3. Potassium phosphate 0.5 g
(2) Dissolve 2, 3, 4 and 5 in (1). 4. MgS04 0.3 g
(3) Distribute in 50.0 cc. lots. 5. NaoCOs 0.5 g
(4) Add 0.0, 0.1, 0.2, 0.4, 0.5 or 0.6 g. of 6. NaCl 0.5 g
NaaCOa to each flask (1.0% gives best Preparation

results). (1) Distill water two times with "chama-
(5) Iron may be added. leon."
Sterilization: Not specified. (2) Dis-solve 2, 3, 4 and 5 in 1. The
Uses: To study nitrate production from Na2C03 should be water free, heated
nitrites. The author found that the to a weak glowing.
(3) Adjustment of reaction not specified. 3. K2CO3 5.0 g.

(4) Flask in 50.0 cc. lots. 4. NaNOs 2.0 g.
Sterilization: Sterilize in the autoclave. Preparation
Use: To study nitrification. (1) Dissolve 2, 3, and 4 in 1.
Added nutrients and variants : The author (2) Distribute in 30 cc. lots in flasks.
added glucose, urea, asparagin or peptone Sterilization: Not specified.
to the solution to determine the efTect of Use: To study the oxidation of nitrite by
organic materials on nitrification. Dis- fungus. Nitrite was found to be still
tribute in 50.0 cc. lots and add: present after four weeks.
(a) 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2.0, 2.4 or Reference: Gartner (1898 p. 4).
4.0 g. glucose.
(b) 0.05, 0.1, 0.2, 0.4, 0.8, 1.2 or 1.6 g.
92. Wimmer's Sodium Nitrite Solution
peptone. Constituents:
(c) 0.1, 0.2, 0.4 or 0.8 g. urea. 1. Distilled water.. 1000.0 cc.
(d) 0.1, 0.2 or 0.4 g. asparagin. 2. Natrium nitro-
(e) 5.0 cc. of bouillon. sum puriss
The presence of organic materials gen- (NaNOo) 1.0, 2.0 or 50.0 g.
erally tends to slow up the oxidation. Preparation: (1) Dissolve 2 in 1.
WINOGRADSKY
AND
OMELIANSKY
Distilled water 1000.0 cc. 1000.0 cc. 1000.0 cc. 1000.0 cc.
NaN02 1.0 g. 2.0 g. 25.0 cc. 2.0% l.Og.
solution
Potassium phosphate 0.5 g. 1.0 g.
MgS04 0.3 g. 0.3 g. 0.5 g. 0.3 g.
NaCl 0.5 g. 0.5 g. l.Og. 0.5 g.
NaoC03 1.0 g. 0.3 g.
K2HPO4 1.0 g. l.Og.
K2CO3 0.5 g.
KCl 0.1
Peptone in small amounts tends to Sterilization: Not specified.

hasten oxidation. Same is true of urea. Use : To study nitrification by soil bacteria.
Asparagin and glucose even in small Raference: Wimmer (1904 p. 140).
amounts slow up the oxidation.
Variants: The modifications shown in the
93. Beijerinck and van Delden's Basal
Nitrite Solution
table have been described. Their prepa-
ration is like the original solution. Constituents:
Stutzer and Hartleb bubbled air con- 1. Water 1000.0 cc.
taining CO2 thru their solution. Carbo- 2. KNO2 0.5 g.
nates or CO 2 were essential for nitrate 3. K0HPO4 0.5 g.
production. Preparation
References: Winogradsky (1896 p. 423), (1) Dissolve 2 and 3 in 1.
Stutzer and Hartleb (1897 p. 244), Wino- (2) Add one of the added nutrients to (1).
gradsky and Omeliansky (1899 p. 378), Sterilization:Not specified.
Stutzer (1901 p. 169), Lohnis (1913 p. 97) Use: Tostudy denitrification by 5. subtilis,
Giltner (1921 p. 375). B. mesenlericus and Azo. chrooincoccum.
B. subtilis gave no ammonia test. B.
91. Gartner's Sodium Nitrite Solution
mesenlericus gave ammonia using man-
Constituents nitol as a carbon source. Chroococcum
1. Water 1000.0 cc. gave ammonia using either mannitol or
2. Potassium phosphate 0.5 g. malate as a carbon source.
Added The authors used either

nutrients: Giltay-Aberson's Basal Nitrate Solu-
20.0 g. mannitol or 20.0 g. calcium
of tion (Stoklasa) 99
malate as a carbon source. Henneberg's Basal Nitrate Solution. 100
Reference: Beijerinck and van Delden A3. Salts of mono, di and tri valent cations
(1902 p. 41). added.
Bi. Calcium salts added.
94. Gottheil's Nitrite Solution No. VIII Stoklasa and Vitek's Basic Nitrate
Salt Solution 101
Constituents. Harvey's Basal Nitrate Solution 102
Bo. Calcium salts not added.
2. Potassium phosphate 1-0 g.
Beijerinck and van Delden's Nitrate
3. CaClo 0.1 g.
Solutions, B, E, F, H, N 103
4. MgSOi 0.3 g. Kita's Basal Nitrate Solution 104
5. NaCl 0.1 g.
Stoklasa's Basal Nitrate Solution . . . 105
6. Iron trace 106
Lantzsch's Basal Nitrate Solution. . .
7. KNO2 0.05 g.
8. Soda 0.05 g. 96. Beijerinck's Basal Nitrate Solution
Preparation : Dissolve 2, 3, 4, 5, 6, 7 and 8
Constituents:
in 1.
1. Water 1000.0 cc.
2. KNO3 1.0 g.
Use: Cultivation of bacteria found in soil
3. K2HPO4 0.2 g.
on roots and rhizomes.
Preparation
Reference: Gottheil (1901 p. 432).
(2) Add one of the materials listed under
95. Beijerinck and van Delden's Potassium
added nutrients.
Nitrite Solution A
Constituents: Use: To study denitrification by Azoto-
1. Water (tap) 100.0 cc. bacter chroococcum.
2. KCl 0.01 g. Added nutrients and variants
3. K2HPO4 0.02 g. (a) The author added 20.0 to 100.0 g.
4. Kx\02 0.01 g. mannitol.
Preparation (b) Beijerinck and van Delden used 0.2 g.
(1) Dissolve 2, 3 and 4 in 1. KNO3 in the basic solution without
(2) The reaction is slightly alkaline. additional nutrients for the cultiva-
Sterilization: Not given. tion of Bacillus oligocarbophilus
Use: Bacillus oligocarbophilus grows in a (c) Beijerinck and van Delden used 0.2 g
dry thin snow white scum on the surface KNO3 and 0.2 g. Na2HP04 in the
of the medium. basic solution and added 0.2 g. KCl for
Reference: Beijerinck and van Delden the cultivation of Bacillus oligocarbo-
(1903 p. 41). philus.
(d) Beijerinck and van Delden used 0.1 g.
KNO3 and 0.1 g. K2HPO4 in the basic
Inorganic basal solutions of known com- solution and added 0.1 g. Na2C08 for
positions; nitrogen supplied as nitrates; the cultivation of Bacillus oligo-
incomplete solutions requiring the addition carbophilus.
of other nutrients. (e) Beijerinck and Minkman used 1.0%
A Saltsof monovalent cations only added.
I.
KNO3 and 0.05% K.2HPO4 in the
Beijerinck's Basal Nitrate Solution. 96 basic solution and added 2.0% man-
Stoklasa and Vitek's Basal Nitrate nitol, glycerol, sodium acetate or
Solution 97 sodium propionate to study deni-
A2. Salts of mono and divalent cations trification.
added. (f ) Beijerinck and van Delden used 0.5 g.

Czapek's Basal Nitrate Solution KNO3 and 0.5 g. K2HPO4 in the basic
(Waksman) 98 solution and added 2.0 g. of calcium
malate or mannitol to study deni- maltose sodium salts of:

trification. mannitol tartaric acid
References: Beijerinck (1901 p. 575), Beij- inulin oxalic acid
erinck and van Delden (1902 p. 41), glucose lactic acid
(1903 p. 41), Beijerinck and Minkman cellulose in form of Whitman filter paper
(1910 pp. 35, 36). 1.0 cc. of 0.5% cellulose suspension.
Reference: Waksman (1919 p. 317).
97. Stoklasa and Vitek's Basal Nitrate
Solution 99. Giltay-Aberson's Basal Nitrate Solution
Constituents: (Stoklasa)
1. Water 1000.0 cc. Constituents:
2. NaNOs 8.5 g. 1. Water 1000.0 cc.
Preparation 2. NaNOs 2.0 g.
(1) Dissolve 2 in 1. 3. MgS04 2.0 g.
(2) Add a suitable carbon source. 4. K2HPO4 2.0 g.
Sterilization: Not specified. 5. NaCl 0.2 g.
Use: To study nitrate reduction by Bad. 6. Iron chloride less than 0.2 g.
Hartlebi and other bacteria. Preparation
Added nutrients The author suggested the
: (1) Dissolve 2, 3, 4, 5 and 6 in 1.
addition of the following carbon sources: (2) Adjust to slightly alkaline reaction
xylose 15.0 g. by adding Na2C03.
glucose 18.0 g. (3) Add one of the combinations given
sucrose 34.2 g. in added nutrients.
Reference: Stoklasa and Vitek (1905 p. Sterilization: Not specified.
205). Use: To study denitrification and influence
of carbohydrates on denitrification
98. Czapek's Basal Nitrate Solution by soil forms.
(Waksman) Added nutrients: The author added one of
Constituents: the following combinations:
1. Water 1000.0 cc. (a) Citric acid 5.0 g. and glucose 2.0 g.
2. K2HPO4 1.0 g. (b) Glucose 2.0 g.
3. MgS04 0.5 g. (c) Glucose 2.0 g. with stable manure.
4. KCl 0.5 g. (d) Glucose 2.0 g. and citric acid 5.0 g.
5. FeS04 0.01 g. with stable manure.
6. NaNOa 2.0 g. (e) Glucose 2.0 g. with Chilisaltpeter.
Preparation (f) Glucose 2.0 g. and citric acid 5.0 g.
(1) Prepare a solution of 1, 2, 3, 4, 5 and 6 with Chilisaltpeter.
using chemically pure materials. (g) Chilisaltpeter, peat, or soil rich in
(2) Add 20.0 g. of one of the test ma- humus.
terials. Reference: Stoklasa (1907 p. 28).
Sterilization: Autoclave at 15 pounds pres-
sure for 15 minutes.
100. Henneberg's Basal Nitrate Solution
Use: To study the metabolism of actino- Constituents:
mycetes. The author found that the 1. Water 1000.0 cc.
Actinomycetes utilized the materials in 2. MgS04 1.0 g. or 2.0 g.
order listed. Starch, glucose, lactose, 3. KNO3 2.0 g. or 3.0 g.
maltose, glycerin, sucrose, cellulose and Preparation:
then organic acids. (1) Dissolve 2 and 3 in 1.
Added nutrients: The author used 20.0 g. (2) Add one of the test materials to (1).
of one of the following carbon sources: Sterilization: Not specified.
arabinose starch Use: To determine the fermentation of
glycerol sodium salts of acetic acid bacteria, B. oxydans, B.
lactose acetic acid acetosum, B. aceti, B. Kutzingianum, B.
sucrose malic acid Pasteurianum, B. acetigenum. B. oxy-
dans ferments arabinose, levulose, dex- Reference: Stoklasa and Vitek (1905 p.
trose, galactose, maltose, dextrin, ethyl 104) (1901 p. 262).
and propyl alcohol, erythritol. B. aceto-
sum ferments arabinose, dextrose, galac- 102. Harvey's Basal Nitrate Solution
tose, ethyland propyl alcohol. B. aceti, Constituents:
B. Kiitzingianum, B. Pasteurianum and 1. Distilled water 1000.0 cc.
B. acetigenum ferment dextrose and 2. NaNOa 1.0 g.
ethyl and methyl alcohol. 3. NaCl 0.02 g.
Added nutrients: The author added the 4. MgS04 0.02 g.
following carbon sources: 5. K2HPO4 0.5 g.
arabinose inulin 6. FeCla trace
levulose methyl alcohol Preparation
glucose ethyl alcohol (1) Dissolve 2, 3, 4, 5 and 6 in 1.
galactose propyl alcohol (2) Add one of the added nutrients.
sarobose amyl alcohol Sterilization: Not specified.
sucrose glycerol Use: Study of nitrate reduction.
maltose erythritol Added nutrients: The author added 10.0 g.
lactose mannitol glucose or 10.0 g. glycerol.
dextrin dulcitol Variants : The author used the following
starch melampyrit solution:
glycogen quercitol 1. Distilled water 3000.0 cc.
Reference: Henneberg (1898 p. 19). 2. KNO3 7.0 g.
3. K0HPO4 1.5 g.
101. Stoklasa and Vitek's Basal Nitrate 4. MgS04 1.5 g.
Salt Solution 5. NaCl 1.5 g.

6. CaS04 5.0 g.
Constituents: 7. FeCl 3 solution few drops
1. Water 1000.0 cc Reference: Harvey (1921-22 p. 104).
2. NaNOs 2.0 g.
3. K2HPO4 103. Beijerinck and van Delden's Nitrate
1.25 g.
4. K2SO4 Solution B, E, F, H, N
0.2 g.
5. CaCl2 0.05 g. Constituents
6. MgCU 0.05 g. Solution B:
7. NaiCOs 0.1 g. 1. Distilled water 1000.0 cc.
8. FeP04 0.05 g. 2. KCl 0.2 g.
Preparation 3. KNO., 1.0 g.
(1) Prepare solutions as indicated in the 4. K2HPO4 0.2 g.
table by dissolving the salts in water. Solution E:
(2) Add 2.0 or 2.5 g. of test material. 1. Distilled water 1000.0 cc,
Sterilization: Not specified. 2. KCl 0.1 g.
Use: To study reduction of nitrates by 3. KNO3 1.0 g.
denitrifying organisms. 4. K2HPO4 0.2 g.
Added nutrients: The
author added a Solution F:
variety of carbohydrates (2.0 o;- 2.5 g.) 1. Distilled water 1000.0 cc.
or 2.0 g. of organic acids. Neutralize 2. KNO3 0.1 g.
the acids with NaoCOs. 3. Na2HP04 0.2 g.
butj-ric acid glucose Solution H:
succinic acid levulose 1. Distilled water 1000.0 cc.
lactic acid galactose 2. KCl 0.2 g.
citric acid arabinose 3. KNO3 0.2 g.
malic acid xjdose 4. Na2HP04 0.2 g.
tartaric acid saccharose Solution N:
asparagin lactose 1. Distilled water 1000.0 cc.
Variants: The authors substituted 0.25 g. 2. KNO3 0.1 to 1.0 g.
Na2HP04 for 1.25 g. K2HPO4. 3. K2HPO4 0.1 g.
Preparation Added nutrients: The author added 20.0 g.
(1) Dissolve the salts in water as indi-

of any carbohydrate.
cated in the table. Reference: Stoklasa (1911 p. 479).
(2) Add one drop of Beijerinck and van

106. Lantzsch's Basal Nitrate Solution
Delden's mineral solution (see Med.
25) to each 100.0 cc. of (1). Constituents
Sterilization: Not specified. 1. Water (tap) 100.0 cc.
Use: Bacillus oligocarbophilus grows as a 2. K2HPO4 (0.01%) = 0.01 g.
dry thin snow white scum on the surface 3. NaN03 (0.01%) = 0.01 g.
of the medium. 4. MgS04 trace

Reference: Beijerinck and van Delden 5. FeCls trace
(1903 p. 41). Preparation

(1) Dissolve 2, 3, 4 and 5 in 100.0 g. tap
lOi. Kita's Basal Nitrate Solution water or in 29 parts tap water + 80
Constituents parts distilled water.
1. Water 1000.0 cc. (2) Place in an atmosphere of one of the
2. MgS04 0.25 g. test materials.
3. KH2PO4 5.0 g. Sterilization: Not specified.

4. FeCls solution few drops Use : To study utilization of gaseous carbon
5. KNO3 50 g. compounds.
Preparation Added nutrients: The author prepared the
(1) Dissolve 2, 3, 4 and 5 in 1. various atmospheres of test materials as
(2) Add 5.0% of any carbohydrate. follows:
Adjustment of reaction not specified. (a) Decompose 0.6 g. lead formate with
(3)
Sterilization: Not specified. H2SO4, in a 2500.0 cc. flask.
molds, Aspergillus (b) 2.0 cc. of a 4.0% formalin solution in a

Use: Cultivation of
albus, Aspergillus Okazaki, Aspergillus 2000.0 cc. flask.
candidus, Aspergillus tamarii, Pseudo- (c) 0.125 g. CH3COOH in 2500 cc. flask.
rhizopus, Aspergillus glaucus. (d) 0.1 g. acetone in a 2000.0 cc. flask.

Added nutrients: The author did not (e) 0.2 cc. butyric acid in 5 liter flask.
specify the carbohydrates used. (f) 0.12 cc. of benzol or xylol in 2000.0
Reference: Kita (1913 p. 434). cc. flask.
Reference: Lantzsch (1922 p. 310).
105. Stoklasa's Basal Nitrate Solution
Constituents
1. Distilled water 1000.0 cc. Inorganic liquid media of known compo-

2. K2SO4 10 g. sition; nitrogen supplied as nitrates; com-
3. MgCl2 0.5 g. plete nutrient solutions.

4. Iron sulphate 0.1 g. Ai. Containing oxidizable sulphur.
5. NaNOa Bi. Sulphur added as elementary sulphur.
6. Monodiferric phosphate Beijerinck's Sulphur Nitrate Solution 107
Fe203(P205)3 or monodialu- B2. Sulphur added as thiosulphate.
minum phosphate AI2O3P2O5 1.0 g. Lieske's Thiosulphate Nitrate Solu-
Preparation tion ^(Gehring) 108
4 and one of 6 in 1. Trautwein's Thiosulphate Nitrate
(1) Dissolve 2, 3,
2.0 g. of nitrogen in the form of Solution 109

(2) Add
NaN03 and 20.0 g. of test material Nathansohn's Thiosulphate Nitrate
Solution (Trautwein) 110
to (1).
Distribute in Fernbach flasks.
(3)
A2. Not containing oxidizable sulphur.
Sterilization: Sterilize in the autoclave. Beijerinck's Ammonium Nitrate
Solution Ill
Use: To study the cycle of the phosphate
ion in the soil. Bact. fiuorescens lique- Nabokich and Leedeff's Hydrogen
Nitrate Solution 112
faciens and Bact. Hartlebi were the
organisms used. Bokorny's Calcium Nitrate Solution. 113
107. Beijerinck's Sulphur Nitrate Solution 7. KCl trace

Constituents
Preparation
1. Ditch water or dis-
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
tilled water 100.0 cc.
(2) Distribute in tall glass cylinders.
2. Sulphur (powder) 10.0 g.
Inoculate with waste water filtrate.
(3)
3. KNO3 0.05 g.
4. NasCOa 0.02 g.
Use: Enrichment and isolation of thionic
5. CaCOs 2.0 g.
bacteria (Omeliansky). A slight opal-
6. K2HPO4 0.02 g.
escence formed after 6 days. After a
is
7. MgCl. 0.0 or 0.01 g.
milky turbid culture results, a snow white
Preparation
membrane is formed on the surface.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Variants The author suggested the follow-
:
(2) Completely fill a well stoppered flask

ing solution:
with (1). Distilled water
1. 1000.0 cc.
MgCl2
2. 0.01 g.
Use: To study denitrification with sulphur 3. KNO3 0.1 g.
o.xidation by Thiobacillus denitrificans
4. Na2HP04 0.02 g.
The author found that the nitrate was Na2S203
5. 0.2 g.
reduced, and the sulphur o.xidized to sul-
6. NaHCOa 0.1 g.
phate, with the liberation of nitrogen gas.
Reference: Trautwein (1920 p. 515) (1921
Reference: Beijerinck (1903-4 p. 597).
p. 515).
108. Lieske's Thiosulphate Nitrate Solution 110. Nathansohn's Tliiosulphate Nitrate

(Gehring) Solution (Trautwein)
Constituents Constituents
2. NaaSaOs 0.5 g. 2. NaCl 3.0 g.
3. KxNOs 0.5 g. 3. MgCl. 0.25 g.

4. NaHCOs 0.1 g. 4. KNO3 0.1 g.
5. K2HPO4 0.02 g. 5. Na2HP04 0.05 g.

6. MgCl2 0.01 g. 6. NaoSoOs 0.2 or 0.1 g.
7. Fe.Cle trace Preparation: (1) Dissolve 2, 3, 4, 5 and 6

8. CaCl2 trace in 1.
Prepj ration Sterilization: Not specified.
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.

Use: Cultivation of thionic bacteria (Ome-
(2) Distribute in test tubes 45 cm. high.
liansky).
Sterilization: Not specified. Reference: Trautwein (1921 p. 518).
Use: Isolation and cultivation of denitri-

fying thiosulphate bacteria, Thiobacillus
111. Beijerinck's Ammonium Nitrate
Solution
denitrificans.
Reference: Gehring (1914 p. 405), Traut- Constituents
wein (1921 p. 518). 1. Water (tap) 100.0 cc.
2. NH4NO3 0.2 g.
3. Potassium bi-phosphate 0.05 g.
109. Trautwein's Thiosulphate Nitrate
Preparation: (1) Dissolve 2 and 3 in 1.
Solution
Constituents: Use: Cultivation of gonidia of algae.
1. Distilled water 100.0 cc. Variants: Beijerinck used 0.02 g. NH4NO3
2. Na2S203 0.5 g. and 0.02 g. K2HPO4 in 100.0 cc. of tap
3. KNO3 0.5 g. water to cultivate Chlorophijcae, Chloro-
4. NaaCOa 0.1 g. coccum and Cyanophycee.
5. K2HPO4 0.02 g. References: Beijerinck (1893 p. 371), Beij-
6. MgClj 0.01 g. erinck (1901 p. 563).
112. Nabokoch and Lebedeff's Hydrogen supplied. The sections recognized may be
Nitrate Solution differentiated as follows:
Constituents Key to the sections of Subgroup I-C

1. Water 1000.0 cc.
Ai. Chemical composition of all constit-
2. Na,HP04 0.5 g.
uents known.
3. KNO, 2.0 g.
Bi. Nitrogen inorganic, carbon organic.
4. MgSO, 0.2 g.
Ci. Nitrogen present as free or elementary
5. NaHCOa 1.0 g.
nitrogen only. Section 1 (Med. 114-190)
.
6. FbsCU some
C2. Nitrogen present as ammonium salts.
7. Hydrogen
Section 2 (Med. 191-323)
Preparation
Cj. Nitrogen present as nitritesj.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) The reaction is slightly alkaline
C4. Nitrogen present as nitrates.
(about 0.050 g. H2SO4 to 100.0 cc. of
solution).
B2. Nitrogen organic.
(3) Distribute in 100 or 150.0 cc. lots in
Ci. Nitrogen present as amino acids.
vacuum flasks with side tubes.
(4) Inoculate.
C2. Organic nitrogen other than amino
(5) Remove all the air by means of an
acids present Section 6 (Med. 480-515)
.
pump.
. .
oil
A2. Chemical composition of one or more
(6) Replace the air by a mi.xture of oxygen
constituents not definitely known.
and hydrogen ("Knallgase") contain-
Bi.* Containing digests.
ing carbonic acid.
Ci.* Containing commercial peptones or
digests.
Use: To study the oxidation of hydrogen
Di. Additional if any,
constituents,
in-
by microorganisms.
organic Section 7 (Med. 516-555)
Reference: Nabokich and LebedefT (1907
D2. Containing additional organic constit-
p. 352), Lohnis (1913 p. 106).
uents.
113. Bokomy's Calcium Nitrate Solution El. Chemical composition of all additional
materials known.
Constituents
Fi. No additional nitrogen compounds
1. Water 100.0 cc.
added.
2. Ca(N03)2 0.1 g.
Gi. Basal solutions. Employed with the
3. MgS04 (crystalline) 0.02 g.
addition of various nutrients.
4. KH2PO4 0.02 g.
B2. Complete nutrient solutions.
6. KCl 0.0 or 0.05 g.
F2. At least one additional nitrogenous
in 1.
constituent.
Section 10 (xMed. 648-683)
Use: To study the metals required for the
E2. Composition of one or more additional
growth of Spirogyra majuscula. It was
materials not definitely known.
found that potassium was necessary.
Fi. Additional organic material exclusively
Reference: Bokorny (1912 p. 125).
of plant origin
SUBGROUP I-C F2. Containing additional materials of
animal origin; plant derivatives may also
Liquid Media with One or More be present.
Constituents Organic G:. Not containing extracts or infusion.
The liquid media in which one or more G2. Containing extracts or infusion.
constituents are organic may be divided Section 13 (Med. 748-977)
conveniently into sections based upon the
character of the nitrogen and carbon sources See page 37 for B2 and C2.
Cj. Containing non-commercial digests. Gerlach and Vogel's Basal Glucose

Section 14 (Med. 978-1140) Salt Solution 123
Bj. Not containing digests. Henneberg's Basal Glucose Di-so-
Ci. Containing plant constituents of un- dium Phosphate Solution 127
known composition. Beijerinck's Glucose Salt Solution. . . 128
Section 15 (Med. 1141-1228) Kisch's Basal Glucose Salt Solution. 129
C2. Containing animal constituents of un- Beijerinck's Phosphate Glucose Salt
known composition. Solution (Harvey No. 1) 130
Section 16 (Med. 1229-1376) Laborde's Basal Invert Sugar Tar-
C3. Containing extracts of soil, ashes, etc. tarate Solution 131
Section 17 (Med. 1377-1394) Calmette, Massol and Breton's Basal
Glucose Salt Solution 132
SUBGROUP I-C. SECTION 1 Lohnis' Basal Glucose Salt Solution . . 133
G2. Disaccharides added.
Liquid media or basal solutions with con-
Will's Basal Sucrose Salt Solution. . 134
known chemical composition
.
stituents of
Henneberg's Basal Sucrose Salt Solu-
containing free or elementary nitrogen
tion 135
only, carbon organic.
Czapek's Sucrose Salt Solution
Ai. Inorganic salts not added.
(Waksman) 136
Bi. Carboh3'drates added.
von Bronsart's Basal Sucrose Salt
Pringshein's Glucose Cellulose Solu-
Solution 137
tion 114
Bokorny's Basal Sucrose Salt Solu-
Waksman and Joffe's Basal Glucose
tion 138
Solution 115
Smith's Sucrose Salt Solution 139
Buchanan's Sucrose Solution 116
B2. Alcohols added.
Munter's Basal Galactose Salt Solu-
Waksman and tion 140
Joffe's Basal Glycerol
Solution 117 Buchanan's Basal Maltose Salt Solu-
Buchanan's Glycerol Solution I 118 tion 141
A2. Inorganic salts added. Qs. Polysaccharides Added.

Bi. Potassium salts, only, added. Omeliansky's Basal Cellulose Salt
Ci. Containing carbohydrates. Solution 142
Beijerinck and van Delden's Phos- F2.* Carbon present as alcohols.
phate Glucose solution 119 Gi. Glycerol added.
Beijerinck's Phosphate Sucrose Solu- Percival's Glycerol Phosphate Solu-
tion 120 tion 143
C2. Containing alcohols. Buchanan's Glycerol Salt Solution II. 144
Beijerinck's Phosphate Mannitol Gerlach and Vogel's Basal Glycerol
Solution 121 Salt Solution 145
C3. Containing salts of organic acids, Kuhne's Basal Lactate Solution.
van Delden's Basal Lactate Solution. 122 (Proskauer and Beck) 146
Beijerinck and van Delden's Ace- Calimard and Lacomme's Basal Glyc-
tate Solution 123 erol Salt Solution (KoUe-Wasser-
B2. Other salts added. man) 147
Ci.* Magnesium salts added.
G2. Mannitol added.
Di. Phosphates not added. Bijerinck's Modified Mannitol Salt
Gage's Glucose Salt Solution 124 Solution. (Omeliansky and Sswe-
Stoklasa's Phosphorous Free Glucose rowa) 148
Salt Solution 125
Murray's Mannitol Salt Solution... 149
Dj. Phosphates added.
Percival's Mannitol Phosphate Solu-
El.* Calcium salts not added. tion 150
Fi. Carbon present as carbohydrates.
Capaldi and Proskauer's Basal Man-
Gi. Monosaccharides added.
nitol Salt Solution 151
*See next page for C2, E2. * See ne.xt page for F3.
F3. Carbon present as organic acids or their Winogradsky's Mannitol Salt Solu-
salts. tion (Lantzsch) 175
Percival's Succinate Solution 152 G2. Glycerol added.
E2. Calcium salts added. Koser and Rettger's Basal Glycerol
Fi. Carbon present as carbohydrates. Salt Solution 176
Gi. Glucose added. F3. Carbon present as organic acids
Hi. Glucose only organic carbon source. or their salts.
Winogradsky's Salt Glucose Solution. 153 Puriewitsch's Tartaric Acid Salt
Stutzer, Burri and Maul's Glucose Solution 177
Salt Solution 154 C2. Magnesium salts not added.
Stoklasa's Basal Glucose Salt Solu- Di. Phosphates not added.
tion 155 Omeliansky's Cellulose Solution 178
Charpentier's Salt Glucose Solution. 156 Killer's Mannitol Solution 179
Krzemieniewska's Glucose Salt Solu- Ringer's Salt Solution 180
tion (Vogel) 157 D2. Phosphates added.
Kriiger and Schneidewing's Basal Ei. Calcium salts not added.
Glucose Salt Solution (Heinze) 158 Bijerinck's Glucose Salt Solution. . . . 181
Charpentier's Basal Glucose Salt Stoklasa's Glucose Salt Solution. . . . 182
Solution 159 Gag's Maltose Salt Solution 183
Stoklasa's Basal Glucose Salt Solu- Sohngen's Colloid Mannitol Solution. 184
tion 160 E2. Calcium salts added.
Henneberg's Basal Glucose Salt Solu- Fi. Carbon present as carbohydrates.
tion 161 Bijerinck's Phosphate Glucose Solu-
H2. Glucose with other organic carbon. tion 185
Gottheil's Carbohydrate Glycerol Buhlert and Fickendey's Phosphate
Solution 162 Glucose Solution 186
Harvey's Citric Acid Glucose Solu- F2. Carbon present as alcohols or acids.
tion 163 Wojtkiewicz's Mannitol Salt Solu-
Munter's Basal Mannitol Salt Solu- tion 187
tion 164 Kranisky's Mannitol Salt Solution 188 . . .
G2. Sucrose added. Peklo's Modified Bijerinck's Man-

Chraszcz's Basal Sucrose Salt Solu- nitol Salt Solution 189
tion 165 Butcher's Basal Chinate Salt Solu-
Maze's Sucrose Salt Solution 166 tion 190
Linde's Basal Sucrose Salt Solution. . 167
114. Pringsheim's Glucose Cellulose
Gs. Polysaccharides added.
Solution
Lohnis and Lochhead's Basal Cellu-
lose Salt Solution 168 Constituents:
van Iterson's Basal Cellulose Salt 1. Water 1000.0 cc.
Solution 169 2. Cellulose 25.0 g.
Carbon present as alcohols. 3. Glucose 0.2 or 0.4 g.

F2.*
Preparation: (1) Add 2 and 3 to 1.
Gi. Mannitol added.
Jones' Modified Ashby's Mannitol
170
Use: To study nitrogen assimilation meth-
Solution
ane fermenting bacteria.
Fred and Loomis' Mannitol Salt Solu-
Variants: The author used the following
tion 171
combinations to study nitrogen assimila-
Krzemieniewska's Mannitol Salt
tion by Clostridium and azotobacter:
Solution (Vogel) 172
(a) 10.0 g. cellulose +0.4 g. de.xtrose in
Harvey's Mannitol Salt Solution. 173 . . .
500.0 cc. solution

Sackett's Modified Lipman's Man-
(b) 5.0 g. cellulose +0.4 g. dextrose in
nitol Salt Solution 174
250.0 cc. solution
(c) 10.0 g. cellulose +0.2 g. dextrose in
See next column for F3. 1000.0 cc. solution
(d) 5.0 g. cellulose +0.2 g. dextrose in distilled water. Repeat the precipita-
1000.0 cc. solution tion 5 times. Finally dry the precipi-
(e) 2.5 g. cellulose +0.2 g. dextrose in tate. Saccharose may be used in higher
500.0 cc. solution concentrations.
(f) 5.0 g. cellulose +0.2 g. dextrose in Reference: Buchanan (1909 p. 371).
500.0 cc. solution
117. Waksman and Joflfe's Basal Glycerol
(g) 2.5 g. cellulose +0.2 g. dextrose in
Solution
500.0 cc. solution
(h) 5.0 g. cellulose + 0.2 g. mannite in Constituents
1000.0 cc. solution 1. Water to make 1000.0 cc.
Reference: Pringsheim (1909 p. 303), (1910
2. Glycerol 30.0 g.
p. 224).
Preparation
115. Waksman and Joffe's Basal Glucose (2) Add one of the test materials (added
Solution nutrients) to (1). When using casein
Constituents: and egg albumin, dissolve casein and
1. Water 1000.0 cc. egg albumin in N/10 NaOH. In using
2. Glucose 30.0 g. fibrin, add fibrin to each tube.
Preparation (3) Solutions vary in pH = 7.2 to 7.7,
(1) Dissolve 2 in 1. urea pH = 8.0.
(2) Add 2.0 g. of one of the added nu- (4) Tube in 10-12 cc. lots.
trients. Sterilization: Autoclave for 15 minutes at
(3) Tube in 10 to 12 cc. lots. 15pounds pressure.
Sterilization: Sterilize at 15 pounds for 15 Use: To study change in reaction in
minutes. Actinomycetes metabolism.
Use: To study reaction changes in Actino- Added nutrients: The authors added 5.0 g.
mycetes metabolism. of one of the following nitrogenous com-
Added nutrients: The authors added 2.0 g. pounds:
of one of the following nitrogen sources: fibrin leucin
(NH4)2S04 (pH of solution = 5.8) casein glycocoll
(XHOsCOa (pH of solution = 6.8) egg albumin urea
Urea (pH of solution = 7.4) peptone acetamide 5.0 g.
Reference: Waksman and Joffe (1920 p. 39). asparagin
Variants: The authors used 3.0 g. of glyc-
116. Buchanan's Sucrose Solution
erol and added 2.0 g. of NaNOa, NaNOs,
Constituents or (NH4)2S04 as inorganic nitrogen
1. Water (tap) 1000.0 cc. sources.
2. Sucrose (2.0%) 20.0 g. Reference: Waksman and JofTe (1920 pp.
Preparation 36-37).
(1) Prepare a 2.0% saccharose solution
118. Buchanan's Glycerol Solution I
in tap water.
(2) Distribute in 500.0 cc. lots in liter Constituents:
1. Water (tap) 1000.0 cc.
Sterilization: Method not given. 2. Glycerol 5.0 cc.
Use: Development of gum produced by Preparation: (1) Dissolve 2 in 1.
Bacillus radicicola. The author found Sterilization: Not specified.

that 1.0% N/1 malic acid or citric acid Use: Cultivation of Monascus purpurens
was sufficientcompletely inhibit
to from silage.
growth. 1.0% N/1 NaOH stimulated Variants: The author used a 10.0, 20.0 or
growth and gum production, while 2.0% 40.0% glycerol solution instead of 5.0%.
prevented growth. To isolate the gum Growth in the 5.0 and 10.0% solution, but
add 100.0 cc. of 95% alcohol, to the 2 little or no growth in the 20.0 or 40.0%
week old culture, filtering the white pre- solutions.
cipitate, wash in alcohol and dissolve in Reference: Buchanan (1910 p. 102).
119. Beijerinck and van Delden's Phosphate and suggested that CaCOs might be
Glucose Solution added if desired.
(d) Percival specified the use of tap
Constituents
water.
1. Water 1000.0 cc.
(e) Beijerinck and van Delden used 0.5 g.
2. Glucose 20.0 g.
3. K2HPO4 0.5 g.
K2HPO4 instead of 0.2 g. K0HPO4.
(f Stoklasa specified Moldau river water
and used 0.5 g. potassium phosphate
Sterilization: Not given.
Use: To study nitrogen assimilation by
instead of 0.2 g. K2HPO4.
Reference: Beijerinck (1901 pp. 568, 578),
chroococcum.
Jacobitz (1903 p. 101), Christensen (1907
Variants: Stoklasa prepared a solution of
p. 110), Percival (1920 p. 179), Beijerinck
25.0 g. of glucose and 1.5 g. K2HPO4 in
He and Van Delden (1902 p. 8), Stoklasa
1000.0 cc. of Moldau river water.
(1908 p. 489).
studied the relation between the assim-
ilation of phosphoric anhydride and 122. van Delden's Basal Lactate Solution
nitrogen by azotobacter. It was deter-
Constituents
mined that 1.0 g. of phosphoric acid
1. Water (ditch) 1000.0 cc.
anhydride and 2 to 2.3 g. elementary N
were assimilated.
2. K2HPO4 0.2 g.
3. Sodium lactate 1.0 g.
Preparation
(1902 p. 8), Stoklasa (1911 p. 461).
120. Beijerinck's Phosphate Sucrose (2) Additional nutrients may be added
Solution as indicated below.
Constituents: Sterilization : Not specified.
1. Water 1000.0 cc. Use Cultivation and enrichment of Micro-

:
2. Sucrose 20.0 g. spira desulfuricans Used also to study

.
3. K2HPO4 0.2 g. sulphate reduction.

Preparation: (1) Dissolve 2 and 3 in 1. Variants
Sterilization: Not specified. (a) The author used 0.5 g. sodium lactate.
Use: Growth of Bacillus radicicola. (b) The author used 0.5 g. K2HPO4 and
Reference: Beijerinck (1901 p. 575). 1.0 g. of calcium lactate.
Added nutrients:
121. Beijerinck's Phosphate Mannitol (a) The author did not specify the use of
Solution ditch water, and added (NH4)2S04
Constituents andO.Og. MgS04-7H20.
1. Water 1000.0 cc. (b) The author did not specify the use of
2. Mannitol 20.0 g. ditch water and added 0.2 g. glycocoU
3. K2HPO4 0.2 g. andO.Og. MgS04-7H20.
Preparation (c) The author used 0.5 g. sodium lactate
(1) Dissolve 2 and 3 in 1. with 0.5 g. K2HPO4 and added 1.0 g.
(2) Reaction is slightly alkaline. asparagin and 0.0, 0.3, 0.6, 0.9, or
(3) Inoculate with 0.1 to 0.2 g. earth. 1.2 g. gypsum.
Sterilization: Not specified. (d) The author specified the use of tap
Use: Used for the enrichment of Azotobac- water, used 0.5 g. sodium lactate with
ter chroococcum and for nitrogen assim- 0.5 K2HPO4, added NH4CI and 1.2 g.
ilation studies. gypsum.
Variants (e) The author specified the use of tap
(a) Beijerinck specified the use of ditch water, used 0.5 g. of sodium lactate
water. with 0.5 g. K2HPO4 and added 0.5 g.
(b) Jacobitz specified the use of distilled asparagin.
water and studied nitrogen fixation (f) The author specified the use of tap
hy Bacillus ellenhachensis a (Caron). water, used 0.5 g. of sodium lactate
(c) Christensen specified distilled water with 0.5 g. K2HPO4 added 1.0 g.
asparagin and 1.0 g. gypsum or 4.0 g. gen organisms was very poor in this
fi.xing
MgS04-7H20. solution. Phosphorus is required for

(g) The author specified the use of tap luxuriant and rapid development.
water, used 0.5 g. of sodium lactate Variants
with 0.5 g. K2HPO4 added 2.5 g. (a) The author suggested the addition
asparagin and 4.0 g. MgS04-7H20. of 1.0 g. of nitrogen in the form of
(h) The author specified the use of tap (NH4)2S04or NaNO.,.
water, used 10.0 g. sodium lactate, (b) The author suggested the addition of
added 1.0 g. of asparagin and 2.0 or 0.1 g. of aluminum sulphate and 1.0 g.
5.0 g. of MgS04-7H20. of one of the following phosphorus
Reference: van Delden (1903-04 p. 85). compounds:
dicalcium phosphate CaHP04-2H20.
123. Beijerinck and van Delden's Acetate
Monodiferric phosphate Fe203(P205)3.
Solution
Monodialuminum phosphate AI2O3-
Constituents: (P205)2-8H20.
1. Water 1000.0 cc. Tricalcium phosphate Ca3(P04)2-2H20.
2. K2HPO4 0.5 g. Ditriferric phosphate (Fe03)4(P205)5-
3. Sodium acetate 5.0 g. 3H2O.
Preparation: (1) Dissolve 2 and 3 in 1. Fe203P205 + 4H2O.
Triferric phosphate
Sterilization: Not specified. Trialuminum phosphate AloOj-PjOs +
Use: Cultivation of chroococcum and 4H2O.
granulobacter. The medium was in- (c) The author used variant (b) and sub-
oculated with 20.0 g. of fresh earth. stituted arabniose for d-glucose.
Reference: Beijerinck and van Delden (d) The author added aluminum sulphate
(1902 p. 29). and the equivalent of 0.2 g. of phos-
phoric anhydride in the form of
124. Gage's Glucose Solution
monomagnesium phosphate, mono-
Constituents: dialuminum phosphate, monodiferric
1. Water 1000.0 cc. phosphate or trimagnesium phos-
2. Glucose 5.0 g. phate to the original solution.
3. MgS04 0.25 g. References: Stoklasa (1911 pp. 492, 441,
4. CaCl2 0.01 g. 490).
5. Iron sulphate solution 1 drop
126. Gerlach and Vogel's Basal Glucose
Sterilization: Not specified. Salt Solution
Use To study the fixation of nitrogen by

: Constituents:
Pseudomonas radicicola and nitroso bac- 1. Distilled water 1000.0 cc.
teria. It was found that nitrites and 2. Glucose 5.0 g.
possibly nitrates were produced by Bacil- 3. Potassium phosphate 0.5 g.
lus radicicola in this medium. 4. MgS04 0.3 g.
Reference: Gage (1910 p. 34). 5. NaCl 0.5 g.
6. Na.COs 0.5 g.
125. Stoklasa's Phosphorus Free Glucose 7. FeS04 0.2 g.
Salt Solution Preparation
Constituents (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
1. Distilled water 1000.0 cc. (2) Add one of the nitrogen sources
2. d-glucose 25.0 g. added nutrients.
listed in
3. K2SO4 1.0 g. (3) Adjustment of reaction not given.
4. MgCl2 0.5 g. Sterilization : Method not specified. When
5. Iron sulphate 0.1 g. utilizing urea as nitrogen source sterilize
Preparation (1) Dissolve
: 2, 3, 4 and 5 in 1. the urea at 100C. dry heat.
Sterilization: Not specified. Use: Cultivation of albumin formers from
Use : To study the role of phosphorus in the soil and stable manure. These organ-
soil. It was found that growth of nitro- isms grow very well on agar, gelatin and
bouillon which contain 0.3% NaNOs. Variants

The medium is changed to
nitrate in this (a) Peklo used the solution to grow plant
nitrite after a few days and then quanti- actinomyces. He specified that the
tively to insoluble albuminous materials. solution might be neutralized by the
Ammonia salts and urea also are suitable addition of 0.5 g. NaoCOs.
nitrogen sources for albumin production (b) Malenkovic used 100.0 g. glucose,
by these organisms. Albumin produc- 1.0 g. K2HPO4 and added 2.0 g. of
tion using urea however goes on very KNO3or2.0g.NH4H2PO4. He culti-
slowly. vated coniophora cerebello (Corti-
Added nutrients : The author employed the cium putaneum) and reported
following nitrogen sources: NH4H2PO4 a better nitrogen source.
NaNOa (c.p. Merck) 3.0 g Reference: Beijerinck (1900 p. 7), Peklo
(NH4)2S04 3.0 g (1910 p. 514). Malenkovic (1906
NH4NO3 2.0 g p. 412).
NH4CO3 2.0 g
urea 20.0 g 129. Kisch's Basal Glucose Salt Solution
Reference : Gerlach and Vogel (1901 p. 612) Constituents:
1. Water 1000.0 cc.
127. Henneberg's Basal Glucose Disodium
2. K2HPO4 1.0 g.
Phosphate Solution
3. MgS04 0.5 g.
1. Water 1000.0 cc. 5. FeS04 trace
2. Na2HP04 3.0 g. 6. Calcium phosphate trace
3. MgS04 2.0 g. 7. Glucose 10.0 g.
4. NaCl 2.0 g. Preparation
5. Glucose 20.0 g. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Preparation (2) Add Na2C03 until the reaction is
(1) Dissolve 2, 3, 4 and 5 in 1. slightly alkaline to litmus.

(2) Add one of the added nutrients (3) Add one of the nitrogen sources listed
(amounts not specified). under added nutrients to (2).
(3) Adjustment of reaction not given. Sterilization: Not specified.
Sterilization: Method not given. Use: To study the nitrogen requirements
Use: To study the food requirements for of members of the colon-typhoid group.
acetic acid organisms, B. Pasteurianum, Added nutrients: The author added one
B. oxydans, B. aceti and B. Kiltzingi- of the following nitrogen sources.
anum. The author found that KNO 3 was (a) KNO3 0.29 g.
the poorest source of nitrogen and Bad. paratyphi B. shows growth.
(NH4)2S04 was the best. Bad. enter itidis Gartner little
Added nutrients The author employed one
: growth.
of the following salts or carbon sources: (b) KNO2 2.43 g.
KNO3 asparagin No growth.
(NH4)2S04 peptone (c) Urea 8.6 g.
ammonium tartrate Bad. coli and Bad. enteritidis Gart-
Reference: Henneberg (1898 p. 18). ner show some growth.
(d) Urea 8.6 g.
128. Beijerinck's Glucose Salt Solution
KNO2 2.4 g.
Constituents B. coli shows growth.
1. Water 1000.0 cc. (e) (NH4)2S04 1.9 g.
2. KH2PO4 0.5 g. B. paratyphi shows good growth.
3. MgS04 0.5 g. B. coZt shows good growth. B. enteri-
4. Glucose 10.0 g. tidis Gartner, and dysentery bacillus
Preparation: (1) Dissolve 2, 3 and 4 in 1. show good growth.
Sterilization: Not specified. (f) NH4H2PO4 1.42 g.
Use: Growth oi Streptothrix chromogena. Bad. paratyphi shows good growth.
Bad. shows good growth. B.

colt 3. Tartaric acid 2.5 g.
enteritidis Gartner and dysentery 4. Potassium tartrate
bacillus show good growth. (neutral) 0.75 g.
<g) NH4CI 1.63 g. 5. Mg3(P04)2 1.0 g.
Bad. coli and B. enteritidis show 6. H2SO4 0.1 g.
slight growth. Also B. 'paratyphi. 7. Iron and zinc sulphate trace
<h) (NH4)2C03 1.1 g. 8. KoSiOs trace
Bad. coli and B. enteritidis show Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7
slight growth. Also B. paratyphi. and 8 in 1.
(i) Ammonium lactate 2.94 g. Sterilization: Not specified.

B. typhi, B. paratyphi, B. enteri- Use: To study the nitrogen requirements
tidis, Gartner, B. typhi murum and of a mold, Eurotiopsis Gayoni.
dysentery show slight growth. Bad. Added nutrients: The author added 0.2 g.
coli grows well. of nitrogen in the form of one of the
(j) Ammonium tartrate 2.62 g. following:
B. paratyphi. Bad. coli, B. enteri- NaNOa casein
tidis Gartner show good growth. KNO3 asparagin
B. dys. Flexner show growth. Ammonium tar- urea
(k) Asparagin 1-9 g. trate gelatin
Nearly all strains show growth. NH4CI fibrin
(1) Nucleic acid (from yeast (NH4)2S04 blood albumin
Merck 2.6 g. Ammonium phos- egg albumin
Nearly all except B. paratyphi A phate yeast extract
show growth. NH4NO2 peptone
Reference: Kisch (1918-19 p. 32). gluten
Reference: Laborde (1897 p. 3).
130. Beijerinck's Phosphate Glucose Salt
Solution (Harvey No. I) 132. Calmette, Massol and Breton's Basal
Glucose Solution
Constituents:
1. Distilled water 1000.0 cc. Constituents:
2. K2HPO4 1.0 g. 1. Water 1000.0 cc.
3. MgS04 0.2 g. 2. FeS04 0.04 g.
4. NaCl 0.02 g. 3. MgS04 0.05 g.
5. 1-1000 MnS04 1.0 cc. 4. K2HPO4 1.05 g.
6. 1-1000 FeS04 1.0 cc. 5. NaCl 8.5 g.
7. Glucose 20.0 g. 6. Glucose 10.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 7. Na2C03 10.0 g.
in 1. Preparation
Sterilization: Not specified. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Use: Study of nitrogen fixation. (2) Add one of the added nutrients to (1)
Variants Sterilization: Not specified.

(a) Giltner used 0.5 g. MgS04, 0.01 g. Use: To determine constituents essential
Fe2(S04)3, 0.01 g. MnS04 and 0.01 for growth of tubercle bacilli. The
g. NaCl. authors reported that generally inorganic
(b) Heinemann used MgS04, 0.01
0.5 g. nitrogen did not support the growth of
g. FeS04, 0.01 g. MnS04 and 0.01 g. the tubercle bacillus.
NaCl. Added nutrients: The authors prepared
Reference: Harvey (1921-22 p. 105), Gilt- the following solutions.
ner (1921 p. 370), Heinemann (1922 p. 39). (a) Basal solution + 10.0 g. Na2C03 +
5.0 g. of one of the nitrogen sources.
131. Laborde's Basal Invert Sugar Tartrate (b) Basal solution + 10.0 g. NaoCOs +
Solution 40.0 g. of glycerol + 5.0 g. of one of
Constituents: the nitrogen sources.
1. Water 1000.0 cc. (c) Basal solution + 40.0 g. glycerol +
2. Invert sugar 50.0 g. 5.0 g. of one of the nitrogen sources.
The following nitrogen sources were Sterilization: Method not given.

given Use: Cultivation of yeast.
NaNOs Added nutrients: The author added one of
NaNOa the following combinations to the basic
(NH 4)280 4 solution:
(NH4)2HP04 (a) asparagin 20.0 g.
Ammonium acetate dextrin 30.0 g.
Ammonium tartrate (b) peptone 20.0 g.
Ammonium citrate dextrin 30.0 g.
Ammonium malate Variants: Peklo used a solution contain-
Ammonium succinate ing 5.0 g. KH2PO4, 2.0 g. MgS04 and 20.0
Succinamide g. of sucrose per liter to grow plant
Succinimide actinomyces.
Asparagin Reference: Will (1896 Peklo (1910
p. 754),
Leucin
p. 551).
Tyrosine
Reference: Calmette, Massol and Breton 135. Henneberg's Basal Sucrose Salt
(1909 p. 581).
Solution
133. Lohnis' Basal Glucose Salt Solution Constituents:
Constituents 1. Distilled water 1000.0 cc.
1. Distilled water 1000.0 cc. 2. K2HPO4 (0.5%) 5.0 g.
2. K2HPO4 0.5 g. 3. MgS04 (0.2%) 2.0 g.
3. MgS04 0.02 g. 4. Sucrose 50.0 g.

5. FeoCle trace (1) Dissolve 2, 3 and 4 in 1.
6. Glucose (1.0%) 10.0 g. (2) Add one of the added nutrients.
Preparation Sterilization: Not specified.
(1) Dissolve 2, 3, 4 and 5 in 1. Use: Cultivation of yeast.

(2) Dissolve 1.0% glucose and 0.1% of Added nutrients: The author added 0.3%
one of the added nutrients in (1). of one of the following as nitrogen sources
Sterilization: Not specified. ammonium tartrate
Use: To study nitrogen assimilation. (NH4)2S04; NH4CI
Added nutrients: The author added 0.1% ammonium phosphate, NH4NO3
of one of the following: Reference: Henneberg (1907 ^40-50) (1909
Urea (NH4)2S04 p. 104).
sodium hippurate ammonium acetate
asparagin ammonium butyrate 136. Czapek's Sucrose Salt Solution
ammonium lactate NaNOs (Waksman)
uric acid
Reference: Lohnis (1913 Constituents
p. 99).
1. Water 1000.0 cc.
134. Will's Basal Sucrose Solution 2. K2HPO4 10 g.
Constituents 3. KCl 0.5 g.
1. Water 1000.0 cc. 4. MgSOi 0.5 g.
2. KH2PO4 25.0 g. 5. FeS04 0.01 g.

6. Sucrose 30.0 g.
3. MgS04 8.5 g.
4. Sucrose 5.0 g. Preparation
5. Citric acid 5.0 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Preparation (2) Place in 100.0 cc. portions in flasks.
(1) Dissolve 2, 3 and 4 in 1. Sterilization : Autoclave at 15 pounds pres-

(2) Add- 0.5% citric acid to (1). sure for 15 minutes.
(3) Add one of the combinations given Use: Cultivation of bacteria found in soil.
under added nutrients. Reference: Waksman (1918 p. 479).

137. von Bronsart's Basal Sucrose Salt of MgS04, 0.3 g. to 20.0 g. for the
Solution basic solution. The author reported
Constituents that 2.0% MgS04 did not inhibit the
1. Water 1000.0 cc. growth of yeast, but 0.03% sufficed
2. Sucrose (4.0%) 40.0 g. for normal growth.
3. MgS04 (0.25%) 2.5 g. (d) peptone 25.0 g.
4. KH2PO4 (0.25%) 2.5 g. (e) aspartic acid amount not given,

Preparation (f) leucineamount not given.
(1) Dissolve 2, 3 and 4 in 1. (g) tyrosine amount not given,
(2) Add one of the nitrogen sources given
(h) glycocoll amount not given.
in the added nutrients. (i) Bokorny used 4.0 g. KH2PO4 in the
Sterilization: Not specified. basic solution and added one of the
Use: To study the availability of various following:

nitrogen sources for Xylaria, Xylaria asparagin 25.0 g.
arbuscula, Xylaria polymorpha, Xylaria aspartic acid 25.0 g.
hypoxylon. The author found that as- peptone 25.0 g.
paragin was a better nitrogen source (j) Bokorny used 4.0% sucrose in the
than peptone. KNO3 was found to be basic solution, and added 0.25%
the best inorganic source. aspartic acid,
Variants: The author used 0.5% asparagin (k) Bokorny used 1.0 g. KH2PO4, 0.3 g.
with 1.0, 4.0, 8.0, 16.0, 32.0 or 50.0% MgS04 and added a trace of CaCU
sucrose. He found that sugar concen- to the basic solution. To this solu-
trations above 16.0% inhibited develop- tion he added:

ment of the Xylaria listed above. There KNO3, 0.1, 0.2 or 0.5 g.
(a)
was slight growth using 32.0% sucrose, KNO3, 0.2 and asparagin 2.0 g.
(b)
but no growth using 50.0% sucrose. (c) KNO3, 0.1 g. and asparagin 0.3 g.
Added nutrients: The author employed Reference: Bokorny (1911 p. 183; 1912 pp.
the following nitrogen sources: 121; 148; 1917 p. 364; 1917 p. 47; 1920 p. 24).
asparagin 1 .0%
139. Smith's Sucrose Salt Solution
peptone 0.5%
Constituents
NH4NO3 1.0%
1. Triple distilled water 1000.0 cc.
(NH4)2S04 0.5%
2. Sucrose 5.0 cc.
Reference: von Bronsart (1919 p. 61).
3. KH2PO4 2.0 cc.
4. MgS04-7H20 0.1 cc.
138. Bokorny's Basal Sucrose Salt Solution
5. NaCl 0.5 cc.
Constituents: Preparation
1. Water 1000.0 cc. (1) Dissolve 2, 3, 4 and 5 in 1.
2. Sucrose 100.0 g. (2) All apparatus should be scrupulously
3. MgS04 1.0 g. clean and only chemicals of known
4. KH2PO4 5.0 g. purity be used.
(1) Dissolve 2, 3 and 4 in 1. Use: Cultivation of bacteria.

(2) Add one of the materials listed under Reference: Smith (1905 p. 198), Tanner
added nutrients. a919p. 65).
140. Miinter's Basal Galactose Salt Solution
Use To study yeast growth.
:
Added nutrients and variants: One of the Constituents

following nitrogen sources or combina- 1. Water 1000.0 cc.
tions was added: 2. MgS04 0.5 g.
(a) asparagin 25.0 g. 3. NaCl 0.5 g.
(b) (NH4)2S04 5.0 g. 4. K2HPO4 1.5 g.
(c) asparagin 5.0 g. with 1.0 g. KH2PO4, 5. CaCl2 0.1 g.
a trace of CaCU and varying amounts 6. FeCl3 drops
7. Glucose 10.0 g. 142. Omeliansky's Basal Cellulose Salt

8. Galactose 2.0 g. Solution
Preparation
Constituents
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
2. NaCl trace
(3) Add 0.25 g. of one of the added nu-
trients to each 50.0 cc. lot.
3. MgS04 0.5 g.
5. Filter paper, straw, lilac pith, or cab-
Use: Cultivation of Actinomycetes. Actin-
bage
omyces odorifer, Act. chromogenes and
Preparation
others. Alanin and tyrosin were found
to be the best nitrogen sources for the
actinomycetes studied. The remainder
(3) Add one of the cellulose sources.
of the compounds, however, were good
sources for some of the actinomycetes.
Added nutrients: The author added 0.25 g. Use: To study fermentation of cellulose
of one of the following to each 50.0 cc. by amylobacter and organisms found in
of solution: the mud of rivers.
urea Added nutrients and variants:

sulphocarbamide (a) The author added 1.0 g. of one of the
alanin following to the basic solution:

tyrosin (NH4)2S04
dicyandiamide ammonium phosphate
Reference: Munter (1913 p. 377).
peptone
asparagin
(b) Kroulik specified the use of 1.0 g.
141. Buchanan's Basal Maltose Salt
Solution
K2HPO4 in the basic solution and
added 1.0 to 2.0% cellulose and added
Constituents 1.0 g. (NH4)2S04, 0.5% CaCOa, and
1. Distilled water 1000.0 cc. 1.0% MgCOs. He used the medium
2. KH2PO4 2.0 g. for the cultivation of cellulose split-
3. IMgS04 0.1 g. ting thermophiles.
4. Maltose (1.0%) 10.0 g. (c) Brussoff specified the use of K2HPO4
Preparation in the basic solutionand added 1.0 g.
(1) Add 2, 3, and 4 to 1. (XH4)2S04, 2.0 g. CaCOs and filter
(2) Add 1.0% of one of the added nu- paper. The solution was distributed
trients. in 20.0 cc. lots and a strip of filter
Sterilization: Sterilizefor 20 minutes on paper (about 0.1 g.) was added to
each of 3 successive days in streaming each lot. The medium was used to
steam. Then incubate at room tempera- study cellulose decomposition by iron
ture for one week to test sterility. bacteria {Ferribacterium duplex).
Use To study gum formation by Bacillus
: He reported that gas bubbles were
radicicola. Growth and gum production formed after 2 days and a yellow
generally good. Poorest results were ob- membrane formed after 4 days when
tained when ammonium citrate was inoculated with sludge.
present in the medium. (d) Khouvine specified the use of K2HPO4
Added nutrients: The author added 1.0% in the basic solution and added 1.0 g.
of one of the following: (NH4)2S04 0.0 or 2.0 g. of glucose
Ammonium succinate and cellulose. The medium was
Ammonium citrate tubed in 5.0 cc. lots and 1 square
Ammonium asparaginate centimeter of Berzilius filter paper,
Asparagin or a little cellulose precipitated
Peptone (Witte's) after having been dissolved in
Reference: Buchanan (1909 p. 393). Schweitzer's reagent to each tube.
The medium was used for isolation

and enrichment of B. cellulose dis-
solvens.
(e) Giltner specified the use of K2HPO4
and added
in the basic solution 1.0 g.
(XH4):S04 and added 2.0 g. of filter
paper, cotton straw or starch. The

medium was used to cultivate anaer-
obic cellulose fermenters. 1000.0
Reference: Omeliansky (1902 p. 226),
Kroulik (1912-13 p. 340), Brussoff (1918
p. 193), Giltner (1921 p. 373), Khouvine
(1923 p. 713).
143. Percival's Glycerol Phosphate Solution

Constituents:
2. Magnesium phosphate 0.1 g.
3. KH2PO4 2.0 g.
4. Glycerol 10.0 g.
Preparation
(2) Add 4 to (1).

(3) Heat to boiling.
(4) Filter.
(5) Adjustment of reaction not specified.
(6) Tube in 10 to 15.0 cc. quantities.
(7) After sterilization inoculate with
contents of a nodule of a pea, runner
bean, red clover and broad bean by
means of a sterile knife.
Sterilization : Steam on each of 3 successive
days.
Use: Growth of bacteria from the roots of
leguminous plants, (Bacteroids).
Reference: Percival (1920 p. 204).
144. Buchanan's Glycerol Salt Solution II
Constituents
1. Water 1000.0 cc.
2. KH2PO4 (0.2%) 2.0 g.
3. MgSO4(0.01%) 0.1 g.
4. Glycerol (1.0, 3.0 or 5.0%) 10.0, 30.0 or
50.0 g.
Preparation :
(1) Heat 2, 3 and 4 in 1 to boiling.

(2) Cool on ice.
(3) Filter to remove insoluble precipi-
tates.
(4) Tube.
Sterilization: Sterilize intermittently in
flowing steam on each of 3 successive days.
Use: Cultivation of Bacillus radicicola
bacteroids.
Lohnis added 0.1% of urea sodium hip- (d) tyrosine 1 -0 g.

purate, uric acid, asparagin, ammonium asparagin 2.0 g.
sulphate acetate, butyrate lactate or taurine 0.5 g.
NaNOs. Lohnis also specified that (e) tyrosine 10 g.
other carbohydrates, alcohols and acids asparagin 2.0 g.
might be used instead of glycerol. ammonium mucate 2.0 g.
Reference: Gerlach and Vogel (1901 p. (f leucine 4.0 g.
612). Lohnis (1913 p. 94), Cunningham tyrosine 1 -0 g.
(1924 p. 153). asparagin 3.0 g.
(g) leucine 4.0 g.
146. Kuhne's Basal Lactate Solution tyrosine 1 -0 g.
(Proskauer and Beck) ammonium mucate 2.0 g.
(h) leucine 4.0 g.
Constituents
1. Water 600.0 cc. asparagin 2.0 g.
2. MgS04 3.5 g.
ammonium mucate 2.0 g.
3. Gypsum (burned) 1-5 g. (i) tyrosine 1-0 g.
4. NaCl 16.0 g. asparagin 2.0 g.
5. Magnesium (burned).. 2.5 g. .

6. Potash (dried) 62.13 g. (j) leucine 4.0 g.

7. Soda 7.35 g. tyrosine 1 g.
8. Ferrum reductum 6.2 g. taurine 0.5 g.
9. H3PO4 (Sp. G. 1.3). .. 95.0 g. (k) leucine 4.0 g.
10. Lactic acid (Sp. G. 1.2). 50.0 to 60.0 g. asparagin 2.0 g.
11. Glycerol taurine 0.5 g.
Preparation (1) tyrosine 1-0 g.
(1) Dissolve 2, 3, 4, 5, 6, 7, 8, 9 and 10 in 1. asparagin 2.0 g.
(2) Add 12.0 cc. of (1), 40.0 g. glycerol, taurine 0.5 g.

5.0 g. NaCl and 1000.0 cc. of water (m) leucine 4.0 g.
to one of the combinations listed ammonium mucate 2.0 g.
under added nutrients. taurine 0.5 g.
Sterilization: Not specified. (n) tyrosine 1.0 g.
Use: To study the requirements for the ammonium mucate 2.0 g.
growth of tubercle bacilli. Growth gen- taurine 0.5 g.
erally occurred. The authors stated that asparagin 2.0 g.
(0)
12.0 cc. of the basic solution was the ammonium mucate 2.0 g.
equivalent of 10.0 g. of commercial taurine 0.5 g.
Liebig's meat extract.
(p) leucine 4.0 g.
Added nutrients: The authors use 12.0 cc.
tyrosine 1 g.
of (1) basic salt solution and add 1000.0
asparagin 2.0 g.
cc. water, 40.0 g. glycerol and 5.0 g. of
NaCl. The following materials were
taurine 0.5 g.
added to this solution:
(a) tyrosine 10.0 g (q) alanine 4.0 g.
asparagin 2.0 g.
asparagin 2.0 g
ammonium mucate 2.0 g (r) alanine 4.0 g.
taurine 0.5 g asparagin 3.0 g.
(b) leucine 4.0 g ammonium mucate 3.0 g.
asparagin 2.0 g (s) Used 7.35 soda instead of 6.35 g. in

ammonium mucate 2.0 g the basal solution and added:
taurine 0.5 g leucine 4.0 g.
(c) tyrosine 1 -0g alanine 4.0 g.
ammonium mucate 2.0 g glycocoll 4.0 g.
taurine 0.5 g glucose 2.0 g.
(t) glycocoU 3.0 g. 5. MnS04 (1:1000) 1.0 cc.

glycerol 40.0 g. 6. FeS04 (1:1000) 1.0 cc.
taurine 5.0 g. 7. Mannitol 20.0 g.
(u) Tomasczewski used 7.35 soda in the Reference: Omeliansky and Sswerowa
basal solution and added: (1911 p. 645), Harvey (1921-22 p. 105).
tyrosine 1 -0 g 149. Murray's Mannitol Salt Solution
leucine 4.0 g Constituents:
asparagin 2.0 g 1. Water 1000.0 cc.
ammonium mucate 3.0 g 2. K2HPO4 0.2 g.
taurine 0.5 g
3. MgS04 0.2 g.
Reference: Proskauer and Beck (1894 p 4. NaCl 0.5 g.
129-131, 134-135), Tomasczewski (1899
5. Mannite 20.0 g.
p. 249).
6. FeCl. 1 drop of 10% sol.
147. Calimard and Lacommes' Basal Preparation

Glycerol Salt Solution (Kolle and (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Wassermann) (2) Neutralize with KOH
using phenol-
phthalein as indicator.
Constituents
1. Water 1000.0 cc.
Use: To study the fixation of nitrogen
2. NaCl (0.5%) 5.0 g.
under anaerobic and aerobic conditions
3. MgS04 (0.05%) 0.5 g.
by bacteria from the soil.
4. Glycerol potassium phos-
Reference: Murray (1916 p. 604).
phate (0.2%,) 2.0 g.
5. Glycerol (1.5%,) 15.0 g. 150. Percival's Mannitol Phosphate Solution
Preparation: (1) Dissolve 2, 3, 4, 5 and one Constituents
of the added nutrients in 1.
Sterilization: Not specified. 2. KH,P04 2.0 g.
Use: General culture medium. 3. Magnesium phosphate 10 g.
Added nutrients: The author added one of 4. Mannitol 20.0 g.
the following: Preparation:
urea, amines, leucine, tyrosine, etc., etc.
Reference: Kolle and Wassermann (1912 p. Add 4 to (1).
(2)
394). Heat to boiling.
(3)
148. Beijerinck's Modified Mannitol Salt (4) Filter.
Solution (Omeliansky and Sswerowa) (5) Adjustment of reaction not specified.

(6) Tube in 10 to 15.0 cc. quantities.
Constituents: After sterilization inoculate with
(7)
1. Distilled water 1000.0 cc. contents of a nodule of a pea, runner
2. Mannitol 20.0 g bean, red clover, or broad bean, by
3. Potassium phosphate.. 1.0 g means of a sterile knife.
4. MgS04 0.5 g Sterilization: Sterilize on 3 successive
5. NaCl 0.1 g days (time not given).
6. Iron sulphate 0.1 g Use: Cultivation of bacteria from the roots
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 of leguminous plants (bacteroids).
in 1.
Use: To study the pigment production of 151. Capaldi and Proskauer's Basal Man-
Azotobacter chroococcum. nitol Salt Solution
Variants: Harvey used the following vari- Constituents:
ant of this solution to study nitrification: 1. Water 1000.0 cc.
1. Distilled water 1000.0 cc. 2. NaCl (0.02%) 0.2 g.
2. K2HPO4 1.0 g. 3. MgS04 (0.01%) 0.1 g.
3. MgS04 0.2 g. 4. KH2PO4 (0.2%) 2.0 g.
4. NaCl 0.02 g. 5. Mannitol (0.2%) 20.0 g.
Preparation 152. Percival's Succinate Solution

(1) Dissolve 2, 3, 4 and 5 in 1.
Constituents:
(2) Add a suitable nitrogen source.
2. KH2PO4 2.0 g.
Use: To study acid production by B. colt. 3. Magnesium phosphate 0.1 g.
Added nutrients The authors added one of
:
4. Sodium succinate 5.0 g.
the following nitrogen sources: Preparation
glycocoll
alanine Add 4 to (1).
(2)
leucine Heat to boiling.
(3)
oxamide (4) Filter.
succinamide Adjustment of reaction not specified.
(5)
tyrosine Tube in 10 to 15.0 cc. quantities.
(6)
urea (7) After sterilization inoculate with
sulfocarbamid contents of a nodule of a pea, runner
guanidinchlorhydrate bean, red clover, or broad bean by
guanine means of a sterile knife.
creatine Sterilization: Sterilize on 3 successive days
sarcosine (time not given).
allantoin Use: Cultivation of bacteria from the roots
alloxantin of leguminous plants, (bacteroids).
alloxan Reference: Percival (1920 p. 204).
theobromine
caffein 153. Winogradsky's Glucose Salt Solution
potassium urate Constituents
glycocoll + urea 1. Distilled water 1000.0 cc.
ammonium formate 2. Glucose 20.0 g.
ammonium acetate 3. Potassium phosphate .. . 1.0 g.
ammonium butyrate 4. MgS04 0.2 g.
ammonium isobutyrate 5. NaCl small amount
ammonium valerate 6. FeS04 small amount
ammonium stearate 7. MnS04 small amount
ammonium oleate 8. Chalk
ammonium lactate Preparation
ammonium pyroracemate (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
ammonium oxalate (2) Adjustment of reaction not specified.

ammonium malonate (3) Distribute into Drechsel's flasks with
ammonium succinate aground cover.
ammonium tartrate (4) Add an excess of chalk to each flask.
ammonium fumarate Sterilization: In the autoclave.
ammonium mucate Use Used by Winogradsky to study nitro-
:
ammonium citrate gen fixation by Clostridium Pastoriunum.

ammonium aconitate Other investigators used other nitrogen
ammonium itaconate fixing bacteria.
ammonium citraconate Variants
ammonium amidobenzoate (a) von Freudenreich used 20 to 40.0 g.
ammonium hippurate glucose, 0.5 g. of potassium phosphate
ammonium orthonitrozimmtate instead of 0.2 g., and specified 0.01
ammonium piperate to 0.02 g. of NaCl, FeS04 and MnS04.
ammonium carbamate (b) Jacobitz used the same variation as
ammonium cyanurate (a) but specified distilled water.
ammonium carbonate (c) Smith used 20 to 40.0 g. glucose,
Reference: Capaldi and Proskauer (1S96 0.5 g. MgS04, 0.01 to 0.02 g. NaCl,
p. 456). 0.1 to 0.2 g. iron sulphate, instead of
FeS04, omitted the MnS04 and used and alfalfa plant nodules and found that
30.0 to 40.0 g. CaCOs per liter. little, if any, nitrogen was assimilated
(d) Bredemann specified 1.0 g. K2HPO4, after 60 days incubation at 28 to 30'^C.
0.02 g. NaCl, 0.1 g. FeS04, and 0.01 g. Reference: Stutzer, Burri and Maul (1896
MnS04 but did not specify distilled p. 669).
water.
155. Stoklasa's Basal Salt Solution
(e) Percival specified 1.0 g. K..HP04,
used 30.0 g. glucose and 0.5 g. MgS04. Constituents
(f) Lantzsch specified 1.0 g. K,HP04, 1. Water 2300.0 cc
0.01 g. NaCl, 0.01 g. FeS04 and omit- 2. Sodium phosphate 0.5 g
ted the MnS04. He did not specify 3. K.SO4 0.2 g.
distilled water. 4. MgClo 0.05 g.
(g) Lantzsch specified the use of 0.01 g. 5. Glucose 2.5 g.
NaCl, 0.2 g. FeS04 used 50.0 g. 6. CaCOs 10.0 g.
CaCOa, and omitted the MnS04. Preparation
This solution was employed as a basic (1) Dissolve 0.5 g. sodium phosphate,
solution and added one of the follow- 0.2 g. potassium sulphate and 0.05 g.
ing to each 100.0 cc. of solution: magnesium chloride in 500.0 cc. of
(a) 2.0, 5.0 or 10.0 cc. of a 2.0% NaOH water.
(soil extract). (2) Dissolve 2.5 g. glucose in 500.0 cc.
(b) 0.28 g. or 0.56 g. animal charcoal. water.
(c) 0.4 g., 1.0 g. or 3.0 g. of Bolus alba (3) Mix 200.0 cc. (1) with 100.0 cc. (2).
(pure kaolin). (4) Dissolve 6 and one of the added
(d) 0.25% or 1.0% gelatin. nutrients in (3).
This medium was used for the cultivation (5) Dilute to 20C0.0 cc.
amylobacter spores.
of Sterilization: Method not given.
References: Winogradsky (1902 p. 49), von Use: To study utilization of nitrogen by
Freudenreich (1903 p. 516), Jacobitz B icillus megatherium, {Bacillus Ellen-
(1903 p. 100), Smith (1905 p. 199), Brede- The author reported
bichii) or "Alinit."
mann (1909 p. 496), Percival (1920 p. 174), that more nitrogen was found to be in
Lantzsch (1921 p. 2), Lantzsch (1921 p. 5). solution after incubation.These organ-
isms decomposed nitrogenous material to
154. Stutzer, Burri and Maul's Glucose a form that may be absorbed by the root
Salt Solution hairs.
Constituents: Added nutrients: The author added 50.0 g.

1. Water 1000.0 cc. of glutin or fibrin.
2. Asbestos Reference: Stoklasa (1898 p. 508).
3. Potassium phosphate 10 g.
156. Charpentier's Glucose Salt Solution
4. MgS04 0.5 g.
5. NaCl 1.0 g. Constituents:
6. CaCU 0.1 g. 1. Water 1000.0 cc.
7. Dextrose 20.0 g. 2. K2HPO4 0.25 g.
Preparation 3. KH2PO4 0.25 g.
(1) Dissolve 3, 4, 5, 6 and 7 in 1. 4. MgS04 0.37 g.
(2) Heat long fibre asbestos to glowin?. 5. Iron phosphate trace
(3) To a 2 liter flask add 10.0 g. (2) 25.0 6. CaS04 trace
water.
cc. (1), 25.0 cc. sterile 7. NaCl 0.2 g.
(4) After sterilization add a large plati- 8. Glucose
num loop of a gelatin streak culture Preparation:
of an organism to 100.0 cc. sterile (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
water. Add 2.0 cc. of (5) to (4). (2) Distribute in flasks.
Sterilization: Method not given. (3) Add 0.075 g. glucose to each flask.
Use: To determine nitrogen fixation. Amount of medium in the flask not
Authors used the bacteria from mustard specified.
(4) Pass a current of air thru H2SO4 and 3. K2HPO4 2.0 g.

then thru the culture. 4. CaCl2 0.1 g.
Sterilization: Not specified. 5. FeS04 0.05 g.
Use: Nitrogen fixation by algae; Cysto- 6. Glucose 10.0 g.
coccus humicola did not fix free nitrogen. Preparation
Reference: Charpentier (1903 p. 322). (1) Dissolve one of the added nutrients
in water.
157. Krzemieniewska's Glucose Salt
(2) Dissolve 2, 3, 4, 5 and 6 in the re-
Solution (Vogel)
mainder of the water.
Constituents (3) Mix sterile (1) and (2) under aseptic
1. Water 1000.0 cc. conditions.
2. MgS04-7H20 0.25 g. Sterilization: Sterilize (1) by filtration and
3. CaHP04 0.25 g. (2) in the autoclave.
4. Glucose 13.5 g. Use: To study assimilation of nitrogen by
5. K2SO4 0.0 to 0.872 g. algae, Cystococcus humicola. Author
Preparation reported that Ca(N03)2 was a more
(1) Dissolve 2, 3 and 4 in 1. suitable nitrogen source than asparagin
(2) Add from 0.0 to 87.2 mg. of K2SO4 or peptone.
to(l). Added nutrients and variants:
Sterilization: Not specified. (a) The author added 1.8 g. of peptone or
Use: Nitrogen fixation. The author found asparagin as a nitrogen source.
1.8 g.
that the most nitrogen was fixed in the (b) Miiller omitted the FeS04, used 0.1%
presence of 0.872 g. K2SO4. K2HPO4 and 0.2% MgS04 in the
Reference: Vogel (1911-12 p. 417). basic solution and added one of the
following nitrogen sources. He culti-
158. Kriiger andSchneidewind's Basal vated Bacterium Giintheri.
Glucose Salt Solution (Heinze) KNO3
Constituents ammonium tartrate
1. Water 1000.0 cc. aspn-ragin
2. Glucose (1.0%) 10.0 g. peptone
3. K3PO4 (0.2%) 2.0 g. References: Charpentier (1903 p. 333),
4. MgS04 (0.04%) 0.4 g. Miiller (1907 p. 472).

5. CaCl2 (0.02%) 0.2 g.
160. Stoklasa's Basal Glucose Salt Solution
6. FeCh (20.0% soln.) 1 drop
Preparation Constituents:
(2) Add one of the nitrogen sources listed 2. d-glucose 25.0 g.
in added nutrients. 3. Phosphoric acid anhydride. 0.2 g.
Sterilization: Not specified. 4. K2SO4 1.0 g.
Use: To study nitrogen assimilation by 5. MgCl2 0.5 g.
green algae, Chlorella, Chlorothecimn, 6. Iron sulphate +
Stichococcus . The author reported that 7. Aluminum sulphate 0.1 g.
nitrogen was not assimilated. Poor 8. CaCl2 0.1 g.
growth unless nitrogen was present. Preparation
Variants: The solution may be used with- (1) Dissolve 2, 4, 5, 6, 7 and 8 in 1.
out the addition of any nitrogen source. (2) Add one of the added nutrients to (1).
Added nutrients: The author added 0.25% (3) Add 0.2 g. of phosphoric acid anhy-
of (NH4)2S04or NaNOs. dride in the form of monomagnesium
Reference: Heinze (1906 p. 647). phosphate, monodialuminum phos-
phate, monodiferric phosphate or
159. Charpentier's Basal Glucose Salt
trimagnesium phosphate to (2).
Solution
(4) Inoculate 8 days following steri-
Constituents lization.
1. Water 1000.0 cc. Sterlization : In the autoclave.
2. MgS04 1.0 g. Use: To study role of phosphorus in the
soil . Development of the bacteria hardly 3. Citric acid 5.0 g.

visible when phosphoric anhydride was 4. MgS04 2.0 g.
omitted. 5. K2HPO4 2.0 g.
Added nutrients: The author added 1.0 g. 6. CaCl2 0.2 g.
nitrogen in the form of (NH4)2S04 or 7. FeCla trace
(NH4)N03. Preparation
Reference: Stoklasa (1911 p. 490). (1) Dissolve 2, 3, 4, 5, 6 and 7 in distilled
water.
161. Henneberg's Basal Glucose Salt Reaction should be neutral (indicator
(2)
Solution
not specified).
Constituents: Sterilization: Not given.
1. Distilled water 1000.0 cc. Use: Nitrogen fixation by aerobic nitro-
2. KH2PO4 (0.1%) 1.0 g. gen fixing bacteria.
3. MgS04 (0.02%) 0.2 g. Reference: Harvey (1921-22 p. 104).
4. CaCl2 (0.01%) 0.1 g.
164. Miinter's Basal Mannitol Salt Solution
5. Glucose (2.0%) 20.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and 1.0% Constituents:
of one of the added nutrients in 1. 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Mannitol 2.0 g.
Use: To study nitrogen requirements for 3. Glucose 10.0 g.
lactic acid bacteria. Urea, asparagin or 4. MgS04 0.5 g.
ammonium phosphate were found not to 5. NaCl 0.5 g.
be suitable as nitrogen sources. 6. K2HPO4 2.0 g.
Added nutrients: The author added 1.0% 7. CaCl2 0.1 g.
of one of the following: 8. FeCls trace
peptone asparagin Preparation
casein urea (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
gluten ammonium phosphate (2) Add 0.02 or 0.1 g. of nitrogen in the

Reference: Henneberg (1903 p. 7). form of one of the added nutrients.
(3) Adjustment of reaction not given.
162. Gottheil's Carbohydrate Glycerol
Solution
Constituents: Use: To study availability of nitrogen for
1. Distilled water 1000.0 cc. Actinomycetes odorifer, Act. chromo-
2. Potassium phosphate 1-0 g. genes, Act. albus. NH4CI, NH4NO3 and
3. CaCl2 0.1 g. asparagin were found to be equally suit-
4. MgS04 0.3 g. able nitrogen sources for the actinomy-
5. NaCl 0.1 g. cetes studied.
6. Iron trace Added nutrients: The author used the
7. Dextrose 5.0 g. following nitrogen sources:
8. Cane sugar 5.0 g. NH4CI
9. Glycerin 5.0 g. NH4NO3
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8 asparagin
and 9 in 1. Reference: Miinter (1913 p. 368).
Use: Used as a general culture medium. 165. Chrzaszcz's Basal Sucrose Salt
Author used bacteria found in the soil Solution
and on the roots of plants. Constituents:
Reference: Gottheil (1901 p. 432). 1. Water 1000.0 cc.
2. Sucrose 100.0 g.
163. Harvey's Citric Acid Glucose Solution
3. MgS04 2.0 g.
Constituents: 4. K2SO4 2.0 g.
1. Distilled water 1000.0 cc. 5. Na2HP04 5.0 g.
2. Glucose 2.0 g. 6. CaCOa 1.0 g.
Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Add one of the added nutrients to (1).
Use: To study growth requirements of
yeasts. Author reported that yeast grew
more luxuriantly in a medium containing
peptone than asparagin. Growth best,
however, in sugar beer wort medium.
Added nutrients: The author added 5.0 g.
of asparagin or 5.0 g. of peptone.
Reference: Chrzaszcz (1904 p. 148).
166. Maze's Sucrose Salt Solution

Constituents:
2. Sucrose 20.0 g.
4. NaCl 1.0 g.
5. FeS04 trace
6. MgS04 trace
7. ZnCl2 trace
8. Calcium saccharate trace
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.

Sterilization: Sterilize at 120C. (time not
given).
Use: Cultivation of bacteria from the
nodules of leguminous plants, nitrogen
fixing bacteria.
Reference: Maze (1898 p. 12).
167. Linde's Basal Sucrose Salt Solution
Constituents
1. Distilled water 1000.0
2. K2HPO4
3. MgS04-7H20
4. NaCl
5. CaCl2
6. Sucrose (0.2%)
Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Add 0.15% of one of the nitrogen

sources given in added nutrients.
Use: Cultivation of Cladothrix. The
author reported that the organism grew
better on meat extract media.
Added nutrients: The author added 0.15%
of one of the following:
KNO3 (NH4)3P04
NH4CI (NH4)2S04
Reference: Linde (1913 p. 387).
CULTUEE MEDIA FOR CULTIVATION OF MICROORGANISMS 55
(3) When using KNO3 distribute by 171. Fred and Loomis' Mannitol Solution
filling 200.0 cc. flasks. When using
Constituents:
other nitrogen sources distribute in
thin layers in flasks.
2. Mannitol 10.0 g.
(4) Inoculate with canal water contain- 3. MgS04 0.2 g.
ing some mold or decay (moder).
4. KH2PO4 0.2 g.
Sterilization: Not specified. 5. NaCl 0.2 g.
Use: To study the decomposition of cellu- 6. CaS04 0.1 g.
lose by denitrifying organisms. The Preparation
author reported denitrification and de- (1) Dissolve 2, 3, 5 and 6 in 1.
composition of cellulose with the pro- (2) Dissolve phosphate separately in a
duction of N2 and CO2. little water and add to (1).
Added nutrients: The author used the (3) Make (2) neutral to phenolphthalein
following nitrogen sources: with N/1 NaOH.
KNO3 2.5 g. (4) After sterilization add NaOH and
NH4CI 1.0 g. H2SO4 to obtain pH value from 2.7
KNO2 amount not given to 11.1.
MgNH4P04 amount not given Sterilization: Method not specified.
peptone amount not given Use: To show the effect of hydrogen ion
Variants The author sprinkled MgNH4P04
: concentration on growth of Rhizobium.
between two Swedish filter papers and Maximum growth at pH 7.2. None in
placed them in a flask. Then he poured pH 2.77 and 3.1. Yamagata and Itano
a solution of 0.5 g. K2HPO4 in a liter of found the optimum pH for azotobacter
water over the paper. He found that to be between 6.6 and 7.7 depending on
Bacillus ferr ugineus from the soil decom- the type of azotobacter.
posed the cellulose. Variants: Yamagata and Itano used 20.0 g.
Reference: van Iterson (1903-4 p. 690). of mannitol instead of 10.0 g.
Reference: Fred and Loomis (1917 p. 629),
170. Jones' Modified Ashby's Mannitol Yamagata and Itano (1923 p. 522).
Solution
172. Krzemieniewska's Mannitol Salt
Constituents: Solution (Vogel)
2. Mannitol 20.0 g.
1. Water 1000.0 cc.
3. IV2HPO4 0.2 g.
2. K2HPO4 2.5 g.
4. MgS04 0.2 g.
3. MgS04 2.5 g.
5. NaCl 0.2 g.
4. CaS04 2.5 g.
6. CaS04 0.1 g.
5. Mannitol 20.0 g.
7. CaCh 5.0 g
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 Sterilization: Not specified.
in 1.
Use: Cultivation of Azotobacter.
Sterilization: Not specified. Variants: The author used 2.5 g. CaHPOi
Use: Cultivation of Azotobacter. instead of 2.5 g. CaS04, or omitted the
Variants K2HPO4 and added 2.5 g. CaHPO* instead
(a) Bonazzi specified the use of tap of CaS04.
water, used 0.408 g. MgS04-7H20; Reference: Vogel (1911-12 p. 416).
0.127 g. CaS04; 0.2 g. CaCOs and
specified that Ca(N03)2 might be 173. Harvey's Mannitol Salt Solution
added if desired (amount not given). Constituents:
(b) Giltner used 15.0 g. of mannitol and 1. Distilled water 1000.0 cc.
added 1 drop of 10.0% Fe2Cl6 solution. 2. Mannitol 15.0 g.
Reference: Jones (1913 p. 14), Bonazzi 3. MgS04 0.2 g.
(1921 p. 357), Giltner (1921 p. 376). 4. KH2PO4 0.2 g.
5. NaCl 0.2 g. and pass N2 that has been washed in

potassium pyrogallic acid solution
6. CaS04 0-1 g-
three times, then in H2SO4 and
7. CaCOs 5.0 g.
Preparation NaOH, thru the flasks.
distilled water. Sterilization: Not specified.
(1) Dissolve 4 in a little
(2) Neutralize (1) to
phenolphthalein. Use: To study nitrogen fixation by Bacil-
lus amylobacter, Bad. pneumoniae and
(3) 2, 3, 5, 6 and 7 to (2) and make
Add
up to 1000.0 cc. torula under anaerobic and aerobic con-
Sterilization: Not specified. ditions.
Use Cultivation of aerobic nitrogen fixing

:
Variants: The author suggests theûse of
bacteria and protozoa. glucose instead of mannitol and specifies
Reference: Harvey (1921-22 p. 104). that 10.0 or 20.0 g. of calcium humate
may or may not be added to the glucose
174. Sackett's Modified Lipman's Manni- medium.
tol Salt Solution Reference: Lantzsch (1921 p. 2).
Constituents
176. Koser and Rettger's Basal Glycerol
1. Water (surface) 1000.0 cc.
Salt Solution
2. Mannitol 15.0 g.
3. K2PO4 0-5 g- Constituents

4. MgS04 0-2 g- 1. Distilled NH3 free water. . 1000.0 cc.
5. CaCl2 002 g. 2. NaCl 5.0 g.
6. 10.0% FeCU solution 1 drop 3. MgS04 0.2 g.

4. CaCl2 0.1 g.
Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1000.0 cc.
5. KH2PO4 1-0 g.
of surface water. 6. K2HPO4 10 g.
(2) Neutralize with N/1 NaOH to

phenol- 7. Glycerol 30.0 g.
phthalein. Preparation
(1) Dissolve 2, 3, 4, 5, 6 and 7 in
1.
Erlenmeyer flasks. (2) Add one of the added nutrients to (1).
Sterilization: Autoclave for 5 minutes at (3) pH = 6.6.
(4) Medium is clear and colorless and

120C.
Use: Study nitrogen fixation by bacteria requires no filtration.
found in soil. (5) Distribute into test tubes.
Variants: Heinemann used 0.2 g. K2HPO4 Sterilization Autoclave at 10 to 12 pounds

:
instead of 0.5 g. K3HPO4 and 0.01 g. pressure for 15 minutes.

FeoCle. Use: To study bacterial nutrition.
Reference: Sackett (1912 p. 87), Heine- Added nutrients: The authors used 1.0 g.
mann (1922 p. 39). of one of the following:

valin
175. Winogradsky's Mannitol Salt Solution glutamic acid
(Lantzsch) tyrosin
Constituents phenylalanin
1. Water 1000.0 cc. lysin picrate
2. Mannitol 20.0 g. urea
3. K2HPO4 10 g. uric acid
4. MgS04 0.2 g. hypoxanthin
5. FeS04 0.01 g. allantoin
6. NaCl 0.01 g. creatin
7. CaCOa 5.0 g. glycocoU
Preparation aspartic acid
(1) Dissolve one of 2, 3, 4, 5, 6 and 7

in 1. asparagin
(2) To prepare anaerobic culture after
tryptophan
inoculation, stopper the flasks with leucin
2 holed stoppers, seal with paraffin histidin monochloride
lysine dichloride 3. KCl 0.2 g.

taurine 4. CaCl2 0.2 g.
Variants: The authors found that com- 5. Sodium bicarbonate 0.1 g.
binations of amino acids possessed little 6. Glucose 1.0 g.
value over single amino acids. Preparation: (1) Dissolve 2, 3, 4, 5 and 6

Reference Koser and Rettger (1919 p. 304),
: in 1.
Koser (1918, p. 12). Not specified.

Sterilization:
Use: Diluent and a basis solution to which
177. Puriewitsch's Tartaric Acid Salt carbonaceous and nitrogenous material
Solution (Heinze)
may be added. (See medium 600.)
Constituents Reference: Park, Williams and Krumwiede
1. Water 1000.0 cc (1924 p. 122).
2. Tartaric acid 3.0 g.
181. Beijerinck's Glucose Salt Solution
3. MgS04 0.2 g.
Constituents:
4. CaCli 0.4 g.
5. KH2PO4 0.4 g.
Preparation: Dissolve 2, 3, 4 and 5 in 1.

2. Glucose 0.05 g.
(1)
Sterilization: Not specified. 3. Potassium phosphate 0.1 g.
4. FeCls several drops
Use: Study of nitrogen assimilation by
molds, Aspergillus niger and Penicillum 5. NasCOs 0.5 g.
glaucum. Preparation
(1) Dissolve 2, 3, 4 and 5 in ditch water
Reference: Puriewitsch (1895 p. 342 from
an abstract by Heinze 1903 p. 27). ("Grabenwasser") containing 45 mg.
SO3 per liter.
178. Omeliansky's Cellulose Solution (2) Distribute into flasks or beaker.
Constituents: Cover the beaker with a glass plate
1. Water or the flask full to reduce the sur-
fill
2. Chalk face exposed to the air.

3. Paper. Sterilization: Not specified.
Preparation: (1) Inoculate a flask of water Use: To study sulphate reduction by
containing chalk and paper with some Spirillum, desulfuricans. US and H2SO3
river mud. can be detected after 12 hours.
Use: To study cellulose fermentation by Reference: Beijerinck (1895 p. 57).
organisms found in mud of rivers. The
182. Stoklasa's Glucose Salt Solution
paper was completely destroyed after
Constituents:
3.5 years.
Reference: Omeliansky (190 p. 229). 1. River water 1000.0 cc.
2. Glucose 20.0 g.
179. Killer's Mannitol Solution 3. K2HPO4 0.5 g.
Constituents 4. NaoCOa 0.25 g.
2. Mannitol 20.0 g. (1) Dissolve 2, 3 and 4 in 1.
3. Potassium phosphate 0.5 g. (2) Adjustment of reaction not given.

Preparation: (1) Dissolve 2 and 3 in 1. (3) Distribute in thin layers in large
Sterilization: Not specified. flasks.
Use: To study nitrogen assimilation by Sterilization: Sterilize thoroughly in the

soil bacteria and protozoa. autoclave.
Variants : Dilute the medium 5 to 10 times Use: To study the utilization of carbo-
for cultivation of protozoa. hydrate and nitrogen assimilation by
Reference: Killer (1913 p. 522). Azotobacter chroococcum Beijerinck.
Reference: Stoklasa (1908 p. 503).
180. Ringer's Salt Solution (Park, Williams
and Krumwiede) 183. Gage's Maltose Salt Solution
Constituents Constituents:
1. Water 1000.0 cc. 1. Water nitrite free 1000.0 cc.
2. NaCl 10.0 g. 2. KH2PO4 1.0 g.
3. NaCl 0.5 g. Sterilization: Not specified.

4. Maltose 1.0 g. Use: Growth of mesonitrophilic organisms,
Preparation Azotobacter agilis, Granulobacter spher-
(1) Dissolve 2, 3 and 4 in 1000.0 g. of icum. When inoculated with pasteurized
nitrite free water. garden soil after 2 or 3 days a fermenta-
(2) Distribute in 100.0 cc. lots in 150.0 tion takes place. The odor of ethyl or
cc. flasks. propyl alcohol is given off.
Sterilization: Not specified. Variants: (1) Bonazzi specified the use of
Use: To study the fixation of nitrogen by deep well water and added 0.5 g. CaCOa
nitroso bacteria. No nitrogen was fixed for each 100.0 cc. of medium. He also
by the culture studied. specified that 1.246 g. Ca(N03)2 might be
Reference: Gage (1910 p. 19). added if desired.
Reference: Beijerinck (1901 p. 573), Bo-
184. Sohngen's Colloid Mannitol Solution
nazzi (1921 p. 336).
Constituents
1. Water 1000.0 cc.
186. Buhlert and Fickendey's Phosphate
2. Mannitol 20.0 g.
Glucose Solution
3. K2HPO4 0.5 g. Constituents:
4. Iron aluminum silicon about 0.1 g. 1. Water 1000.0 cc.
5. Colloidal material 2. K2HPO4 1.0 g.
Preparation 3. Glucose 20.0 g.
(1) Dissolve 2, 3 and 4 in 1. 4. NaCl 1.0 g.
(2) After sterilization add one of the 5. CaCOs 4.0 g.
following sterile materials to 100.0 Preparation
cc. of (1). (1) Dissolve 2, 3, 4 and 5 in 1.
(a) 2.0 g. raw humus (prepared by (2) Distribute in large Erlenmeyer flasks.
sieving and washing soil, moisten Sterilization: Not specified.
with several drops of soda solution) Use : To study nitrogen fixation by bacteria
(b) 400 mg. of humus acid containing from the soil.
traces of iron, aluminum and Variants
silicon. (a) Barthel used 10.0 g. CaCOs.
(c) 500 mg. colloidal silicon dioxide as (b) Percival used 5.0 g. CaCOs.
a sol. References: Buhlert and Fickendey (1906
(d) 400 mg. Thompson phosphate. p. 403), Barthel (1910 p. 122), Percival
(e) 200 mg. purified potassium humate. (1920 p. 209).
500 mg. of iron oxide as a
(f) sol.
187. Wojtkiewicz's Mannitol Salt Solution
Use: To study the effect of colloids on bac- Constituents:
terial processes (nitrogen assimilation) 1. Water 1000.0 cc.
by azotobacter. The most nitrogen was 2. Mannitol 20.0 g.
assimilated in the presence of raw humus 3. K2HPO4 0.2 g.
and silicon dioxide. 4. Chalk 5.0 g.
Reference: Sohngen (1913 p. 627). Preparation
185. Beijerinck's Phosphate Glucose
Solution
Erlenmeyer flasks.
Constituents: Sterilization: Not specified.
1. Water 1000.0 cc. Use: To study nitrogen assimilation by
2. Glucose 20.0 g. bacteria from the soil.
3. K2HPO4 0.2 g. Variants
4. CaC03 20.0 g. (a) Hanzawa used 0.5 g. K2HPO4 and
Preparation did not specify the amount of chalk
(1) Dissolve 2 and 3 in 1. to be added.
(2) Add 4 to (1). (b) Hanzawa suggested the use of soil
extract instead of water in variant Added nutrients: The author added 0.5 g.
(a). Nitrogen assimilation was bet- NH4CI or 0.5 g. peptone.
ter in plain solution however, Reference: Butcher (1924 p. 295).
(c) Hanzawa suggested the addition of
humus or saltpeter in varying SUBGROUP I-C. SECTION 2
amounts to variant (a).
Liquid media or basal solutions with con-
References: Wojtkiewicz (1914 p. 255),
stituents of known chemical composition;
Hanzawa (1914 p. 574).
nitrogen supplied as ammonium salts,
188. Krainsky's Mannitol Salt Solution carbon organic.
Ai.* Media primarily for growth of yeast,
Constituents:
fungi, or algae; not bacteria.
1. Water 1000.0 cc.
KH2PO4 Bi. Ammonia supplied as ammonium
2. 0.5 g.
chloride.
3. NaCl 0.5 g.
Wilder's Sugar Salt Solution
4. CaCOa 0.5 g.
(Chrzaszcz) 19I
5. FeSOj trace
6. Mannite 10.0 g. to 20.0 g.
LaGarde's Sucrose Ammonium Chlo-
ride Solution 192
Fulmer and Nelson's Sucrose Am-
in 1.

monium Chloride Solutions 193
Use: Enrichment Azotobader chro-

Bj.t Ammonia Supplied as ammonium ni-
of
trate.
ococcum.
Ci. Organic carbon supplied only as carbo-
Reference: Krainsky (1908 p. 726).
hydrates.
189. Peklo's Modified Beijerinck's Man- Di. Mono or disaccharides added.
nitol Salt Solution El. Calcium salts added.
Mayer's Sucrose Ammonium Nitrate
Constituents:
Solution (Pringsheim) 194
1. Water (river) 1000.0 cc.
2. Mannitol 25.0 g.
Bokorny's Sucrose Ammonium Ni-
trate Solution 195
3. K^HPOi 8.0 g.
E2. Calcium salts not added.
4. K2CO3 4.0 g.
Hollborn's Sucrose Ammonium Ni-
5. CaCOa
trate Solution 196
Preparation
Laborde's Sucrose Ammonium Ni-
(1) Dissolve 2, 3 and 4 in Moldau river
trate Solution 197
water.
Ono's Sucrose Ammonium Nitrate
(2) Add CaCOa, amount not specified.
Solution 198
D2. Polysaccharides added.
Use: Cultivation of plant actinomyces.
Behren's Cellulose Ammonium Ni-
Reference: Peklo (1910 p. 514).
trate Solution 199
190. Butcher's Basal Chinate Salt Solution C2. Organic carbon not supplied exclusively
as carbohydrates.
Constituents:
Raulin's Solution (Smith) 200
1. Water 1000.0 cc.
Behrens' Tartrate Ammonium Ni-
2. K2HPO4 0.5 g.
trate Solution 201
3. Calcium chinate 10.0 g.
Behrens' Citrate Ammonium Nitrate
4. FeCls 0.1 g.
Solution 202
Preparation: (1) Dissolve 2, 3, 4 and one of
von Tubeuf's Cellulose Ammonium
Nitrate Solution 203
B3. Ammonia supplied as ammonium sul-
Use: Differentiation of colon-aerogenes
phate.
group. Author reported that a solid
medium was preferred to a liquid medium.
Bad. aerogenes gave a black coloration *See next page for A2.
to the medium, while Bad. coli did not. tSee B3, B4, Bs, Be and B7
Ci. Only one type of organic carbon added. Bs. Ammonia present as ammonium salts
Di. Organic carbon supplied as carbo- of organic acids.
hydrate. Ci. No other types of organic carbon added.
El. Monosaccharides added. Bokorny's Ammonium Tartrate Solu-
Charpentier's Glucose ammonium tion 222
Sulphate Solution 204 C2. Other types of organic carbon added.
Artari's Cane Syrup Ammonium Sul- Pasteur's Sucrose Ammonium Tar-
phate Solution (Owen) 205 trate Solution (Pringsheim) 224
Ei. Disaccharides added. Bokorny's Sucrose Ammonium Tar-
Bokorny's Sucrose Ammonium Sul- trate Solution 225
phate Solution (acid) 206 Pringsheim's Sucrose Ammonium
Mortensen's Sucrose Ammonium Sul- Tartrate Solution 226
phate Solution 207 A2. Media Primarily for growth of bacteria.
Woltje's Sucrose Ammonium Sulphate Bi. Ammonia supplied as ammonium hy-
Solution (Zikes) 208 droxide.
D2. Organic carbon supplied as alcohols. Hulton-Frankel, Barber and Pyle's
Bokorny's Ethyl Alcohol Ammonimn Acetic Acid and Ammonia Solu-
Sulphate Solution 209 tion 227
Bokorny's Glycerol Ammonium Sul- B2. Ammonia supplied as ammonium chlo-
phate Solution 210 ride.
Dj. Organic carbon supplied only as acids Ci. Only one type of organic carbon
or their salts. supplied.
Behrens' Basal Malic Acid Ammo- Di.* Organic carbon supplied as carbo-
nium Phosphate Solution 211 hydrate.
C2. More than one type of organic carbon Wherry's Levulose Ammonium Chlo-
added. ride Solution 228
Di. Containing carbohydrates and organic Schardinger's Sucrose Ammonium
acids. Chloride Solution 229
Dombrowski's Glucose Ammonium Klecki's Lactose Ammonium Phos-
Sulphate Solution 212 phate Solution 230
Bokorny's Tartrate Ammonium Sul- Krainsky's Starch Ammonium Chlo-
phate Solution 213 ride Solution 231
Raulin's Solution (Lode) 214 Vierling's Cellulose Ammonium Chlo-
D2. Containing alcohols and organic acids. ride Solution 232
Proskauer and Beck's Basal Citrate D2. Organic carbon supplied as alcohols.
Ammonium Sulphate Solution. Proskauer and Beck's Basal Glycerol
(Mendel) 215 Ammonium Chloride Solution 233
B4. Ammonia supplied as ammonium salts Higgins's Glycerol Ammonium Chlo-
of phosphoric acid. ride Solution 234
Ci. Organic carbon present as carbohy- Revis' Glycerol Ammonium Chloride
drates (other types of organic carbon may Solution 235
also be present). Proskauer and Beck's Mannitol Am-
Pasteur's Glucose Ammonium Phos- monium Chloride Solution 236
phate Solution (Simanowsky) 216 D3. Organic carbon supplied as salts of
Omeliansky's Glucose Ammonium organic acids,
Phosphate Solution 217 van Delden's Lactate Ammonium
Henneberg's Sucrose Ammonimn Chloride Solution 237
Acid Phosphate Solution 218 Sohngen's Basal Ammonium Chloride
Kossowicz's Sucrose Ammonium Salt Solution 238
Phosphate Solution (Will) 219 Wherry's Ammonium Acetate Solu-
Schukow's Glucose Ammonium Phos- tion 239
phate Solution 220 D4. Organic carbon supplied as hydro-
C^. Organic carbon supplied as fat. carbons.
Rahn's Fat-Ammonium Phosphate
Solution 221 *See D2, D3 and D4.
Sohngen's Hydrocarbon Ammonium Heller's Starch Ammonium Sulphate

Chloride Solution 240 Solution 259
C2. More than one type of organic carbon Heinze's Glycogen Ammonium sul-
supplied. (Containing alcohols together phate Solution 260
with carbohydrates or organic acids.) McBeth's Cellulose Ammonium Sul-
Proskauer and Beck's Glucose Am- phate Solution (Sanbom) 261
monium Chloride Solution 242 Lohnis' Ammonium Sulphate Cellu-
Conn's Glycerol Ammonium Chloride lose Solution 262
Solution 243 Lohnis' Pectin Ammonium Sulphate
Proskauer and Beck's Mannitol am- Solution 263
monium Chloride Solution 244 Beijerinck's Starch Ammonium Sul-
Bj. Ammonia supplied as ammonium ni- phate Solution 264
trate. D2. Organic carbon supplied as alcohols.
Mayer's Ammonium Nitrate Solution Proskauer and Beck's Mannitol Gly-
(Tanner) 244a cerol Ammonium Sulphate Solution. 265
Capaldi and Proskauer's Mannitol Proskauer and Beck's Bisal Glycerol
Ammonium Nitrate Solution 245 Ammonium Sulphate Solution 266
Proskauer and Beck's Glycerol Am- D3. Organic carbon supplied as acids or
monium Nitrate Solution 246 their salts.
B4 Ammonia supplied as ammonium Doryland's Acetic Acid Ammonium
carbonate. Sulphate Solution 267
Heraeus' Glucose Ammonium Car- Proskauer and Beck's Basal Citrate
bonate Solution 247 Ammonium Sulphate Solution 268
Lowenstein's Glycerol Ammonium C2. More than one type of organic carbon
Carbonate Solution 248 supplied.
Proskauer and Beck's Basal Glycerol Peglion's Sucrose Ammonium Sul-
Ammonium Carbonate Solution 249 . . . phate Solution 269
B5. Ammonia supplied as ammonium sul- Higgins' Succinate Ammonium Sul-
phate. phate Solution 270
Ci. Only one type of organic carbon Lockemann's Citrate Ammonium
supplied. Sulphate Solution 271
Di. Organic carbon supplied only as carbo- Be. Ammonia supplied as a salt of phos-
hydrates. phoric acid.
Ei. Monosaccharides only added. Ci.* Only one type of organic carbon
Bijerinck's Glucose Ammonium Sul- supplied.
phate Solution 250 Di.* Organic carbon supplied only as car-
Doryland's Glucose Ammonium Sul- bohydrate.
phate Solution 251 El. Monosaccharides only added.
Winogradsky's Glucose Ammonium Bijerinck's Glucose Ammonium Phos-
Sulphate Solution 252 phate Solution 272
Gage's Glucose Ammonium Sulphate Ayers' Glucose Ammonium Phos-
Solution 253 phate Solution 273
Stoklasa's Glucose Ammonium Sul- Kendall, Walker and Day's Glucose

phate Solution 254 Ammonium Phosphate Solution 274 . . .
Percival's Glucose Ammonium Sul- Wherry's Levulose Sodium Ammo-

nium Phosphate Solution 275
phate Solution 255
Wolffin's Glucose Ammonium Phos-
Lipman, Waksman & JoflFe's Glucose
phate Solution 276
Sulphur Solution 256
E2. Disaccharides only added.
Ej. Disaccharides only added.
Stutzer's Sucrose Ammonium Mag-
Bokorny's Sucrose Ammonium Sul-
nesium Phosphate Solution 277
phate Solution (Basic) 257
Henneberg's Sucrose Ammonium
Ej. Polysaccharides added. (Other carbo-
hydrates may be added.)
E3. Polysaccharides only added.
Wellman's Starch Ammonium Sul-
phate Solution 258 * See ne.xt page for C2, D2, D3 and D*.
Botkin's Starch Ammonium Phos- Ci. No additional organic carbon supplied.

phate Solution 279 Di. Acetates used.
Waksman & Carey's Ammonium Wherry's Ammonium Acetate Salt
Phosphate Cellulose Solution 280 Solution 298
Percival's Cellulose Ammonium Harvey's Ammonium Acetate Solu-
Phosphate Solution 281 tion 299
D2. Organic carbon supplied only as alde- D2. Tartrates used.
hydes. Proskauer and Beck's Basal Ammo-
Loew's Formaldehyde Ammonium nium Tartrate Solution 300
Phosphate Solution 282 Carapelle's Methylene Blue Ammo-
D3. Organic carbon supplied only as nium Tartrate Solution 301
alcohols. Meyers' Basal Ammonium Tartrate
Ei. Monoatomic alcohols, only, added. Solution 302
Loew's Methyl Alcohol Ammonium Naegeli's Ammonium Tartrate Solu-
Phosphate Solution 283 tion (Smith) 303
Omeliansky's Ethyl Alcohol Ammo- Cohn's Ammonium Tartrate Solu-
nium Phosphate Solution 284 tion (Smith) 304
Bijerinck's Alcohol Ammonium Phos- D3. Lactates used.
phate Solution 285 Nicolle and Zia Bey's Ammonium
E2. Polyatomic Alcohols, only, added. Lactate Solution 305
De Schweintiz's Glycerol Ammonium Braun and Cahn-Bronner's Ammo-
Phosphate Solution (Gage and nium Lactate Solution 306
Phelps) 286 D4. Citrates used.
Lowenstein's Glycerol Ammonium Lohnis' Iron Ammonium Citrate
Phosphate Solution 287 Solution 307
Capaldi and Proskauer's Mannitol Ds. Succinates used.
Ammonium Phosphate Solution 288 . . .
Gessard's Ammonium Succinate Solu-
Omeliansky's Mannitol Ammonium tion 308
Phosphate Solution 289 Kuntze's Ammonium Succinate Solu-
D4. Organic carbon supplied only as acids tion 309
or their salts. Fermi's Basal Ammonium Succinate
Loew's Formate Ammonium Phos- Solution 310
phate Solution 290
C2. Additional organic carbon supplied.
Stuzer's Citrate Ammonium Phos-
Di. Containing only one additional organic
phate Solution 291
carbon sucrose.
Koser's Citrate Sodium Ammonium
El. Carbohydrates only added.
Fischer's Glucose Ammonium Tar-
Koser's Citrate Ammonium Phos-
trate Solution (Muller) 311
phate Solution 293
Naegeli's Sucrose Ammonium Tar-
Ds. Organic carbon supplied only as hydro-
trate Solution (Fermi) 312
carbon.
Groenewege's Sucrose Ammonium
Tausz and Peter's Paraffin Magne-
Tartrate Solution 313
sium Ammonium Phosphate Solu-
294
Fermi and Monteasano's Sucrose Am-
tion
Sohngen's Methane Magnesium Am-
monium Tartrate Solution 314
monium Phosphate Solution 295 E2. Alcohols only added.
C2. More than one type of organic carbon

Proskauer and Beck's Glycerol Am-
supplied.
monium Lactate Solution 315
Laurent's Sucrose Ammonium Phos- Stutzer and Hartleb's Glycerol Am-

phate Solution (Pringsheim) 296 monium Citrate Solution 316
Janke's Alcohol Ammonium Phos- Kuntz's Glycerol Ammonium Succi-

phate Solution 297 nate Solution 317
B7 Ammonia supplied only as salts of Wherry's Glycerol Ammonium Ace-

organic acids. tate Solution 318
Proskauer and Beck's Basal Glycerol 193. Fulmer and Nelson's Sucrose Ammo-
Ammonium Tartrate Solution 319 nium Chloride Solutions
Uschinsky's Glycerol Ammonium Lac-
Constituents
tate Solution 320
1. Water 1000.0 cc.
Fermi and Montesano's Glycerol Am- NH4CI
2. 1.88 g.
monium Tartrate Solution 321
3. Sucrose 100.0 g.
D2. Containing more than one additional
4. K2HPO4 1.0 g.
carbon source.
Preparation
Gottheil's Ammonium Tartrate Solu-
No. VI
tion. 322
MacKensie's Ammonium Tartrate
Solution (Smith) 323
Use: Continuous cultivation of Saccharo-
myces cerevisiae. Authors found that
191. Wilder's Sugar Salt Solution these solutions gave continuous growth.
(Chrzaszcz) Variants
(a) Authors added 1.0 g. CaCl2 to above
Constituents:
solution.
1. Water 1000.0 cc.
(b) Authors added 1.0 g. CaClo and 6.0 g.
2. Sugar 100.0 g.
dextrin to the above solution.
3. MgS04 2.5 g.
Reference: Fulmer and Nelson (1923 p.
4. KCl 2.5 g.
132).
5. NH4CI 2.5 g.
6. Na2HP04 2.5 g.
Mayer's Sucrose Ammonium Nitrate
194.
7. CaCOa 0.5 g.
Solution (Pringsheim)
Preparation
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. Constituents
(2) A white precipitate of Ca3P04 forms 1. Water 1000.0 cc.
when CaCO'; is added. This may or 2. Sugar 100.0 g.
may not be filtered out. 3. KH2PO4 5.0 g.
(3) Distribute in flasks or tubes. 4. MgS04 5.0 g.
Sterilization: Not specified. 5. Ca3(P04)2 0.5 g.
Use: To study growth requirements of 6. NH4NO3 7.5 g.
yeasts. The filtered medium showed less Preparation: (1) Dissolve 2, 3, 4, 5 and 6
growth of yeast than the non-filtered in 1.
material. Sterilization : Not specified.
Reference: Chrzaszcz (1904 p. 145). Use: To study bios requirements of yeast.

Growth and fermentation occurred after
LaGarde's Sucrose Ammonium Chlo- one day.

192.
ride Solution
Reference: Pringsheim (1906 p. 114).
Constituents 195. Bokomy's Sucrose Ammonium Nitrate

1. Water 1000.0 cc. Solution
2. Cane sugar 30.0 g. Constituents
3. NH4CI 6.0 g. 1. Water 1000.0 cc.
4. MgS04 0.5 g. 2. NH4NO3 (0.47%) 4.7 g.
5. KH2PO4 0.5 g. 3. rNH4)2S04 (0.005%) 0.05g.
4. Cane sugar (9.0%) 90.0 g.
Sterilization: Not specified. 5. Dipotassium phosphate
Use: Cultivation of Phycomyces nilens, (0.035%) 0.35 g.
Mucor Mucedo, Mucor Rouxii, Mucor 6. MgS04(0.036%) 0.36 g.
corymbifer, Mucor spinosus, Mucor race- 7. CaCl2 (0.0015%) 0.015 g.
mosus, Mucor rhizopodiformis, Mucor Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
stolonifer. in 1.
Reference: LaGarde (1911-12 p. 248). Sterilization : Not specified.

Use: To study yeast growth. 7. FeS04 trace

Reference: Bokorny (1911 p. 182). 8. CUSO4 solution
Preparation
196. HoUbom's Sucrose Ammonium Nitrate
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Solution
Constituents (3) Add CUSO4 so that the flasks will
1. Water 1000.0 cc. contain from 1/250 to 1/32,000 Mol
2. Sugar 50.0 g. CUSO4.
3. (NHONOs 10.0 g. Sterilization : Sterilize in the steamer.
4. KH2PO4 5.0 g. Use: To study the effect of small amounts
5. MgS04 2.5 g. of CuS04 on the growth of Aspergillus
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. niger. It was found that small amounts
Sterilization: Not specified. of CUSO4 tend to increase growth.
Use: Cultivation of Tricophyton rodens Higher concentration than 1/250 tend to
causing "Alopecia areata," one kind of hinder growth.
baldness. Growth similar to that in Reference: Ono (1902 p. 155).
bouillon. Growth on the surface first 199. Behrens' Cellulose Ammonium Nitrate
olive green and later darker green.
Solution
Similar medium used to study fermenta-
tion by Aspergillus niger. Constituents:
Variants: Currie recommended the use of 1. Water 1000.0 cc.
the following medium to study citric acid 2. NH4NO3 10.0 g.
fermentation of Aspergillus niger: 3. Potassium phosphate 5.0 g.
1. Water 1000.0 cc. 4. MgS04 2.5 g.
2. Sucrose 125.0-150.0 g. 5. Filter paper (Swedish) 50.0 g.
3. NH4NO3 2.0-2.5 g. Preparation
4. KH2PO4 0.75-1.0 g. (1) Dissolve 2, 3 and 4 in 1.
5. MgS04-7H20 0.2-0.25 g. (2) After sterilization add 5.0 g. of puri-
6. HCl to give pH between 3.4^3.5 (4-5 Swedish filter paper containing
fied
cc. N/5 HCl). some of the mycelium.
Sterilization Method not given.
:
197. Laborde's Sucrose Ammonium Nitrate

Use: Cultivation of Pseudo-Demaiophora.
Solution
Growth was lu.xuriant. Utilized cellulose,
Constituents as a source of carbon.
1. Water 1000.0 cc. Reference: Behrens, (1897 p. 640).
2. Sugar 50.0 g.
200. Raulins Solution (Smith)
3. NH4NO3 2.0 g.
4. Potassium phosphate 1.0 g. Constituents
5. MgS04 0.1 g. 1. Water 1500.0 cc
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 2. Sugar 70.0 g
Sterilization : Not specified. 3. Tartaric acid 4.0 g
Use: Cultivation of the mold Enrotiopsis 4. NH4NO3 4.0 g
Gayoni. 5. Ammonium Phosphate 0.6 g
Reference: Laborde (1897 p. 13). 6. K2CO3 0.6 g
7. MgS04 0.4 g
198. Ono's Sucrose Ammonium Nitrate
8. (NH4)3S04 0.25 g
Solution
9. ZnS04 0.07 g,
Constituents 10. FeS04 0.07 g,
1. Water 1000.0 cc. 11. KSiOs 0.07 g

2. Cane sugar 85.0 g Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8,
3. NH4NO3 16.0 g 9, 10 and 11 in 1.
4. MgS04 3.0 g Sterilization : Not specified.

4. KCl 3.0 g Use: Cultivation of Aspergillus niger and
6. NH4H2PO4 30 g other molds.
Variants 5. K2HPO4 2.0 g.

(a) Bezangon did not specify the type 6. MgS04 1.0 g.
of ammonium phosphate, and speci- 7. Tannin 0.0, 1:1000, 1:500, 1:100 or
fied that iron sulphate was to be used. 1:50.
(b) Harvey omitted the MgCOs and did Preparation :
not specify the type of ammonium (1) Dissolve 2, 3, 4, 5 and 6 in 1.
phosphate. (2) Distribute in 50.0 cc. lots.
(c) Dopter and Sacquepee, and Heine- Sterilization: Method not given.
mann, did not specify the type of Use: Cultivation of Botrytis. Tannin in-
ammonium phosphate. hibits growth, 0.5 and 1.0 g. per flask
(d) Tanner omitted the K2CO3. showed no growth of the spores.
(e) Harvey also gave the following Variants: The author added 0.0, 0.05 g.
solution: (1:1000), 0.1 g. (1:500), 0.5 g. (1:100) or
1. Distilled water 1500.0 cc. 1.0 g. (1:50) of tannin to each flask before
2. NH4NO3 4.5 g. sterilization.
3. Sucrose 70.0 g. Reference: Behrens' (1898 p. 642).
4. Potassium tartrate 6.5 g.
5. K2HPO4 0.6 g.
202. Behrens' Citrate Ammonium Nitrate
6. MgCOs 0.4 g.
Solution.
7. K2SO4 0.25 g.
8. FeS04 0.07 g. Constituents
9. ZnS04 0.07 g. 1. Water 1000.0 cc.
10. KaSiOa 0.07 g. 2. Cane sugar 100.0 g.
(f Betrand and Javillier gave the follow- 3. NH4XO3 20.0 g.
ing modification: 4. Citric Acid 10.0 g.
1. Water 1000.0 cc. 5. Potassium phosphate 10.0 g.
2. Sugar 46.6 g. 6. MgSOi 5.0 g.
3. Tartaric acid 2.66 g. Preparation

4. K2CO3 0.4 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1 liter of
5. NH4NO3 2.66 g. water rich in potassium.

6. Ammonium phosphate. . 0.4 g. (2) Distribute in 50.0 cc. lots in 300.0 cc.
7. (NH4)2S04 0.155 g. flasks (28 flasks).
8. MgS04 0.71 g. Sterilization: Method not given.

9. Iron aluminum 0.081 g. Use: To determine influence of chemicals
10. K2Si03 0.046 g. on growth of Pseudodetnatephaca.
(g) Roux and Rochaix did not specify Growth takes place in copper sulphate
the type of ammonium phosphate 1:100,000, 1:10,000 and 1:5000. In iron
used, used 0.4 g. MgCOs instead of sulphate 1:100,000 to 1:500; in sodium
MgS04, and 0.07 g. K2SO4 instead of fluoride 1 100,000,
: and in potassium
K2Si03. sulfocarbonate 1:100,000 to 1:500.
References: Smith (1905 p. 197), Roux and Added materials: The author added the
Rochaix (1911 p. 104), Brenner (1914 following materials:
p. Bezangon (1920 p. 20), Besson
556), (a) 1.0 and 0.1% solutions of copper
(1920 p. 36) Harvey (1921-22 p. 103), sodium fluoride
vitrol, iron vitrol or
Dopter and Sacquepee (1921 p. 16), Tan- so that the concentration would be
ner (1919 p. 66), Heinemann (1922 p. 42), 1:100, 1:200, 1:500, 1:1000, 1:5000,
Betrand and Javillier (1912 p. 244). 1:10,000, 1:100,000 (previous to ster-
ilization).
201. Behrens' Tartrate Ammonium Nitrate
(b) 1.0% and 0.1% sterile solutions of
Solution.
potassium sulphocarbonate so that
Constituents the concentration would be 1:100,
1. Water 1000.0 cc. 1:200, 1:500, 1:1000, 1:5000, 1:10,000,
2. Sucrose 50.0 g. 1:100,000 (added to the sterilized
3. Tartaric acid 5.0 g. medium under aseptic conditions).
4. NH4NO3 . 5.0 g. Reference: Behrens' (1897 p. 745).
203. von Tubeuf s Cellulose Ammonium Preparation

Nitrate Solution. (1) Dissolve 2, 3, 4 and 5 in 1. This is
Artari's solution.
Constituents
1. Water 1000.0 cc. (2) Prepare the following modifications
2. NH4NO3 10.0 g.
with cane syrup. Preparation of
syrup or exact composition not
MgS04 given (may be 50.0% cane sugar
4. 1.0 g.
solution).
5. Lactic acid 2.0 g.
6. Cellulose
(a) Make no additions to cane syrup.
Preparation (b) To 2 parts cane syrup, add 1 part
(2) Distribute in 50.0 cc. lots. (c) Add an equal amount of (1) to cane
syrup.
(3) Prepare a series of flasks by adding
10.0 g. filter paper (Swedish) or 10.0 g.
(d) To 1 part cane syrup add 2 parts
(1).
ordinary filter paper, or 10.0 g. of
pine shavings or 10.0 g. of wadding (3) Tube in about 10.0 cc. lots.
(10.0 g. sucrose with 5.0 g. gelatin

Sterilization : Steam for 15 minutes on each
were also added).
Use To study effect of density on growth of
:
Use: Cultivation of Merulius lacrymans Saccharomyces Zopfii. Cells are smaller

(causing dry rot). Good growth using
in solutions of greatest density. Yeast
Swedish filter paper as carbon source. grown in thick syrups seem to retain
their fermentation ability when subjected
Little growth on shavings or wadding.
to high temperatures, more than yeast
Good growth on ordinary malt and meat
extract containing cane sugar and citric
grown in a medium of lesser density.
Reference: Owen (1913 p. 473).
acid 1 to 3%.
Reference: v Tubeuf (1902 p. 130). 206. Bokorny's Sucrose Ammonium Sul-
204. Charpentier's Glucose Ammonium phate Solution (acid)
Sulphate Solution Constituents
1. Water 1000.0 cc. 2. Cane Sugar 50.0 g.
2. Glucose 10.0 g. 3. KH2PO4 1.0 g.
3. FeS04 trace 4. MgS04 1.0 g.

4. (NH4)2S04 0.5 g. 5. (NH4)2S04 5.0 g.
5. CaCl2 0.023 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
7. MgS04 1.0 g. Use: Growth of yeast.
Preparation Reference: Bokorny (1902 p. 56), (1917 p.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. 369).

207. Mortensen's Sucrose Ammonium
Sulphate Solution
Use : Nitrogen assimilation by Algae, Cys-
tococcus humicola. Constituents
Reference: Charpentier (1903 p. 330). 1. Water 100.0 cc,
2. Cane sugar (10.0%) 10.0 g.
205. Artari's Syrup Ammonium Sulphate 3. (NH4)2S04 (0.5%) 0.5 g.
Solution (Owen) 4. Cobalt salt
1. Water 1000.0 cc (1) Dissolve 2 and 3 in 1.
2. NasHPOi (0.15%) 0.15 g (2) Add cobalt salts in varying amounts
3. MgS04 (0.1%) 0.1 g from 4 to 1/32%.
4. (NH4)2S04 (0.15%) 0.15 g Sterilization: Not specified.
5. Grape sugar (2.0%) 2.0 g Use: To study the effect of cobalt salts on
6. Cane syrup the growth of Aspergillus niger. The
author found that the toxic properties of Preparation

cobalt are different in different media. (1) Dissolve 2, 3, 4 and 5 in 1.
Reference: Mortensen (1909 p. 532). (2) Distribute in 10.0 cc. lots.
208. Woltje's Sucrose Ammonium Sulphate trients to each tube.
Solution (Zikes) Sterilization: Usual method. (Method not
Constituents: given.)
1. Water 100.0 cc. Use: Cultivation of Penicillium glaucum

2. (NH4)2S04 1.0 g. and Botrytis vulgaris. Growth was more
3. MgS04 0.25 g. lu.xuriant in medium containing pectin
4. Saccharose 7.5 g. than in arabinose medium.
5. K2HPO4 0.5 g. Added nutrients: The author added 1.0 g.
Preparation: Dissolve 4 and 5 in 1. of arabinose or calcium pectate (from
(1) 2, 3,
flax) to each 10.0 cc. lot.
Use: To study perithecium formation by Variants: The author made no additions to
Aspergillus oryzae. the basic solution.
Reference: Zikes (1922 p. 340).
Reference: Behrens (1898 p. 552).
209. Bokomy's Ethyl Alcohol Ammonium

212. Dombrowski's Glucose Ammonium
Sulphate Solution.
Sulphate Solution
Constituents:
Constituents
1. Water 1000.0 cc.
1. Water 1000.0 cc.
2. K2HPO4 1.0 g.
2. Ethyl alcohol (0.2%) 20.0 g.
3. MgS04 0.5 g.
3. (NH4)2S04 (0.02%) 0.2 g.
4. Dextrose 100.0 g.
4. K2HPO4 or KH2PO4 (0.05%) 0.5 g.
5. Lactic acid
5. MgS04 0.2 g.
6. (NH4)2S04 0.5 to 5.0 g.
Preparation
Use: Growth of fungi, Schizomycetes,
(2) Acidify slightly with lactic acid
molds and yeast.
(3) Add 0.05, 0.1, 1.0, 2.0 or 5.0 g. of
(NH4)2S04 to 100.0 cc. of (2).
210. Bokomy's Glycerol Ammonium Sul- Sterilization: Not specified.
phate Solution Use: To study nitrogen requirements for

milk yeasts, Saccharoinyces Inciis and
Constituents: Zygosaccharomyces lactis. Peptone was
1. Water 1000.0 cc.
found to be the best nitrogen source.
2. Glycerin 5.0 g.
Variants: The author substituted 20.0 to
3. KH,P04 0.1 g.
50.0 g. of asparagin or 2.0 to 50.0 g. of
4. Bittersalts MgS04 0.1 g.
Witte's peptone for the (NH4)2S04.
5. (NH4)2S04 0.5 g.
Reference: Dombrowski (1910 p. 393).
Sterilization: Not given. 213. Bokorny's Tartrate Ammonium

Use: Cultivation of yeast. Sulphate Solution
Reference: Bokorny (1911 p. 182). Constituents:
1. Water 1000.0 cc.
211. Behrens' Basal Malic Acid Ammonium Cane sugar (10.0%)
2. 100.0 g
Phosphate Solution Ammonium tartrate
3. (0.5%) 5.0 g.
Constituents: 4. K2HPO4 (0.035%) 0.35 g.
1. Wâter 1000.0 cc. 5. MgS04 (0.006%) 0.06 g.
2. (NH4)2S04 20.9 g. 6. CaCla (0.0015%o) 0.015
3. Malic acid 90.0 g. 7. (NH4)2S04 (0.0061%) 0.061g.
4. MgS04 5.0 g. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
5. Potassium phosphate 10.0 g. in 1.
Sterilization: Not given. 216. Pasteur's Glucose Solution

Use: Cultivation of yeast. The culture (Simanowsky)
may or may not be treated with a current
Constituents
of air.
1. Water 1000.0 cc.
2. KH2PO4 5.0 g.
3. (NH4)H2P04 5.0 g.
214. Raulin's Solution (Lode)
4. Glucose 80.0 g.
Constituents Preparation: (1) Dissolve 2, 3 and 4 in 1.
1. Water 1500.0 cc Sterilization : Not given.

2. Sugar candy 70.0 g Use: Cultivation of yeast.
3. Tartaric acid 4.0 g Variants: The author substituted 80.0 g. of
4. Ammonium phosphate 0.6 g sucrose for glucose.

5. CaCOs 0.6 g Reference: Simanowsky (1886 p. 22).
6. MgCOs 0.4 g
217. Omeliansky's Glucose Ammonium
7. (NH4)2S04 0.25 g
Phosphate Solution
8. FeS04 0.07 g
9. ZnS04 0.07 g Constituents
10. Calcium silicate 0.07 g 1. Water 1000.0 cc.
11. Ammonium acetate 4.0 g 2. Glucose 100.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8, 3. Ammonium phosphate 2.0 g.
9, 10 and 11 in 1. 4. KCl 0.1 g.
Sterilization: Not specified. 5. MgS04 0.1 g.
Use: Cultivation of molds, Aspergillus. Preparation
Reference: Lode (1902 p. 125). (1) Dissolve 2, 3, 4 and 5 in 1.
(2) Kind phosphate salt not specified.

of
215. Proskauer and Beck's Basal Citrate (3) Adjustment of reaction not specified.
Ammonium Sulphate Solution (4) Place solution in thin layer in Wino-
(Mendel) gradsky flask.
Method not given.
Sterilization:
Constituents
1000.0 cc. Use: Cultivation of aroma producing yeast
1. Distilled water
and the preservation of stock cultures.
2. (NH4)2S04 3.0 g.
2.0 g.
Gives table wine aroma. Cane sugar
3. Citric acid
cannot replace glucose.
4. MgS04 2.0 g.
Reference: Omeliansky (1923 p. 418).
5. Potassium biphosphate 5.0 g.
6. Glycerin 15.0 g.
218. Henneberg's Sucrose Ammonium Acid
Preparation
Phosphate Solution
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Adjustment of reaction not specified. Constituents

(3) Add 1.0% of one of the carbon sources
1. Water 1000.0 cc.
listed under added nutrients. 2. (NH4)2HP04 (0.2%) 2.0 g.
Sterilization: Sterilize for 30 minutes on 3. KH2PO4 (0.2%) 2.0 g.
each of two successive days. On the 4. MgS04 (0.1%) 1.0 g.
third day add 1.0% of glucose and steri- 5. Sucrose (15.0%) 150.0 g.
lize for 15 minutes. (Method of steriliza- Preparation

tion not specified.) (1) Dissolve 2, 3, 4 and 5 in 1.
Use: Cultivation of Bacterium coli and Sterilization: Not specified.
Bacterium Fitzianus. Use Cultivation of yeast.

:
Added nutrients: The author used 1.0% of Variants: The author suggested the addi-
the following carbohydrates as carbon tion of one of the following:
sources: Gypsum (2.0%) 20.0 g.
glucose maltose Potassium phosphate (2.0%) 20.0 g.
lactose sucrose CaCl2 (0.6%) 6.0 g.
Reference: Mendel (1911 p. 296). Potassium lactate (2.0%) 20.0 g.

K2CO3 (2.0%) 20.0 g. malic acid

Soda (0.6%) 6.0 g. citric acid
K2SO4 (0.6%) 6.0 g. succinic acid
Reference: Henneberg (1909 p. 105). Reference: Schukow (1896 p. 607).
219. Kossowicz's Sucrose Ammonium Phos- 221. Rahn's Fat-Ammonium Phosphate

phate Solution (Will) Solution
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. Saccharose (5.0%) 50.0 g. 2. K2HPO4 5.0 g.
3. KCl (0.4%) 4.0 g. 3. (NH4)3P04 5.0 g.
4. (NH4)2HP04 (0.4%) 4.0 g. 4. MgS04 1.0 g.
5. MgS04 (0.4%) 4.0 g. 5. CaCl2 1.0 g.
6. CaHP04 (0.04%) 0.4 g. 6. FeCls trace
Preparation: 7. HCl trace
(1) Dissolve 2, 3, 4, 5, and 6 in 1. 8. Fat
Use: Cultivation of non-spore forming (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Saccharormjces. (2) Melt fat and pour in an Erlenmeyer
Reference: Will (1908 p. 387). flask and
incline the flask in such a
position so that when it solidifies only
220. Schukow's Glucose Ammonium a portion of the fat will be immersed
Phosphate Solution in the solution.
Constituents
(3) Neutralize (1) with 10.0% NaOH
using litmus as an indicator. Do not
1. Water 1000.0 cc.
filter.
2. Ammonium phos-
Add the flasks containing fat,
(4) (3) to
phate 5.0 g.
solidified on the wall of the flask.
3. Potassium phos-
Do not add enough (3) to immerse
phate 10 g.
all the fat.
4. MgS04 0.5 g.
5. Glucose 10.0 g.
Use: To study fat decomposition by
6. Organic acid 90.0 g. to 100.0 g.
Penicillium glaucum. Fat was decom-
Preparation
Reference: Rahn (1905 p. 423).
(2) Add 9 to 10.0% of an organic acid to
(1).
222. Bokomy's Ammonium Tartrate
Solution
(4) Distribute into fermentation flasks
(sealed with H2SO4). Constituents:
(5) After sterilization and inoculation 1. Distilled water 1000.0 cc.
plug the flasks with a cork and sear 2. Ammonium tartrate . . 2.0 g.
with paraffin. 3. KH2PO4 1.0 g.
Sterilization: Sterilize in the steamer 4. MgS04 0.5 g.
(method not given). 5. Iron chloride trace
Use To study utilization of acids by yeast.
: 6. CaS04 0.0 or 1.0 g.
After 75 days 0.11 g. tartaric, 0.193 g. Preparation: Dissolve 2, 3, 4, 5 and 6 in
malic, 0.34 g. citric and 0.09 g. succinic water that has been distilled in quartz
acid were used. Acid determined by containers.
titration with N/10 NaOH using litmus Sterilization: Not specified.
as an indicator. Use: To study effect of metallic salts on
Variants: The author used one of the fol- j'east. No growth took place in the
lowing organic acids: solution lacking calcium.
tartaric acid Reference: Bokorny (1912 p. 135).
224. Pasteur's Sucrose Ammonium Solution 227. Hulton-Frankel, Barber and Pyle's
(Pringsheim) Acetic Acid and Ammonia Solution
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. Sucrose 100.0 g. 2. H3PO4 M soln
(12.79 g) 129.5 cc.
3. Ammonium tartrate 10.0 g. 3. CH3COOH (1.13 g) M soln. . 18.8 cc.
4. KH2PO4 5.0 g. 4. NH4OH (0.62 g) M soln 17.8 cc.
5. Ca3(P04)2 1.0 g. 5. 0.01% CaClo (0.001 g) 10.0 cc.
6. Mg3(P04)2 1.0 g. 6. 0.01% FeCU (0.001 g) 10.0 cc.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 7. 0.01% MgS04 (0.001 g) . . . . 10.0 cc.
in 1. 8. NaOH M soln
(4.0 g) 100.0 cc.
Sterilization: Not specified. 9. KOH (5.6 g) M soln 100.0 cc.
Use: To study bios requirements of yeast. Preparation
Fermentation took place after one day. (1) Dilute solutions 23, and 4 to 500.0 cc.
Reference: Pringsheim (1906 p. 114). with 1.
(2) Add to (1), 5, 6 and 7 and equal vol-

225. Bokomy's Sucrose Ammonium Tartrate umes of M solution of 8 and 9 to give
Solution pH of 7.0.
(3) Solution is water clear.
Constituents (4) Tube into sterile tubes.
1. Water 1000.0 cc.
(5) pH of medium may be adjusted by
2. Saccharose (Kahlbaum varying amounts
of alkali added.
10.0%) 100.0 g. Medium may be prepared using molar
3. NaK2P04 (0.1%) 1.0 g. solutions or by weight.
4. Ammonium tartrate (0.25%) 2.5 g. Sterilization Steam on each of 3 successive
:
5. MgS04 (0.05%) 0.5 g. days for 15 minutes.

6. KCl Use: General culture medium.
Preparation Variants Hulton-Frankel and Barber used
:

the same solution as a basic medium with
(2) Add 0.0%, 0.025%, 0.05%, 0.1%, litmus or Andrade's indicator to which
0.25%, 0.5% or 1.0%, KCl to 1.
they added 10.0 g. of one of the following:
Sterilization: Not specified. (a) sucrose (c) lactose
Use: To study effect of metalic salts on (b) glucose mannitol (d)
yeast. Potassium reported necessary for References: Hulton-Frankel-Barber and
the growth of the yeast. Pyle (1917 p. 17), Hulton-Frankel and
Reference: Bokorny (1912 p. 123). Barber (1917 p. 17), Harvey (1921-22
p. 104).
226. Pringsheim's Sucrose Ammonium
Tartrate Solution 228. Wherry's Levulose Ammonium
Chloride Solution.
Constituents
1. Water 1000.0 cc. Constituents
2. KH2PO4 2.0 g. 1. Redistilled water 1000.0 cc.
3. MgS04 0.4 g. 2. NH4CI 2.0 g.
4. CaCla 0.2 g. 3. Levulose 2.0 g.
5. Ammonium tartrate 10.0 g. 4. Calcium phosphate 2.0 g.
6. Sucrose 100.0 g. (primary, secondary or tertiary)
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 Preparation
in 1. (1) Dissolve 2, 3 and one of 4 in 1.
Sterilization: Not specified. (2) Adjustment of reaction not given.
Use: To study bios requirements of yeast. Sterilization: Autoclave (time not spec-
The author reported fermentation after ified).
one day in this medium. Use : To study acid proofness of B. tubercu-
Reference: Pringsheim (1906 p. 114). losis. Primary salt no growth; sec-
CULTURE MEDIA FOR CULTIVATION OF MICRO ORGANSIMS 71
ondary salt growth good non-acid 3. CaCOs 3.0

proof rods, some "free spores." Ter- 4. NH4CI 2.0
tiary salt
growth good smaller non- 5. Potassium phosphate 0.0.58
acid proof rods, many "free spores." 6. Ammonium phosphate . . . 0.1053 g,
Variants: The author gave the following 7. MgS04 0.055 g,
variants: 8. (NH4)2S04 0.023 g

(a) Used the same solution but substi- Preparation
tuted primary, secondary, tertiary (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
meta or pyro sodium phosphate for (2) Adjustment of reaction not given.
calcium phosphate. These solutions (3) Distribute in Pasteur fermentation
gave the following results: tubes.
Primary salt growth good at 48 hours Sterilization : Method not given.
part are strongly acid proof, some Use: Cultivation of Bacillus saccharo-
"spores." butyricus. Fermentation takes place after
Secondary salt growth good at 48 hours about 60 hours after inoculation.
part are strongly acid proof, some Reference: Klecki (1896 p. 255).
"spores."
Tertiary salt growth good. Non-acid 231. Krainsky's Starch Ammonium Chloride
proof. Solution.
Meta no growth.
Constituents
Pyrophosphate growth good. Rods
1. Water 1000.0 cc.
and non-acid proof.
are chiefly short
2. Starch (1.0%) 10.0 g.
Some are larger and longer and are
3. NH4CI (0.05%) 0.5 g.
strongly acid proof.
4. K2HPO4 (0.05%) 0.5 g.
(b) Substituted 2.0 g. K2HPO4 for cal-
non-acid
Preparation: (1) Dissolve 2, 3 and 4 in 1.
cium phosphate. Bacilli
Sterilization:Not specified.
proof.
Use: Isolation and enrichment of Actino-
(c) Substituted 2.0 g. KH2PO4 for cal-
myces. After 2 days because of bacterial
cium phosphate. Acid proof rods
increase, a weak coloration and a slight
were often club shaped.
H2S odor appears. The actinomycetes
Reference: Wherry (1913 p. 150).
swim on the surface. Inoculate
colonies
229. Schardinger's Sucrose Ammonium new medium of the
these colonies in a
Chloride Solution same composition and a heavy scum is
Constituents formed.
1. Distilled water 1000.0 cc. Variants: The author substituted 2 to 3.0%
2. Sucrose 50.0 to 80.0 g. 1.0% starch.
cellulose for
3. NH4CI 5.0 g. Reference: Krainsky (1914 p. 657).
4. KH2PO4 1.0 g.
5. MgS04 0.5 g. 232. Vierling's Cellulose Ammonium
6. CaCOs 10.0 to 15.0 g. Chloride Solution
Preparation: (1) Dissolve 2, one of 3, 4, 5
Constituents
and 6 in 1.
1. Water 1000.0 cc.
2. CaC03 (0.1%) 1.0 g.
Use: Study of slime production. Fer-
3. (NH4) CI (0.1%) 1.0 g.
mentation starts after 24 hours. The
4. Filter paper.
slime forms at the bottom of the flask.
Preparation
Variants: The author suggested the use of
(NH4)2S04 instead of NH4CI.
(2) Place strips of filter paper in flasks.
Reference: Schardinger (1902 p. 177).
(3) Pour (1) into each flask containing
230. Klecki's Lactose Ammonium Phosphate filter paper.
Solution (4) Seal the flasks with paraffin after
Constituents inoculation.
1. Water 1000.0 cc. Sterilization: Not specified.
2. Lactose 20.0 g. Use: Decomposition of cellulose by Mycch
bacteria. Growth occurs. Filter paper Sterilization: Not specified.

shows no signs of being attacked. Use: To study stability of physiological
Reference: Vierling (1920 p. 206). properties of colon and coli form organ-
isms. No distinctive changes were pro-
233. Proskauer and Beck's Basal Glycerol
duced in this non-albuminous medium,
Ammonium Chloride Solution
but the vigor of fermentation varied
Constituents : somewhat. The organisms show a greater
1. Water 1000.0 cc. tendency to die off in this medium than
2. Glycerol (1.5%) 15.0 g. in ordinary media.
3. KH2PO4 (0.5%) 5.0 g. Reference: Revis (1910 p. 173).
4. MgS04 (0.25%) 2.5 g.
236. Proskauer and Beck's Mannitol Ammo-
5. NH4CI (0.2%) 2.0 g.
nium Chloride Solution
Preparation :
(1) Dissolve 2, 3, 4 and 5 in 1. Constituents :
(2) Add one of the listed carbon sources. 1. Water 1000.0 cc.
Sterilization not specified. 2. Mannitol (0.6%,) 6.0 g.
Use : To study requirements for growth for 3. NH4CI (0.2%) 2.0 g.
the tubercle bacilli. 4. KH2PO4 (0.5%) 5.0 g.
Added nutrients: The author added the 5. MgS04 (0.25%) 2.5 g.
following carbohydrates and alcohols Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
as carbon sources: Sterilization: Not specified.
maltose 1.0% mannitol 0.6% Use : To study growth requirements of the

1.0%
lactose dulcitol 0.6%, tubercle bacillus. The authors reported
sucrose 1.0% isodulcitol 0.6% no growth unless glycerol was present.
rafhnose 1.0% Reference: Proskauer and Beck (1894 p.
Reference: Proskauer and Beck (1894 p. 147).
146). 237. van Delden's Lactate Ammonium

Chloride Solution
234. Higgins' Glycerol Ammonium Chloride
Solution Constituents :
1. Water 1000.0 cc.

Constituents :
2. NaCl 30.0 g.
1. Water 100.0 cc.
3. K2HPO4 0.05 g.
2. Glycerin 5.0 g.
4. NH4CI 1.0 g.
3. NH4CI 0.8 g.
5. Sodium lactate 10.0 g.
6. MgS04-7H20. (amount not
. . specified)
5. Potassium sulphate or 0.1 g.
Preparation :
in 1.
(1) Dissolve 2, 3 and 4 and 5 in 1.
(2)Neutralize to phenolphthalein.
Use: To study sulphate reduction by M.
Method not given.
Sterilization:
aestuarii.
Use: Cultivation of cholera organisms.
The author used 0.25 g. K2HPO4,
Variants:
Reference: Higgins (1898 p. 666).
0.25 g.NH4CI, specified the use 2.0 g.
235. Revis' Glycerol Ammonium Chloride MgS04-7H20 and omitted the NaCl.
Solution Reference: van Delden (1903-4 pp. 117-
118).
Constituents:
1. Water 1000.0 cc. 238. Sohngen's Basal Ammonium Chloride
2. NH4CI 10.0 g. Salt Solution.
3. Glycerol 10.0 g. Constituents :
4. Potassium phosphate 10 g. 1. Water 1000.0 cc.

5. MgS04 0.2 g. 2. K2HPO4 0.2 g.
6. CaCl2 0.1 g. 3. NH4CI 0.2 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 4. Seignettes salt (sodium po-
in 1. tassium tartrate) 0.2 g.
Preparation 240. Sohngen's Hydrocarbon Ammonium

(1) Dissolve 2, 3, and 4 in 1000.0 cc. of Chloride Solution.
water.
Constituents
(2) Add one of the materials listed under Water
1. lOCO.O cc.
added nutrients.
2. K0HPO4 0.5 g.
3. NH4CI 0.5 g.
(4) Sterilize the solution and colloids
4. CaCOs trace
separately.
5. Paraffin (1.0%) 10.0 g.
(5) Incubate 24 hours to test sterility.
Preparation
2, 3,
Use: To study influence of colloids on bac-
(2) Heat and add 1.0% melted
(1) slightly
terial process of B. fluorescens lique-
paraffin. Shake thoroughly and then
faciens. After two days cultures in (2)
cool quickly. Or add several drops
(a), (c), (e), (h) are turbid white, (d),
of petroleum.
(f), (g) are clear. Growth obtained in
all cases however.
Erlenmeyer flasks.
Added nutrients: The author added one of
(4) Inoculate with garden soil.
the following combinations:
(a) 5.0 g. distilled water
Use: Cultivation of paraffin oxidizers. Soil
(b) 5.0 g. distilled water + 0.5 g. blood
forms. B. fluorescens liquefaciens, B.
charcoal
punctatum, Microcossus paraffmae, B.
(c) 5.0 g. distilled water + 0.5 g. blood
lipolyticum.
charcoal + 0.2 g. CaCOs Variants : Variants used to study the effect
(d) 5.0 g. distilled water + 0.5 g. finely
of a colloid upon oxidation.
divided peat
(a) The author substituted several drops
(e) 5.0 g. distilled water + 0.5 g. finely
ofpetroleum for paraffin.
divided peat + 0.2 g. CaCOa (b) The author substituted 20.0 cc. of
(f) 5.0 g. distilled water + 0.5 g. finely
petroleum for the paraffin and added
cut filter paper
2.0 g. of colloidal silicon dioxide or
(g) 5.0 cc. of a 2.0% colloidal silicum
colloidal iron oxide to the solution.
dioxide solution
He found that the amount of CO2
(h) 5.0 cc. of a 1.0% colloidal iron oxide
formed was increased by the presence
solution
of a colloid, when using Mycobac-
Reference: Sohngen (1913 p. 625).
terium album, Mycobacterium rubrum,
Micrococcus paraffinae.
239. Wherry's Ammonium Acetate Solution. Reference: Sohngen (1913 p. 597), (1913
p. 644.
Constituents
2. NasCOsor NaCl 1.0 g.
242. Proskauer and Beck's Glucose Ammo-
3. KCl 1.0 g.
nium Chloride Solution.
4. CaCla 1.0 g. Constituents:

5. MgS04 1.0 g. 1. Water 1000.0 cc.
6. Ammonium acetate. . 2.0 g. 2. Glucose (1.0%) 10.0 g.
7. NH4CI 2.0 g. 3. Citric acid (0.075%) 0.75 g.
8. KH2PO4 0.0 or 2.0 g. 4. NH4CI (0.2%) 2.0 g.
Preparation 5. Glycerol (1.5%) 15.0 g.
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. 6. KH2PO4 (0.5%) 5.0 g.
(2) Adjustment of reaction not given. 7. MgS04 (0.25%) 2.5 g.
Sterilization: Sterilize in autoclave, time 8. K2SO4 (0.25%) 2.5 g.
not given. 9. NaCl (0.12%) 1.2 g.
Use To study acid proofness of
: B. tubercu- Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8
losis. Medium unfavorable. and 9 in 1.
Reference: Wherry (1913 p. 148). Sterilization: Not specified.

Use: To study growth requirements of 5. KH2PO4 10.0 g.

tubercle bacilli. 6. Sugar (kind not specified).
Variants: The author omitted the citric Preparation
acid. (1) Dissolve 2, 3, 4 and 5 in 1.
Reference: Proskauer and Beck (1894 p. (2) Cool and add sugar (Kind and amount
142). not specified).
243. Conn's Glycerol Ammonium Chloride
Use: General culture medium.
Solution.
Variants: Add 3.0% NaCl if the medium is
Constituents to be used for luminous bacteria, and an

1. Water 1000.0 cc. excess of CaCOs if acid forming bacteria
2. Glycerol 10.0 cc. are to be grown.
3. Malic acid 4.0 g. Reference: Tanner (1919 p. 62).
4. NH4CI 0.5 g.
245. Capaldi and Proskauer's Mannitol
5. K2HPO4 0.5 g.
6. CaCl2 2.5 g.
Ammonium Nitrate Solution
(1) Dissolve 2, 3, 4, 5 and 6 in 1. 1. Water 1000.0 cc.
(2) Adjust the reaction to about neutral 2. NH4NO3 (0.2%) 2.0 g.
(pH = 7.0) by the addition of NaOH 3. Mannite (0.2%) 2.0 g.
(about 10.0 cc. of normal solution). 4. NaCl (0.02%) 0.2 g.
Sterilization: Not specified. 5. KH2PO4 (0.02%) 0.2 g.
Use: Cultivation of Actinomycetes. 6. MgS04 (0.01%) 0.1 g.
Variants: The author omitted the CaCOs Preparation: (1) Dissolve 2, 3, 4, 5 and 6
and used citric acid instead of malic acid. in 1.
Reference: Conn (1921 p. 7). Sterilization: Not specified.

Use: Acid production by B. coli. Authors
244. Proskauer and Beck's Mannitol Ammo- found that B. coli produced acid in this
nium Chloride Solution.
medium.
Constituents Reference: Capaldi and Proskauer (1896
1. Water 1000.0 cc. p. 456).
2. Mannitol (0.6%) 6.0 g.
3. Glycerol (1.5%) 15.0 g.
246. Proskauer and Beck's Glycerol Ammo-
4. NH4CI (0.2%) 2.0 g.
nium Nitrate Solution
5. KH2PO4 (0.5%) 5.0 g. Constituents

6. MgS04 (0.25%) 2.5 g. 1. Water 1000.0 cc.
7. Magnesium citrate (0.25%). 2.5 g. 2. Glucose (1.0%) 10.0 g,
8. Citric acid (0.075%) 0.75 g. 3. Glycerol (1.5%) 15.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7 4. KH2PO4 (0.5%) 5.0 g.
and 8 in 1. 5. MgS04 (0.25%) 2.5 g.
Sterilization: Not specified. 6. NH4NO3 (0.2%) 2.0 g.
Use : To study growth requirements of the Preparation: (1) Dissolve 2, 3, 4, 5 and 6
tubercle bacillus. Authors reported no in 1.
growth unless glycerol was present. Sterilization: Not specified.

Reference: Proskauer and Beck (1894 p. Use : To study food requirements of tuber-
147). cle bacilli.
Reference: Proskauer and Beck (1894 p.
244a. Mayer's Ammonium Nitrate Solution
142).
(Tanner)
247. Heraeus' Glucose Ammonium Car-
Constituents:
bonate Solution
2. MgS04-7H20 10.0 g. Constituents
3. NH4NO3 15.0 g. 1. Distilled water 1000.0 cc.
4. Ca3(P04)2 0.01 g. 2. Potassium phosphate 0.05 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS (O
3. MgS04 0.01 g. 5. Magnesium Citrate (0.25%). 2.5 g.

4. CaCU 0.05 g. 6. (NH4)2C03 (0.3%) 3.0 g.
5. Dextrose 1.0 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6
6. (NH4)2COo 1.0 g. in 1.

(1) Dissolve 1.0 g. potassium phosphates Use: To study constituents essential for
0.2 g. MgS04 and 0.1 g. CaCh in growth of the tubercle bacilli.
1000.0 cc. distilled water Variants
(2) Steam for 1 hour. (a) The authors used 0.2% (NH4)2C03
(3) Allow to stand. instead of 0.3% and substituted 0.4%
(4) Filter and boil again. ammonium citrate for 0.25% magne-
(5) Prepare solutions of dextrose and sium citrate.
(NH4)2C03, the ratio of 1:15. (b) Smith gave the following solution:
(6) Mix 10.0 cc. (4), 1.0 cc. of the dextrose 1. Distilled water 1000.0 g.
solution and 1.0 cc. of (NH4)2C02 2. (NH4)2C03 3.5 g.
solution. 3. KH2PO4 1.5 g.
(7) Dilute to 200.0 cc. with distilled 4. MgS04-7H20 2.5 g.
water. A liter of this solution con- 5. Glycerol 15.0 g.
tains: (c) Tanner gave the same solution as
0.05 g. potassium phosphate Smith but used K3PO4 instead of
0.01 g. MgSO KH2PO4.
0.05 g. CaCl2 References: Proskauer and Beck (1894 p.
1.0 g. dextrose 148), Smith (1905 p. 198), Kolle and
1.0 g. NH4CO3 Wasserman (1912 p. 394), Tanner (1919
(8) Distribute in 50.0 cc. flasks that have p. 68), Klimmer (1923 p. 112).
previously been plugged and sterilized
in the sterilizer.
250. Beijerinck's Glucose Ammonium Sul-
Sterilization : Heat the flasks in the steamer phate Solution
for 30 minutes. Constituents:
Use: General culture medium. 1. Water 100.0 cc.
Reference: Heraeus (1913 p. 224). 2. KH2PO4 0.05 g.
3. (NH4)2S04 0.05 g.
248. Lowenstein's Glycerol Ammonium Glucose
4. 3.0 g.
Carbonate Solution
Constituents Sterilization: Not specified.

1. Water 1000.0 cc. Use: Cultivation of Streptothrix chrom-
2. (NH4)2C04 (Commercial) ... 3.5 g. ogem.
3. Potassium phosphate Reference: Beijerinck (1900 p. 10).
(primary) 1-5 g.
4. MgS04 251. Doryland's Glucose Ammonium Sul-
2.5 g.
5. Glycerin 15.0 g.
phate Solution
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Constituents:
Sterilization: Not specified. 1. Distilled water 500.0 cc.
Use: Cultivation of tubercle bacilli. 2. HCl dilute
Reference: Lowenstein (1913p. 592). Har- 3. MgS04 0.5 g.
vey (1921-22 p. 103). 4. CaO 0.01 g.
5. Fe2(S04)3 0.01 g.
6. MnS04 0.01 g.
Ammonium Carbonate Solution 7. (NH4)2S04 1.00 g.
Constituents 8. H2SO4
1. Water 1000.0 cc. 9. H3PO4
2. Glycerol (1.5%) 15.0 g. 10. Glucose 10.0 g.
3. KH2PO4 (0.5%) 5.0 g. 11. N/ 0.2578 NaOH
4. MgS04 (0.25%) 2.5 g. 12. N/ 0.6205 KOH
(1) Dilute HCl so that 1.0 cc. is not quite (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
neutralized by 1.0 cc. of silicate solu- (2) Adjustment of reaction not specified.
tion made by dissolving 24.0 g. (3) Distribute into Drechsel's flasks with
KsSiOs and 8.4 g. NasSiOa in 500.0 g. a ground cover.
distilled water. (Phenolphthalein as (4) Add an excess of chalk to each flask.
indicator.) (5) After sterilization connect the flask
(2) Add to the HCl the following salts: with a nitrogen generator and after
MgS04 0.5 g. inoculation replace the air with
CaO 0.01 g. nitrogen.
Fe2(S04)3 0.01 g. an autoclave.
Sterilization: Sterilize in
MnS04 0.01 g. Use: Cultivation of Clostridium Pastori-
(NH4)2S04 1.0 g. anum.
(3) Standardize (2) against the silicate Reference: Winogradsky (1902 p. 49).
solution so that 1.0 cc. silicate equiva-
lent to 1.0 cc. (2) using methyl orange 253. Gage's Glucose Ammonium Sulphate
as indicator. Solution
(4) Standardize a solution of H2SO4 in
Constituents:
same way as HCl omitting the salts.
1. Water 1000.0 cc.
(5) Standardize H3PO4 in similar way as
2. (NH4)2S04 4.0 g.
HCl omitting salt and using phenol-
3. Glucose 1.0 g.
phthalein as indicator.
4. KH2PO4 2.0 g.
(6) Mix the acids in the following ratio:
5. MgS04 1.0 g.
HCl 153.5 cc.
6. Iron sulphate 0.8 g.
H2SO4 77.0 cc.
7. NaCl 4.0 g.
H3PO4 116.0 cc.
8. MgCOa
(7) Mix equal quantities of N/0.6205
Preparation
KOH and N/0.2578 NaOH.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
(8) 1.0 cc. of (7) should neutralize 1.0 cc.
of (6), using phenolphthalein as
flasks.
indicator.
(3) To each flask add 1.0 cc. of a heavy
(9) Draw acid and base into separate
suspension of MgCOs.
plugged burettes, allow to stand
several hours to sterilize.
Use: To study nitrification by Nitroso-
(10) Add enough sterile glucose solution
hacter and Pseudomonas radicicola.
to equal amounts of (6) and (7) to give
concentration of glucose of 10.0 grams
per liter to a mixture of equal amounts
254. Stoklasa's Glucose Ammonium
of (6) and (7).
Sulphate Solution
Use: General synthetic culture medium.
Reference: Doryland (1916 pp. 146-148). Constituents:
252. Winogradsky's Glucose Ammonium d-glucose
2. 25.0 g.
Sulphate Solution
3. K2SO4 1.0 g.
Constituents 4. MgCl2 0.5 g.
1. Distilled water 1000.0 cc. 5. Iron sulphate 0.1 g.
2. Dextrose 20.0 g. 6. (NH4)2S04
3. Potassium phosphate 1.0 g. 7. One of the following: 1.0 g.
4. MgS04 0.2 g. Monodiferric-phosphate Fe203(P20s)
5. NaCl ] Monodialuminum-phosphate
'""^^^
6. FeS04 , A1203P205-8H20
^'"*'
7. MnS04 J
Preparation
8. (NH4)2S04 0.01 g. (1) Dissolve 2, 3, 4, 5 and 2.0 g. nitrogen
9. Chalk in the form of (NH4)2S04 in 1.
(2) Add 1.0 g. of one of 7 to (1). Reference: Lipman, Waksman and Joffe
(3) Adjustment of reaction not given. (1921 p. 475) taken from (1922 p. 108).
Sterilization: In the autoclave.
257. Bokorny's Sucrose Ammonium Sul-
phate Solution (Basic)
ion in the soil using Bac. proteus and
vulgaris, Bac. mesentericus vulgatus. Constituents:
PO4 ions were probably formed. 1. Distilled water 1000.0 cc.
Reference: Stoklasa (1911 p. 479). 2. Sucrose 100.0 g.
3. K,HP04 5.0 g.
255. Percival's Glucose Ammonium Sul-
4. MgS04 10 g.
phate Solution
5. (XH4)2S04 5.0 g.
Constituents: Dissolve 4 and 5 in
Preparation: (1) 2, 3, 1.
2. (NH4)2S04 2.0 g.
Use: To study yeast growth.
3. K2HPO4 1-0 g.
Variants Zikes used the modification given
:
4. NaCl 2.0 g.
below to study volutin formation by wine
5. MgS04 0.5 g.
yeast, Saccharomyces Frohberg, Sac-
6. FeS04 0.4 g.
charomyces anamensis, Saccharomyces
7. MgCOs 10.0 g.
ilicis,Saccharomyces, Will (Bajonus)
8. De.xtrose 0.01, 0.03, 1.0 or (3.0%) 0.1,
Pichia membranaefaciens, Cholara myco-
0.3, 3.0, 10.0 or 30.0 g.
derma, Monilia Candida, Oidium laclis,
Preparation
Endomyces Magnusii.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Distribute in 50.0 cc. into conical 100.0 g.
2. Sucrose
flasks.
3. K2HPO4 10 g.
(3) Add 0.5 g. of basic MgCOs to each
4. MgS04 0.3 g.
flask.
5. (NH4)2S04 2.5 g.
To each flask add 0.01, 0.03, 0.3, 1.0
CH =
(4)
(pH = 5.3718, 4.25 X lO--^).
or 3.0% glucose.
Reference: Bokorny (1911 p. 183), Zikes
(1922 p. 29).
Use: To study nitrification by bacteria
from the soil. 258. Wellman's Starch Ammonium Sulphate
Reference: Percival (1920 p. 144). Solution
256. Lipman, Waksman and Joffe's Glucose Constituents:

Sulphur Solution 1. Water 1000.0 cc.
Constituents: 2. (NH4)2S04 2.0 g.
1. Distilled water 1000.0 cc. 3. KNO3 10 g.
2. (NH4)3S04 2.0 g 4. K3PO4 2.0 g.
3. K2HPO4 10 g 5. NaCl 2.0 g.
4. MgS04 0.5 g 6. Starch

5. KCl 0.5 g Preparation
6. FeS04 0.01 g (1) Dissolve 2, 3, 4 and 5 in 1.
7. Glucose 10.0 g (2) Add starch to (1). (Amount not

8. Sulphur 10.0 g given.)
9. Ca3(P04)2 10.0 g Sterilization: Not specified.
Use: Cultivation of Glycobacter proteoly-
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7, and 9 in
8 ticus.
(2) Adjust to pH between 6.0 and 6.2. Reference: Wellman (1912 p. 616).
Sterilization: Sterilize in the steamer the

259. Heller's Starch Ammonium Sulphate
fractional method.
Solution
Use: Cultivation of organisms capable of
o.xidizing sulphur. Constituents
Variants: The authors used only 1.0 g. 1. Water 1000.0 cc.
glucose instead of 10.0 g. 2. Sodium phosphate 0.5 g.

3. Ammonium sulphate 0.5 g. ported that the presence or absence of an

4. Soluble starch 10.0 g. "essential food factor represented by
5. Calcium carbonate 5.0 g. vitamine B (?) exerts a stimulating effect
Preparation upon the growth in physiological effi-
(1) Dissolve 2, 3, 4 and 5 in 1. ciency of C. folia."
(2) Adjustment of reaction not specified. Reference: Sanborn (1926 p. 2).
262. Lohnis' Ammonium Sulphate Cellulose
Use: Enrichment of soil anaerobes.
Solution
Reference: Heller (1921 p. 453).
Constituents
260. Heinze's Glycogen Ammonium Sul-
phate Solution (NH4).2S04
2. 1.0 g.
Constituents: 3. K.2HPO4 1.0 g.
2. KH0PO4 2.0 g. 5. NaCl trace
3. MgS04 0.4 g. 6. CaCOs 20.0 g.
4. CaCl. 0.2 g. 7. Cellulose 5.0 g.
5. Glycogen 10.0 Preparation
6. (NH4)2S04 0.1 (1) Dissolve 2, 3, 4, 5 and 6 in 1.
7. FeCla (dilute solution) ... 20 drops (2) Fill tubes with (1).
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and (3) Add about 0.1 g. of filter paper,
7 in 1. cotton wool or straw to each tube.
Sterilization: Not specified. Sterilization: Not specified.
Use: Cultivation of Aspergillus and other Use: Cultivation of cellulose decomposers.
molds. Author inoculated with rotten dung.
Variants Variants: Cunningham tubed the solution
(a) The author used 2.5 g. glycogen in 15.0 to 20.0 cc. quantities and added a
instead of 10.0 g. small quantity of finely cut filter paper to
(b) The author used 1.3 g. (NH4)2S04 each tube. Fermentation tubes with a
and 2.5 g. of glycogen instead of glass rod inside to hold them above the
0.1 g. (NH4)2S04 and 10.0 g. of glyco- CaCOs were added. The medium was
gen. used to grow anaerobes.
(c) The author used 4.0 g. MgS04, 2.5 g. Reference: Lohnis (1913 p. 93), Cunning-
glycogen and omitted the FeCls ham (1924 p. 142).
solution.
263. Lohnis' Pectin Ammonium Sulphate
Reference: Heinze (1904 p. 182).
Solution
261. McBeth's Cellulose Ammonium Sul-
Constituents
phate Solution (Sanborn) Water (tap) 1000.0 cc.
1.
Constituents: 2. (NH4)2S04 0.5 g.

1. Distilled water 1000.0 cc. 3. K2HPO4 0.5 g.
2. KiHP04 1.0 g. 4. CaCOa 20.0 g.
3. MgS04 1.0 g. 5. Pectin 5.0 g.
4. NaoCOs 1.0 g. Preparation
5. (NH4)2S04 2.0 g. (1) Grate and extract about 500.0 g. of
6. Cellulose carrot roots with warm water con-
Preparation taining chloroform.
(1) Dissolve 2, 3, 4 and 5 in 1. (2) Treat the residue for about 30 minutes
(2) Add a suitable source of cellulose. (without warming) with dilute caus-
Sterilization: Sterilize in the autoclave at tic soda
15 pounds pressure. (3) Wash (2) with water.
Use: Cultivation of Cellculomonas folia. (4) Extract for 30 minutes with dilute
The author used maple leaves and filter HCl.
paper as sources of cellulose. He re- (5) Again wash with water.
CULTURE MEDIA FOR CULTIVATIOX OF MICROORGANISMS 79
(6) Dissolve the pectin by adding dilute (2) Add one of the carbohydrates listed
ammonia and precipitate it from to (1).
solution by the addition of CaCU. Sterilization: Not specified.
(7) Thoroly wash the pectin precipitate Use: To study the constituents essential
with distilled water. for the growth of the tubercle bacillus.
(8) Add 0.1 g. of (7) to a series of flasks. Added nutrients:
(9) Dissolve 2, 3 and 4 in 1. (a) The authors added one of the follow-
(10) Add 2.0 cc. of (9) to each flask of (8). ing carbohydrates.
Sterilization: Not specified. no added carbon
Use: Enrichment of B. amylobader raffinose 1-0%
Reference: Lohnis (1913 p. 92). sucrose .0%1
maltose 1-0%
264. Beijerinck's Glucose Starch Ammo- lactose 1.0%
nium Sulphate Solution
glucose 1.0%
Constituents: manno.se 1-0%
1. Water 1000.0 cc. levulose 1.0%
2. Glucose 30.0 g. dulcitol 0.6%
3. Starch 1.0 g. isodulcitol
4. KH2PO4 0.5 g. (b) Tiffeneau and Marie added 0.6% man-
5. (NH4)2S04 0.5 g. nitol as a carbon source.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Reference: Proskauer and Beck (1894
Sterilization: Not specified. pp. 142-146), Tiffeneau and Marie (1912
Use: Cultivation of Streptothrix chromo- p. 48).
gena. The author found that the medium
supported growth very well.
267. Doryland's Acetic Acid Ammonium
Sulphate Solution
Reference: Beijerinck (1900 p. 11).
Constituents:
265. Proskauer and Beck's Mannitol Glyc-
1. Water 500.0 cc.
erol Ammonium Sulphate Solution 2. NaOH, N/0.2578
Constituents 3. KOH, N/0.6205
1. Water 1000.0 cc. 4. HCl
2. Mannitol (0.6%) 6.0 g. 5. MgSOi 0.5 g
3. Glycerol (1.5%) 1.5 g. 6. CaO 0.01 g
4. KH2PO4 (0.5%) 5.0 g. 7. Fe2(S04)3 0.01 g
5. MgS04 (0.25%) 2.5 g. 8. MnS04 0.01 g
6. (NH4)2S04 (0.2%) 2.0 g. 9. (NH4)2S04 1.0 g
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 10. H2SO4
in 1. 11. H3PO4
Sterilization: Not specified. 12. CH3COOH
Use: To determine constituents essential Preparation
for the growth of the tubercle bacillus. (1) Dilute the HCl so that 1.0 cc. is not
Reference: Proskauer and Beck (1894 quite neutralized by 1.0 cc. of a
p. 147). silicate solution made by dissolving
24.0 g. KaSiOs and 8.4 "g. NaoSiOa
in 500.0 g. water. Phenolphthalein
is used as an indicator.
Constituents: (2)Add to the HCl the following salts:
1. Water 1000.0 cc. MgS04 0.5 g
2. Glycerol (1.5%) 15.0 g. CaO 0.01 g
3. KH2PO4 (0.5%) 5.0 g. Fe2(S04)3 0.01 g
4. Magnesium citrate (0.25%). 2.5 g. MnS04 0.01 g
5. (NH4)2S04 (0.2%) 2.0 g. (NH4)2S04 1.0 g
Preparation (3) Standardize the resulting HCl solu-
(1) Dissolve 2, 3, 4 and 5 in 1. tions against the silicate, using
methyl orange as an indicator, so that 269. Peglion's Sucrose Ammonium Sulphate

1.0 cc. is equivalent to 1.0 cc. of the Solution
silicate solution.
Constituents:
1. Water 1000.0 cc.
the same way as HCl omitting the
2. Cane sugar 50.0 g.
salts.
3. Cream of tartar 10 g.
(5) Standardize H3PO4 and CH3COOH
manner as HCl omitting
4. MgS04 0.3 g.
in a similar
5. (NH4)2S04 0.2 g.
the salts and using phenolphthalein
Preparation : (1) Dissolve 2, 3, 4 and 5 in 1.
as an indicator.
(6) Mix the acids in the following pro-
Use: Cultivation of B. oncidii.
portion:
Reference: Peglion (1899 p. 36).
HCl 153.5 cc.
CHrCOOH 153.5 cc.
H2SO4 77.0 cc. 270. Higgins' Succinate Ammonium Sul-
H3PO4 116.0 cc. phate Solution

Constituents:
KOH and N/0.2578 NaOH. 1. Water 1000.0 cc.
(8) 1 should neutralize 1 cc. of
cc. of (7) 2. Glucose 2.5 g.
(6) using phenolphthalein as indi- 3. Lactose 2.5 g.
cator. 4. Ammonium succinate 2.5 g.
(9) Acids and bases are drawn into 5. (NH4)2S04 1.0 g.
separate burettes and allowed to 6. KCl 1.5 g.
stand to sterilize. 7. KNO3 2.5 g.
(10)Mix equal amounts when ready to use. 8. Ammonium phosphate 2.5 g.
Not specified.
Sterilization: Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7
Use: As a general synthetic culture me- and 8 in 1.
dium. Sterilization: Method not specified.

Reference: Doryland (1916 p. 146). Use: Cultivation of organisms causing
cholera, Picarton cattle disease and other
268. Proskauer and Beck's Basal Citrate
organisms.The author found that this
medium gave approximately the same
Constituents: results as ordinary broth.
1. Water 1000.0 cc. Reference: Higgins (1898 p. 668).
2. KH2PO4 (0.5%) 5.0 g.
3. Magnesium citrate (0.25%). 2.5 g.
271. Lockemann's Citrate Ammonium Sul-
4. (NH4)2S04 (0.25%) 2.5 g.
phate Solution
Preparation
(1) Dissolve 2, 3 and 4 in 1. Constituents
(2) Add 1.0% of one of the listed carbo- 1. Water 1000.0 cc.
hydrates. 2. NaH2P04-H20 3.0 g.
Sterilization: Not specified. 3. KH2PO4 4.0 g.
Use: To determine the constituents essen- 4. (NH4)2S04 5.0 g.
tial for growth of the tubercle bacillus. 5. Magnesium citrate 2.5 g.

Glycerin 20.0 g.
Added nutrients: The authors added 1.0% 6.
of one of the following carbohydrates: Preparation

levulose raffinose (1) Dissolve 2, 3, 4, 5 and 6 in 1.
mannose dulcitol 0.6% (2) The reaction should be acid titer
maltose isodulcitol 0.6% 1/100 N 3.3.

sucrose mannitol 0.6% (3) Distribute in 50.0 cc. lots in flasks.
lactose Sterilization: Not specified.
Reference: Proskauer and Beck (1894 Use: Cultivation of tubercle bacilli.
pp. 144-146). Reference: Lockemann (1919 p. 421).

272. Beijerinck's Glucose Ammonium ferent esters with clear bacteria free
Phosphate Solution culture broth were incubated for 24 hours;
the amount of acid produced (measured
Constituents;
in terms of N/50 NaOH), determined
1. Water 1000.0 g.
lipase production.
2. Glucose 20.0 g.
References: Kendall, Day and Walker
3. K2HPO4 0.2 g.
(1914 p. 434), Kendall, Walker and Day
4. (XH4)2HP04 0.2 g.
(1914 p. 455).
Sterilization: Not specified. 275. Wherry's Levulose Sodium Ammonium

Use: The author found that Azolobocter Phosphate Solution
chrnococcum utilized ammonium salts
Constituents:
readily in this medium.
2. Levulose 2.0 g.
273. Ayer's Glucose Ammonium Phosphate 3. Sodium ammonium phos-

phate 2.0 g.
Solution (Caldwell)
Preparation: Dissolve 2 and 3 in 1.
(1)
Constituents: Sterilization: Sterilize in autoclave (time
1. Distilled water 1000.0 cc. not given).
2. Sodium ammonium phos- Use: To study acid proof ness of B. tuber-
phate 4.0 g. culosis. Author reported good growth,
3. NaHzPOi 2.0 g. but the organisms stain irregularly and
4. Glucose 10.0 g. are not acid proof.
5. Marble Reference: Wherry (1913 p. 150).
Preparation
(1) Dissolve 2 and 3 in 1. 276. Wolffin's Glucose Ammonium Phos-
(2) Prepare a 20% glucose solution in phate Solution
distilled water. Constituents
(3) Mix sterile (1) and sterile (2). 1. Water 1000.0 cc.
(4) Tube in sterile tubes containing a 2. Dextrose 100.0 g.
little ground marble. 3. Potassium phosphate 10 g.
Sterilization: Method not given. 4. Ammonium phosphate 10.0 g.
Use : To study anticolon bacteriophage in a 5. MgS04 0.2 g.
synthetic medium. 6. CaCOs
Reference: Caldwell (1926 p. 123). Preparation
274. Kendall, Day and Walker's Glucose
Distribute in flasks in 250.0 cc. lots.
(3)
Constituents: (4) Add 1.0 g. CaCOs to each flask.
1. Redistilled water 1000.0 cc. Sterilization: Not specified.
2. (XH4)2HP04 4.0 g. Use: To study fermentation by Bacillus
3. NaCl 5.0 g. levans.
4. Glucose 10.0 g. Reference: Wolffin (1894 p. 288).
Preparation:
277. Stutzer's Sucrose Ammonium Magne-
Adjustment of reaction not given.
sium Phosphate Solution
(2)
Sterilization: Not specified. Constituents:
Use: To study the metabolism of the 1. Water 1000.0 cc.
tubercle bacillus. Alizarin, neutral red 2. Sucrose 20.0 g.
and phenolphthalein were used to study 3. Potassium phosphate 1.0 g.
changes in reaction; Ziehl-Nielsen stain 4. Ammonium magnesium
for staining. Kendall, Walker and Day phosphate (dry)
used the solution for the study of lipase (MgNH4P04)." 2.0 g.
production by the tubercle bacillus. Dif- Preparation: (1) Dissolve 2, 3 and 4 in 1.
Sterilization: Not specified. Sterilization: Method not given.

Use: Cultivation of bacteroids from Pis urn Use: Cultivation of Spirochaeta cytophaga
sativum. and other organisms capable of decom-
Reference: Stutzer (1900 p. 902). posing cellulose.
Variant: The authors used the medium
278. Henneberg's Sucrose Ammonium
diluting each flask containing 25.0 cc. of
Phosphate Solution
the medium with 150.0 cc. of water.
Constituents : Reference: Waksman and Carey (1926
1. Distilled water 1000.0 g p. 92).
2. K0HPO4 (0.5%) 5.0 g
3. xMgS04 (0.2%) 2.0
281. Percival's Cellulose Ammonium Phos-
g
phate Solution
4. Cane sugar (5.0%) 50.0 g
5. Ammonium phosphate (0.3%) 3.0 g Constituents:
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1 1. Water 1000.0 cc.
Sterilization: Not specified. 2. KH.PO4 1.0 g.
Use: Cultivation of yeast. 3. Ammonium phosphate 1.0 g.
Reference: Henneberg (1907 pp. 40-45), 4. MgS04 0.5 g.
(1909 p. 104). 5. NaCl a small crystal
6. Filter paper or Blotting
279. Botkin's Starch Ammonium Phosphate paper 3.0 g.
Solution
7. CaCOs 5.0 g.
1. Water 1000.0 cc. (1) Dissolve 2, 3, 4 and 5 in 1.
2. Ammonium phosphate 0.08 g. (2) Pour into 250.0 cc. flasks and add 5.0 g.
3. Potassium phosphate 0.04 g. of CaCOs.
4. MgS04 0.04 g. (3) Cut about 3.0 g. of filter or blotting
5. (NH4)2S04 0.02 g. paper into small pieces and add
6. Starch, potato 30.0 g. to (2).
7. CaC03 20.0 g. (4) Inoculate with a piece of fresh horse
Preparation : dung or mud from the bottom of a
(1) Dissolve 2, 3, 4, 5 and 6 in 1. pond.
(2) Add 7 to (1). (5) Fill the flasks completely and insert
Sterilization: Not specified. a cork thru which passes a bent glass
Use: To ascertain fermentation of starch tube, arranged so as to collect any
by Bacillus hutyricus. The author found gas given off.
that gas was produced from the starch Sterilization: Not specified.
after 24 hours and that the culture Use: Cultivation of B. amylobacter, a
reduced Fehling's solution. cellulose splitting organism. Author re-
Reference: Botkin (1872 p. 431). ported the production of gas after 3 or
4 weeks.
280. Waksman and Carey's Ammonium Reference: Percival (1920 p. 231).
Phosphate Cellulose Solution
282. Loew's Formaldehyde Ammonium
Constituents
Phosphate Solution
1. Distilled water 100.0 cc
2. (NH4)2HP04 5.0 g Constituents
3. MgS04 1.0 g 1. Water 1000.0 cc.
4. FeS04 0.02 g 2. KH2PO4 2.0 g.
5. KCl 1-0 g 3. (NH4)2HP04 1.0 g.
6. Cellulose (filter paper) 4.0 g 4. MgS04 0.1 g.
Preparation : 5. CaClj 0.1 g.
(1) Dissolve 2, 3, 4 and 5 in 1. 6. Sodium salt or formaldehyd-
(2) Grind filter paper. I'| sulfurous acid ("Formalde-
(3) Add 1.0 g. ground filter paper to hyschwefligsaurem Na-
flasks containing 25.0 cc. of sterile (1). trom") 5.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 3. Ammonium phosphate 0.5 g.

in 1. 4. KCl 0.1 g.
Use: Isolation of a red bacillus from the (1) Dissolve 3 and 4 in tap water.
aircapable of utilizing formic acid as a (2) Distribute in 10.0 cc. lots in Frenden-
carbon source. reich flasks.
Reference: Loew (1892 p. 462). (3) After sterilization add 3.0% by
volume of absolute alcohol and suffi-
283. Loew's Methyl Alcohol Ammonium cient acetic acid to each tube to dis-
Phosphate Solution
solve the precipitate.
Constituents: Sterilization: Method not given.
1. Water 1000.0 cc. Use: Cultivation of acetic acid bacteria.
2. Methyl alcohol 5.0 g. References: Beijerinck (1898 p. 214), Janke
3. K2HPO4 0.5 g. (1916 p. 6).
4. (NH4)2HP04 0.5 g.
5. MgS04 0.1%
286. De Schweinitz's Glycerol Ammonium
Preparation
Phosphate Solution (Gage and Phelps)
(1) Dissolve 3, 4 and 5 in 1. Constituents:
(2) After sterilization add 2 to (1). 1. Distilled water 1000.0 cc.
Sterilization: Method not given. 2. MgS04 (0.02%) 0.2 g.
Use: Isolation of bacillus from the air, 3. K2HPO4 (0.1%) 1.0 g.
producing red pigment and capable of 4. Ammonium phosphate
assimilating formic acid. (1.0%) 10.0 g.
Reference: Loew (1892 p. 463). 5. Glycerin (4.0%) 40.0 g.
6. Neutral red 1.0% water
284. Omeliansky's Ethyl Alcohol Ammonium solution (1.0%) 10.0 cc.
Phosphate Solution
Preparation
Constituents: (1) Dissolve 2, 3, 4 and 5 in 1.
1. Distilled water 1000.0 cc. (2) Add 10.0 cc. of a 1.0% water solution
2. Potassium phosphate 1-0 g. of neutral red to (1).
3; MgS04 0.5 g. Sterilization: Not specified.
4. Ammonium phosphate 1-0 g. Use: To observe neutral red reaction by
5. NaCl traces colon types. B. coli changes the color
6. Ethyl alcohol 10.0 cc. from red to yellow.
Preparation Reference: Gage and Phelps (1902 p. 411).
(2) Distribute into long necked flasks

287. Lowenstein's Glycerol Ammonium
with lateral tubes. Phosphate Solution
(3) Add a small amount of chalk to each Constituents
tube. 1. Distilled water 1000.0 cc.
(4) After sterilization cool. 2. Ammonium phosphate 6.0 g.
(5) Add 1.0 part ethyl alcohol to each 3. Glycerol 40.0 g.
100.0 parts medium under aseptic Preparation: Dissolve 2 and 3 in 1.
(1)
conditions. Sterilization: Method not
given.
Sterilization: Method not specified. Use: Cultivation of tubercle bacilli.
Use: Fermentation of ethyl alcohol with Tubercle bacilli develop very poorly on
the production of methane. this medium. Na, K, CI or S is necessary
Reference: Omeliansky (1916 p. 58). for growth.
Variants
285. Beijerinck's Alcohol Ammonium Phos-
Lockemann specified the use
(a) of
phate Solution
(NH4)3P04-3H20 and added 0.6 g.
Constituents: MgSOrTHaO.
1. Water (tap) 1000.0 cc. (b) Lockemann specified the use of
2. Alcohol 30.0 g. (NH4)3P04-3H20 and added 0.6 g.
MgS04-7H20 and 3.0 g. Na.HPOi-

H2O.
References: Lowenstein (1913 p. 592),
Lockemann (1919 p. 421).
288. Capaldi and Proskauer Mannitol

Constituents:
1. Water 1000.0
2. Ammonium phosphate
(0.2%)
3. Mannite (0.2%).
4. NaCl (0.02%)...
5. MgS04 (0.01%)
5. K2HPO4 1.0 g. Use: Isolation and enrichment of Bacillus

6. Sodium citrate 2.0 g. methanicus (utilizing methane).
Preparation References: Sohngen (1905 p. 514), Lohnis
(1) Dissolve 2, 3, 4, 5 and 6 in 1. (Use (1913 p. 105).
2.77 g. sodium citrate 5J H2O.)
(2) pH is about 6.7 to 6.9. 296. Laurent's Sucrose Ammonium Phos-
(3) Distribute in 5.0 to 8.0 cc. quantities. phate Solution (Pringsheim)
Sterilization: In the autoclave. Constituents
Use: Differentiation of the colon-aero- 1. Water 1000.0 cc.
genes group. Bact. coli of fecal origin is 2. Sugar 100.0 g.
unable to utilize citric acid as a source 3. Tartaric acid 1.0 g.
of carbon, while the aerogenes-cloacae 4. KH2PO4 0.75 g.
types utilize citric acid. 5. MgS04 0.1 g.
Variants: Potassium citrate may be used 6. (xNH4)3P04 5.0 g.
instead of sodium citrate. Ammonium Preparation: Dissolve 2, 3, 4, 5 and 6 in 1.
citrate is quite unstable. Sterilization: Not specified.
Reference: Koser (1924 p. 63). Use: To study the bios requirements of
yeast. The author reported that fer-
294. Tausz and Peter's Paraffin Magnesium
mentation occurred after 2 months.
Reference: Pringsheim (1906 p. 114).
Constituents:
1. Water 1000.0 cc. 297. Janke's Alcohol Ammonium Phosphate
2. MgNH4P04 (0.1%) 1.0 g. Solution
3. CaS04 (0.01%) 0.1 g. Constituents:
4. K2HPO4 (0.08%) 0.8 g. 1. Distilled water 1000.0 cc.
5. FeCls trace 2. K2HPO4 0.4 g.
6. KI trace 3. (NH4)2HP04 1.0 g.
7. Paraffin (amount not specified). 4. MgS04 + 7H2O 0.4 g.
Preparation: (1) Mix 1, 2, 3, 4, 5, 6 and 7. 5. Glycerin 5.0 cc.
Complete solution does not take place. 6. Succinic acid 1-0 g.
Sterilization Steam by the Klocker method
:
7. Alcohol (3.0%) 30.0 cc.
on 2 successive days for 30 minutes each Preparation
day. Dissolve 5 and 6 in distilled
(1) 2, 3, 4,
Use: Cultivation of Bacterium aliphat- water.
icum, Bacterium aliphatic um lique- .
Add distilled water to to make

(2) (1)
faciens, paraffin bacteria. 1000.0 cc.
Reference: Tausz and Peter (1919 p. 507). After add
(3) sterilization 3.0% by
295. Sohngen's Methane Magnesium volume of alcohol.
Ammonium Phosphate Solution Sterilization: Method not given.

Use: Cultivation of acetic acid bacteria.
Constituents Variants H3PO4 or acetic acid may be used
:
1. Distilled water 1000.0 cc. instead of succinic acid. The acid is used
2. K2HPO4 0.5 g. for the purpose of keeping the materials
3. MgNH4P04 1.0 g. in solution. Succinic acid does not in-
4. CaS04 0.1 g. hibit growth whereas the other mentioned
5. Methane acids do.
Preparation Reference: Janke (1916 p. 7), (1921 p. 91).
(2) Flask and inoculate (garden earth, 298. Wherry's Ammonium Acetate Salt
dung water or ditch water may be Solution
used for inoculation). Constituents:
(3) Fill the flask with a known mixture 1. Redistilled water
of oxygen and methane. 2. NaCl
4. CaClo 1.0 g. raflfinose 1-0%

5. MgS04 1.0 g. mannose 1.0%
6. KH2PO4 2.0 g. levulose 1.0%
7. Ammonium acetate 5.0 g. (b) Besson used a solution containing
Preparation 10.0 g. ammonium phosphate, 1.0 g.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. potassium phosphate, 0.2 g. MgS04
(2) Adjustment of reaction not specified. and 0.12 g. CaCU per liter and added
Sterilization: In the autoclave (time not 2.0% sucrose or glucose.
given). References: Proskauer and Beck (1894
Use: To study acid proof ness of B. tuber- pp. 144-147), Besson (1920 p. 37).
culosis.
Variants 301. Carapelle's Methylene Blue Ammo-
(a) The KH2PO4 may be omitted. nium Tartrate Solution
(b) Na2C03 naay be substituted for NaCl.
Constituents
(c) 2.0 g. of potassium acetate and 2.0 g.
of calcium acetate may be added.
(d) 2.0 of ammonium acetate with
2. Ammonium tartrate 10.0 g.
g.
2.0 g. or 1.0 g. of KH2PO4 may be
used.
4. MgS04 5.0 g.
5. Tri-basic calcium phos-
Reference: Wherry (1913 pp. 147-148).
phate 5.0 g.
299. Harvey's Ammonium Acetate Solution 6. Methylene blue (1.0%
soln.) several drops
Constituents
Preparation
(1) Dissolve 2, 3, 4 and 5 in (1).
2. Ammonium acetate 10.0 g.
(2) Tube after sterilization.
3. K0HPO4 5.0 g.
(3) Add several drops of a 1.0% methylene
4. MgS04 5.0 g.
blue solution to each tube.
5. Ca3(P04)2 0.5 g.
and
Preparation: (1) Dissolve 2, 3, 4 5 in 1.
Use: To determine reduction ability of
bacteria.
Reference: Harvey (1921-22 p. 103).
Variants: One of the following indicators
300. Proskauer and Beck's Basal Ammo- may replace methylene blue:
nium Tartrate Solution indigo carmine
lackmoid
Constituents:
litmus
1. Water 1000.0 cc.
sodium salt of indigo sulphuric acid
2. KH2PO4 (0.5%) 5.0 g.
Reference: Carapelle (1908 p. 552).
3. MgS04 (0.25%) 2.5 g.
4. Ammonium tartrate (0.2%). 2.0 g.
302. Meyers' Basal Ammonium Tartrate
Preparation
Solution
(2) Add one of the carbon sources listed. Constituents:
Sterilization: Not specified. 1. Distilled water 1000.0 cc.
Use: To study the constituents essential 2. Ammonium tartrate 10.0 g.
for growth of the tubercle bacillus. 3. Anhydrous sodium sulphate. 10.0 g.
Added nutrients and variants: Preparation
(a) The authors employed the following (1) Dissolve 2 and 3 in 1. Flask.
carbon sources: (2) Adjustment of reaction not given.
mannitol 0.6% (3) After sterilization suspend a sterile
dulcitol 0.6% strip of filter paper soaked in 10.0%
isodulcitol 0.6% lead acetate solution in each flask.
sucrose 1.0% Sterilization: Autoclave at 15 pounds pres-
lactose 1.0% sure for 10 minutes.
maltose 1.0%, Use: To determine H2S production by
colon-typhoid group. No growth of cul- References: Smith (1905 p. 197), Roux and
ture studied under aerobic condition. Rochaix (1911 p. 103), Kolle and Wasser-
Modification: The author added 10.0 g. mann (1912 p. 7), Lohnis (1913 p. 44),
glucose. Tanner (1919 p. 63), Besson (1920 p. 37),
Reference: iMeyers (1920 p. 248). Dopter and Sacqu6pee (1921 p. 121),
303. Naegeli's Ammonium Tartrate Solution Giltner (192i p. 369), Klimmer (1923
p. 172).
(Smith)
Constituents: 305. Nicolle and Zia Bey's Ammonium
1. Water 1000.0 cc. Lactate Solution
Constituents:
3. K2HPO4 1.0 g.
4. MgS04 0.2 g.
2. Ammonium lactate . . . 10.0 g.
5. CaClj 0.1 g.
3. MgS04 2.5 g.
4. Potassium phosphate . 0.0 or 5.0 g.
Preparation
Variants
(a) Harvey used 0.12 g. CaCU instead
(2) 5.0 g. of potassium phosphate may
of 0.1 g.
or may not be added.
(b) Harvey used 10.0 g. K2HPO4 instead Sterilization: Not specified.
of 1.0 g., 10.0 g. MgS04 instead of Use: To study pigment and fluorescence
0.2 g., and 0.5 g. Ca3(P04)2 instead
production by Bacillus pyocyaneiis.
of 0.1 g. CaCla.
Phosphate hastened and increased pig-
(c) Harvey used 5.0 g. KH2PO4, 0.5 g.
ment and fluorescence production.
calcium phosphate instead of CaClz
Reference: Nicolle and Zia Bey (1896
and 5.0 g. MgSOi instead of 0.2 g.
p. 671).
(d) Tanner, Devereux and Higgins used
a similar medium with 0.12 g. CaCl2 306. Braun and Cahn-Bronner's Ammonium
for the cultivation of yeast and yeast-
Lactate Solution
like fungi.
References: Smith (1905 p. 197), Harvey Constituents:
(1921-22 p. 103), Tanner, Devereu.x and 1. Water 1000.0 g.
Higgins (1926 p. 62). 2. NaCl 5.0 g.

3. KH2PO4 2.0 g.
304. Cohn's Ammonium Tartrate Solution 4. Ammonium lactate 6.0 g.
(Smith) Preparation
Constituents (1) Dissolve 2 and 3 in about 75.0 cc. of
1. Distilled water 1000.0 cc. water.
2. KH2PO4 5.0 g. (2) Neutralize (1) with sodium carbonate.
3. MgS04 5.0 g. (3) Dissolve 4 in 25.0 cc. water.
4. Ammonium tartrate 10.0 g. (4) Boil (2) and (3) for 1 hour to sterilize.
5. KCl 0.5 g. (5) When cool mix the two solutions.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. (6) Neutralize to litmus by the addition
Sterilization: Not specified. N/1 soda solution and then
of sterile
Use: General culture medium. add 0.7 cc. sterile N/1 soda solution.
Variants Sterilization: See step (4) under prepara-
(a) Roux and Rochaix specified the use tion.
of 10.0 g. potassium phosphate in- Use: To study food requirements for colon
stead of 5.0 g. KH2PO4 and used 0.5 g. typhoid group. Authors reported that
Ca3(P04)2 instead of KCl. colon bacillus and para typhoid B become
(b) Kolle and Wassermann did not specify strict aerobes in this medium.
the type of potassium phosphate Variants: Branham 0.2% K2HPO4
used
used, andsubstituted0.5g. Ca3(P04)2 instead of KH2PO4 and adjusted the
for KCl. reaction so that the final pH be between
(c) Klimmer omitted the KCl. 6.8 and 7.2. He used the medium for
toxin production by Bacterium enteri- (b) Gessard and Vaudremer specified the
tidis. useof K2HPO4, used 5.0 g. ammonium
References: Braun and Cahn-Bronner (1921 succinate and added 1.25 g. CaClj
pp. 4, 197), Branham (1925 p. 299). for the cultivation of tubercle bacilli.
References: Gessard (1892 p. 809), (1901
307. Lohnis' Ferric Ammonium Citrate p. 818); Gessard and Vaudremer (1922
Solution p. 1012).
Constituents 309. Kuntze's Ammonium Succinate

2. Ferric ammonium citrate
Constituents:
(0.05%) 0.5 g.
Preparation
ammonium 2. KH2PO4 10.0 g.
(1) Dissolve 0.05% ferric
3. MgCls 1.0 g.
citrate in water.
the
4. Ammonium succinate 10.0 g.
(2) Distribute in test tubes. Fill
tubes.
Use: To study pigment production.
Use: Cultivation of iron bacteria.
Author reported that when traces of
Variants: Brussoff cultivated Ferribac-
H2SO4 were added to the medium pig-
terium duplex on a medium prepared as
ment was produced, otherwise no pig-
follows. He reported that growth ap-
ment was formed.
peared after several weeks as a very thin
Variants: The author used 1.0 g. K2HPO4
yellow membrane.
instead of 10.0 g. KH2PO4, and from 1.0
(1) Boil tap water.
to 2.0 g. MgS04 instead of 1.0 g. MgCla.
(2) Filter.
ammonium The solution was adjusted to neutrality.
(3) Dissolve 0.05% ferric
Bacillus prodigiosus did not produce pig-
citrate in (2).
ment on this medium.
(4) Distribute in 50.0 cc. lots in small
Reference: Kuntze (1907 p. 306).
Erlenmeyer flasks.
(5) Sterilize in the autoclave for 15 to 20 310. Fermi's Basal Ammonium Succinate
minutes under 1.5 atmospheres pres- Solution
sure.
Constituents
References: Lohnis (1913 p. 116), Brussoff
1. Water 1000.0 cc.
(1916 p. 551).
2. Ammonium succinate
(0.5 to 1.0%) 5.0 to 10.0 g.
308. Gassard's Ammonium Succinate
3. KH2PO4 (0.5%) 5.0 g.
Solution
4. MgS04 (0.5%) 5.0 g.
Constituents 5. K3PO4 (0.05%) 0.5 g.

2. Ammonium succinate 10.0 g. (1) Dissolve2, 3, 4and5in 1.
3. Potassium phosphate 5.0 g. (2) Add one of the added nutrients.
4. xMgS04 2.5 g. Sterilization: Not specified.
Preparation: (1) Dissolve 2, 3 and 4 in 1. Use: Cultivation of Schizomycetes

Sterilization: Not specified. Added nutrients: The author added 5.0%
Use: To study pigment and fluorescence glycerol or 5.0% sucrose.
production by Bacillus pyocyaneus. Reference: Fermi (1892 p. 26).
Also used for tlie cultivation of tubercle

311. Fischer's Glucose Ammonium Tartrate
bacilli.
Solution (Miiller)
Variants
(a) Tlie author used 1.0 g. ammonium Constituents:
succinate, specified the use of 5.0 g. 1. Water 1000.0 cc.
of Na2HP04 or K2HPO4 and added 2. K2HPO4 (0.1%) 1.0 g.
1.25 g. CaCl2 to the solution. 3. MgSO4(0.02%) 0.2 g.

4. CaClj (0.01%) 0.1 g. Use: To study inversion of sugar in syn-

6. Dextrose (1.0%) 10.0 g. thetic medium by bacteria, yeasts and
6. Ammonium tartrate (amount not molds.
given). Variants: Authors substituted 5.0 g. glyc-
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 erol for sucrose.
in 1. Reference: Fermi and Montesano (1895
Sterilization: Not specified. p. 547).
Use: Cultivation of Bad. Giintheri,
Reference: Muller (1907 p. 472). 315. Proskauer and Beck's Glycerol Ammo-
nium Lactate Solution
312. NMgeli's Sucrose Ammonium Tartrate
Constituents:
Solution (Fermi)
1. Water 1000.0 cc.
Constituents: 2. Glycerol (1.5%) 15.0 g.
1. Water 1000.0 cc. 3. KH2PO4 (0.5%) 5.0 g.
2. Ammonium tartrate 10 g. 4. MgS04 (0.25%) 2.5 g.
3. KH2PO4 1.0 g. 5. Ammonium lactate (0.5%).. 5.0 g.
4. MgS04 0.2 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
5. CaCl2 2.1 g. Sterilization: Not specified.

6. Sucrose 50.0 g. Use: To study the constituents essential
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 for growth of the tubercle bacillus.
in 1. Variants
Sterilization: Not specified. (a) The authors added 1.0% glucose,
Use: Cultivation of Schizomycetes. 0.25% K2SO4 and 0.12% NaCl.
Reference: Fermi (1892 p. 26). (b) The authors added 0.3% (NH4)2S04
and omitted 0.25% MgSOi.
313. Groenewege's Sucrose Ammonium (c) Gessard and Vaudremer used 5.0%
Tartrate Solution glycerol and added 0.125% CaClz.
References: Proskauer and Beck (1894
Constituents
pp. 142, 148), Gessard and Vaudremer
1. Water 1000.0 ec.
(1922 p. 1012).
2. Saccharose 20.0 g.
3. Sodium ammonium tartrate. 1.0 g.
4. K2HPO4 0.5 g.
316. Stutzer and Hartleb's Glycerol Ammo-
Dissolve and 4
nium Citrate Solution
Preparation: (1) 2, 3 in 1.

Use: Invertase production by Phytobacter 1. Water 1000.0 cc
lycopersicum n. sp. (causing tomato rot). 2. Ammonium citrate 1-0 g.
Reducing sugars are found to be present 3. Glycerol 10.0 g.
when tested with Fehling's solution. 4. Potassium phosphate 10 g.
Reference: Groenewege (1913 p. 28). Preparation: (1) Dissolve 2, 3 and 4 in 1.
314. Fermi and Montesano's Sucrose Use: Cultivation of a bacterium from a
Ammonium Tartrate Solution case of foot and mouth disease. The
Constituents: authors report that the medium was
1. Distilled water 1000.0 cc. strongly clouded after about 3 weeks.
2. Ammonium tartrate 0.5 g
Reference: Stutzer and Hartleb (1897
3. Potassium phosphate 0.5 p. 382).
g
4. MgS04 0.5 g
5. Calcium phosphate 0.05 g
317. Kuntze's Glycerol Ammonium Suc-
cinate Solution
6. Sucrose 5.0 g
(2) Distribute into flasks. 2. MgS04 2.0 g.
Sterilization : Not specified. 3. KH2PO4 1.0 g.
4. Glycerol (double distilled Use of 0.2% ammonium tartrate with the

Merck) 20.0 g. addition of one of the following:
5. Ammonium succinate 10.0 g. mannitol 0.6%
Preparation glucose 1.0%
(1) Dissolve 2, 3, 4 and 5 in 1. mannose 1.0%
(2) Adjust the reaction to neutral to raffinose 1.0%
litmus. maltose 1 .0%
Sterilization: Method not given. lactose 1.0%

Use: To study pigment production by sucrose 1.0%
Aspergillus niger and Bacillus prodi- dulcitol 1.0%
giosvs. isodulcitol 1.0%
Reference: Kuntze (1907 p. 303). magnesium citrate 1.0%
(NH3)2C03 0.3%
318. Wherry's Glycerol Ammonium Acetate
levulose 1.0%
Solution
K2SO4 0.25%
Constituents: NaCl 0.12%
1. Redistilled water 1000.0 cc. glucose 1 .0%
2. NaCl 1.0 g. Reference: Proskauer and Beck (1894

3. KCl 1.0 g. pp. 144-148).
4. CaCls 1.0 g.
320. Uschinsky's Glycerol Ammonium Lac-
5. MgS04 1.0 g.
tate Solution
6. Ammonium acetate 5.0 g.
7. KH2PO4 2.0 g. Constituents:
8. Glycerin 50.0 g. 1. Water 1000.0 cc.
(Salts to be Kahlbaum's c.p.) 2. Glycerol 40.0 to 50.0 g.
Preparation 3. NaCl 5.0 to 7.0 g.
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. 4. CaClj 0.1 g.
(2) Adjustment of reaction not given. 5. MgSOi 0.2 g.

Sterilization: Sterilize in autoclave, time 6. K2HPO4 1.0 g.
not given. 7. Ammonium lactate . . 10.0 g.

Use: To study acid proof ness of B. tuber- Preparation: (1) Dissolve 2, 3, 4, 5, 6 and
culosis. 7 in 1.
Variants: The author substituted NaCOj Sterilization: Not specified.

forNaCl. Use: General synthetic culture medium.
Reference: Wherry (1913 p. 147). Variants: Gratia suggested the following
solution adjusted to pH = 7.4:
319. Proskauer and Beck's Basal Glycerol 1. Water 1000.0 g.
Ammonium Tartrate Solution
2. Glycerol 30.0 cc.
Constituents: 3. NaCl 5.0 g.
1. Water 1000.0 cc. 4. CaClj 0.1 g.
2. Glycerol (1.5%). 15.0 g. 5. MgS04 0.2 g.
3. KH2PO4 (0.5%). 5.0 g. 6. K2HPO4 2.0 g.
4. MgS04 (0.25%) . 2.5 g. 7. Ammonium lactate 12.0 g.
5. Ammonium tartrate (0.4%). 4.0 g. Reference: Uschinsky (1893 p. 316), Gratia

Preparation (1922 p. 295), Brieger and Cohn (1893
(1) Dissolve 2, 3, 4 and 5 in 1. p. 9).
(2) Add additional carbon compounds as

321. Fermi and Montesano's Glycerol
desired.
Ammonium Tartrate Solution
Use: To study the constituents essential Constituents
for the growth of the tubercle bacillus. 1. Distilled water 100.0 cc.
Added nutrients and modifications: The 2. Ammonium tartrate 0.5 g.
authors suggested the following modifi- 3. Potassium phosphate 0.5 g.
cations: 4. MgSOi 0.5 g.

5. Calcium phosphate 0.05 g. SUBGROUP I-C. SECTION 3

6. Glycerol 5.0 g.
Liquid media with constituents of known
Preparation
chemical composition. Nitrogen supplied
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
as nitrites; carbon organic.
(2) Distribute into flasks.
A I. Organic carbon supplied only as carbo-
hydrates.
Use: Cultivation of bacteria, yeast and
molds. A2. Organic carbon supplied as alcohols.
Reference: Fermi and Montesano (1895 Stutzer and Hartleb's Glycerol

Nitrite Solution 324
p. 547).
Beijerinck and van Delden's Alan-
322. Gottheil's Ammonium Tartrate nitol Nitrite Solution 325
Solution No. VI A3. Organic Carbon supplied only as
Constituents: organic acids.
1. Distilled water 1000.0 cc. Beijerinck and van Delden's Acetate
Nitrite Solution 326
3. CaClj 0.1 g. Beijerinck and van Delden's ]\Ialate
4. MgS04 0.3 g. Nitrite Solution 327
5. NaCl 0.1 g.
6. Ammonium tartrate 10.0 g. 324. Stutzer and Hartleb's Glycerol Nitrite
7. Glycerol 10.0 g. Solution
8. Sucrose 5.0 g.
Constituents:
9. Iron trace
1. Water 1000.0 cc.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7,
8 and 9 in 1.

3. NaCl 1.0 g.
Use: General culture medium. Author

4. MgS04 0.5 g.
used organisms from the soil, and bacteria

5. KCl 0.1 g.
6. Glycerol
found on roots and rhizomes.
7. NaNOa
Preparation
323. MacKensie's Ammonium Tartrate (1) Dissolve 2, 3, 4 and 5 in 1.
Solution (Smith) (2) Distribute in 200.0 cc. lots.
Constituents: (3) Prepare a 2.0% solution of sodium
nitrite.
2. Ammonium tartrate acid. . 1.5 g. (4) After sterilization of (2) and (3) add
3. KH2PO4 2.5 g. under 4 drops of glycerin
5.0 cc. of (3)
4. K2SO4 1.5 g.
to each 200.0 cc. lot of (3) under
5. NaCl 0.5 g. aseptic conditions.
6. Glucose 5.0 g. (5) Stopper with a double hole rubber

7. Lactose 5.0 g. stopper so air may be bubbled thru
8. Glycerol 15.0 g. the medium.
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. Use: To study oxidation of nitrites. The

(2) Adjust alkaline to phenolphthalein. author found that after 14 days neither
Sterilization: Not specified. nitrite nor nitrate test could be obtained.
Use: General culture medium. Nitrogen was in organic form. If more
Variants: Tanner used 2.5 g. acid ammo- NaNOo be added after 3 days nitrate was
nium phosphate and 1.5 g. KH2PO4 present. Air freed of CO2 was bubbled
instead of amounts given by Smith. thru the medium.
Reference: Smith (1905 p. 198), Tanner Reference: Stutzer and Hartleb (1897
(1919 p. 68). p. 311).
325. Beijerinck and van Delden's Mannitol Reference: Beijerinck and van Delden
Nitrite Solution (1902 p. 41).
Constituents
SUBGROUP 1-C. SECTION 4
1. Water 1000.0 cc.
2. K2HPO1 0.5 g. Liquid media or basal solutions of known
3. NaNOa 0.5 g. chemical composition. Nitrogen supplied
4. Mannitol 20.0 g. as nitrates; carbon organic.
Preparation: (1) Dissolve 2, 3 and 4 in 1. Ai. Incomplete or basal solutions, em-
Sterilization: Not specified. ployed with the addition of other nu-
Use: To study denitrification by B. sub- trients.
tilis, B. mesentericus and Azotobacter Stutzer's Basal Citrate Nitrate Solu-
chroococcum. The authors reported that tion 328
B. subtilis ammonia
did not produce Mortensen's Basal Nitrate Salt Solu-
while the other organisms gave ammonia. tion 329
Reference: Beijerinck and van Delden A2. Complete media.
(1902 p. 41). Bi.* Only one type of organic carbon
supplied.
326. Beijerinck and van Delden's Acetate
Ci.* Organic carbon supplied as carbo-
Nitrite Solution
hydrates.
Constituents: Di. Monosaccharides, only, added.
Ei. Containing inorganic salts of mono-
2. KCl 0.2 g.
valent cations only.
3. K2HPO4 0.2 g.
Stutzer's Glucose Nitrate Solution. 330 .
4. KNO2 10 g. Buhlert and Fickendey's Glucose

5. Sodium acetate 0.2 g. Nitrate Solution 331
6. Beijerinck and van Del- E2. Containing inorganic salts of mono and
den's basic salt solution. . 10 drops divalent cations.
(See Med. 25) Fi. Primarily for the cultivation of bac-
Preparation teria.
Beijerinck and Minkman's Glucose
(2) Add drop of Beijerinck and van
1 Nitrate Solution 332
Delden's Basic Salt Solution (see Maze's Glucose Nitrate Solution. 333 . . .
Med. 25) to each 100.0 cc. of 1. Fischer's Glucose Nitrate Solution

(3) Reaction to be slightly alkaline. (Miiller) 334
Sterilization: Not specified. Committee S. A. B. Glucose Nitrate
Use: Cultivation of Bacillus oligocarbo- Solution 335
philus. The organism grew as a dry thin Heraeus' Glucose Nitrate Solution.. 336
snow white scum. Migula's Glucose Nitrate Solution.. 337
Reference: Beijerinck and van Delden Wherry's Levulose Nitrate Solution. 338
(1903 p. 41). F2. Not primarily for cultivation of bac-
327. Beijerinck and van Delden's Malate teria.
Nitrite Solution Went's Glucose Nitrate Solution 339

Charpentier's Glucose Nitrate Solu-
Constituents: 340
tion
1. Water 1000.0 cc.
E.2 Inorganic salts of mono, di and tri-
2. K2HPO4 0.5 g.
valent cations added.
3. NaNOj 0.5 g.
Doryland's Glucose Nitrate Solution 341 .
4. Calcium malate 20.0 g.

D2. Disaccharides, only, added.
Bokorny's Sucrose Nitrate Solution. 342
Stoklasa and Vitek's Sucrose Ni-
Use: To study denitrification by B. sub- trate Solution 343
tilis, B. and Azotobacter
mesentericus
Czapek's Sucrose Nitrate Solution.. 344
The authors reported that
chroococcum.
NH3 was formed only by Azotobacter
chroococcum. * See page 93 for B2 and C2.
D3. Polysaccharides, only, added. 3. KNO3 3.0 g.

Gerlach and Vogel's Cellulose Nitrate 4. K2HPO4 1.0 g.
Solution 345 Preparation
Dubos' Cellulose Sodium Nitrate (1) Dissolve 2, 3 and 4 in 1.
Solution 346 (2) Add one of the organic nitrogen com-
Krainsky's Starch Nitrate Solution. 347 pounds given in added nutrients.
Lohnis' Cellulose Nitrate Solution. 348 . .
C2. Organic carbon supplied only as alco- (4) Distribute in 10.0 cc. lots.
hols. Sterilization: Method not given.
Di. Monoatomic alcohols, only, added. Use: To study denitrification by B. agilis,
Beijerinck and Minkman's Alcohol B. nitrovorus, B. Stutzeri, B. Hartlebi.
Nitrate Solution 349 Author found that nitrate reaction dis-
Bokorny's Alcohol Nitrate Solution. 350 appeared after 6 to 8 days. When pep-
D2. Polyatomic alcohols, only, added. tone or meat extract was added B. Stut-
Kuhne's Glycerol Nitrate Solution zeri and B. Hartlebi decomposed all the
(Proskauer and Beck) 351 nitrate in 3 days. B. nitrovorus and
Weissenberg's Glycerol Nitrate Solu- B. agilis after 6 to 7 days.
tion 352 Added nutrients: Author suggested the
Lantzsch's Glycerol Nitrate Solution. 353
addition of peptone or Schiilke and
Stoklasa's Mannitol Nitrate Solution. 354
Myers beef extract, or a mixture of the
C3. Organic C;arbon supplied as acids or
two. Amounts not specified.
their salts.
Variants: The author used the basic solu-
Doryland's Acetic Acid Nitrate Solu-
tion without additions.
tion 355
Reference: Stutzer (1901 p. 85).
Giltay and Aberson's Citrate Nitrate
Solution (Murray) 356
Percival's Tartrate Nitrate Solution. 357 329. Mortensen's Basal Nitrate Salt
B2. More than one type of organic carbon
Solution
supplied. Constituents:
Ci. Carbon added as carbohydrates and 1. Water 1000.0 cc.
alcohols.
2. KNO3 (0.5%) 5.0 g.
Gottheil's Nitrate Solution No. VII. 358 3. Cobalt salt
C2. Carbon added as carbohydrates and
Preparation
acids.
(1) Dissolve 2 and 10.0% of one of the
Burri and Stutzer's Nitrate Solution. 359
carbon sources listed under added
Kuntze's Nitrate Solution 360
nutrients in 1.
Giltay and Aberson's Nitrate Solu-
(2) Add cobalt salts in varying amounts
tion (Stoklasa and Vitek) 361
from 4 to 1/32%.
Giltay's Nitrate
Sucrose Solution
(Fred) 362
Use: To determine the toxic properties of
Behrens' Cellulose Nitrate Solution. 363
cobalt salts for Aspergillus niger. The
C3. Carbon added as alcohols and organic
author found the toxic properties of
acids.
cobalt were different in different media.
Maassen's Malic Acid Nitrate Solu-
A 0.5% cobalt chloride solution in a
tion (Stoklasa and Vitek) 364
liquid medium was as 1.0%
toxic as
Maassen's Stearate^Nitrate Solution
medium.
cobalt chloride in a gelatin
(Vierling) 365
of one of the following to the basic
328. Stutzer's Basal Citrate Nitrate
solution:
Solution
glucose
Constituents: sucrose
1. Water 1000.0 cc. glycerol
2. Potassium citrate 10.0 g. Reference: Mortensen (1909 p, 523).

330. Stutzer's Glucose Nitrate Solution Preparation

(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Constituents:
(2) Inoculate with soil and incubate in a
1. Water
closed flask.
2. Glucose
3. KNO3
Use: To study denitrification. The au-
Preparation
thors found that 53.0% CO 2, 32.0% H2
(1) Dissolve 2 and 3 in 1. (Amounts
and 15.0% N2 were formed if nitrate was
not specified.)
present. Without nitrate 50.0% of the
gas formed was CO2 and 50.0% H2.
Reference: Beijerinck and Minkman (1910
p. 35).
Use: To study denitrification by B. agilis,
B. nitrovorus, B. Slutzeri and B. Hart- 333. Maze's Glucose Nitrate Solution
lebi. Nitrate was not reduced.
Variants: Beijerinck and Minkman used a Constituents:
Distilled water 1000.0 cc.
2.0% glucose with 1.0% KNO3 solution 1.
to study denitrification. They found a 2. Potassium phosphate 1.0 g.
butyric acid fermentation took place 3. MgS04 0.5 g.
when inoculated with soil. 4. FeS04 0.02 g.
Reference: Stutzer (1901 p. 84), Beijerinck 5. NaCl 0.02 g.
and Minkman (1910 p. 35). 6. CaC03 1.0 g.

7. Glucose 20.0 g.
331. Buhlert and Fickendey's Glucose 8. NaNOa 5.0 g.
Nitrate Solution Preparation : (1) Dissolve 2, 3, 4, 5, 6, 7 and
Constituents: 8 in 1.
2. Glucose (1.5%) 15.0 g. Use: To study denitrification by Bacillus

3. NaNOa (0.3%) 3.0 g. denitrificans The author reported the
.
4. Potassium phosphate (0.1%). 1.0 g. formation of nitrous acid in 24 hours.

Preparation Variants Percival used the following solu-
:
tion to study denitrification by B.

mycoides:
1. Water 1000.0 g.
(3) Inoculate each tube with 5.0 cc. soil
water. 2. K2HPO4 50 g.
Sterilization: Not specified. 3. MgS04 2.5 g.
Use: To study denitrification. 4. NaCl 2.5 g.
Variants: 5. Glucose 2.0 g.
(a) Barthel specified the use of 0.1% 6. NaNOs 2.0 g.
K3PO4. References: Maze (1911 p. 370), Percival
and Harvey specified the use (1921 p. 112).

(b) Percival
tap water and usedO.1% K2HPO4.
of
334. Fischer's Glucose Nitrate Solution
References: Buhlert and Fickendey (1906 (Miiller)
p. 402), Barthel (1910 p. 118), Percival
Harvey (1921-22 p. 108).
Constituents:
(1920 p. 202),
1. Water 1000.0 cc.
332. Beijerinck and Minkman's Glucose 2. K0HPO4 (0.1%) 1.0 g.
Nitrate Solution 3. MgS04 (0.02%) 0.2 g.
Constituents: 4. CaCl2 (0.01%) 0.1 g.
1. Water 100.0 cc. 5. Dextrose (1.0%) 10.0 g.
2. Glucose 2.0 g. 6. KNO3 (amount not specified).
3. Chalk 2.0 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6

4. KNO3 1.0 g. in 1.
5. NH-Cl 0.05 g. Sterilization: Not specified.
6. K2HPO4 0.05 g. Use: To study denitrification by Bacleriuin

Giintheri, Bad. Hartlebi, Bac. pyo- add some more CaCOs. This removes
cyaneous and Bac. fluorescens. all traces of HXO3.
Variants: von Caron used the following (6) Steam in a weighted porcelain dish,
solution to study denitrification: until a solution is obtained contain-
1. Water 1000.0 cc. ing 1 part Ca(N03)2 to five parts
2. Glucose 1.0 g. water.
3. KNO3 0.1 or 3.0 g. (7) Filter and store.
4. MgS04 2.0 g. (8) Prepare solutions of dextrose and
5. K2HPO4 2.0 g. (NH4)2C03 the ratio of 1:15.
CaCla
6. 0.2 g. (9) Mix 25.0 cc. (4), 1.0 cc. of the dextrose
References : MuUer (1907 p. 472), von Caron solution, 2 drops of the (NH4)2C03
(1912 p. 96). solution and 2 drops of (7).
(10) Dilute the 250.0 cc. with distilled
335. Committee S. A. B. Glucose Nitrate
water. A liter of this solution con-
Solution
tains:
Constituents: potassium phosphate 0.1 g
1. Distilled water 1000.0 cc. MgS04 0.02 g
2. CaCIs 0.5 g. CaCl2 0.01 g
3. Nitrate (kind not given). . . 1.0 g. dextrose 0.8 g
4. Glucose 10.0 g. Ca(N03)2 0.8 g
5. K2HPO4 0.5 g. (11) Distribute in 50.0 cc. flasks that have
Preparation Dissolve 2, 3, 4 and 5 in 1.
: (1) previously been plugged and steri-
Sterilization: Method not given. lized.
Use: To study reduction of nitrates. Test Sterilization: Heat the solution and the
for nitrite with sulphanilic acid and a- steamer for 30 minutes.
flasks in the
naphthylamine and for ammonia produc- Use: Culture medium for soil, water and
tion by Thomas' method. air forms.
Variants: Committee S. A. B. specified the Reference: Heraeus (1896 p. 216).
useof l.Og. KNO3.
337. Migula's Glucose Nitrate Solution
Reference: Committee S. A. B. (1922
p. 525), (1923 p. 27). Constituents:
1. Water 1000.0 cc.
336. Heraeus' Glucose Nitrate Solution
2. KNO3 10.0 g.
Constituents 3. Magnesium phosphate 2.0 g.
1. Distilled water 1000.0 cc. 4. CaCl2 l.Og.
2. Potassium phosphate 0.1 g. 5. K2HPO4 l.Og.
3. MgS04 0.2 g. 6. Glucose 20.0 g.
4. CaCls 0.01 g. Preparation
5. Glucose 0.8 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
6. Ca(N03)2 0.8 g. (2) Tube.

7. (NH4)2C03 Sterilization: Boil in the steamer on two
Preparation successive days.
(1) Dissolve 1.0 g. potassium phosphate, Use: Differentiation of typhoid bacilli
0.2 g. MgS04 and 0.1 g. CaCl. in from B. coll.
1000.0 g. distilled water. Reference: Migula (1901 p. 20).
(2) Steam for 1 hour.
338. Wherry's Levulose Nitrate Solution
(3) Allow to stand.
(4) Filter and boil again. Constituents:
(5) Prepare a solution of Ca(N03)2 by 1. Redistilled water 1000.0 cc.
treating a weighed amount of CaC03 2. Na2C03 l.Og.
with dilute HNO3 the solutions being 3. KCl l.Og.
warm. Be sure that there is no free 4. CaCl2 l.Og.
HNO3 in solution. There should be 5. MgS04 l.Og.
some undissolved salt present. Then 6. KNO3 4.0 g.
7. KjHPO* 2.0 g. 341. Doryland's Glucose Nitrate Solution

8. Levulose 0.5 g.
Constituents:
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
2. HCl Dilute
3. MgS04 0.5 g.
Sterilization: Sterilize in autoclave,time
4. CaO 0.01 g.
not given.
5. Fe2(S04)3 0.01 g.
Use: To study acid proofness of B. tubercu-
6. MnS04 0.01 g.
losis. Luxuriant growth with K2HPO4,
7. HNO3 (amount not given)
but no growth without K2HPO4. Acid
8. H2SO4
fast properties of organism not given.
9. H3PO4
Reference: Wherry (1913 p. 151).
10. Glucose 5.0 g.
11. N/0.2578 NaOH
339. Went's Glucose Nitrate Solution 12. N/0.6205 KOH
Constituents Preparation
1. Water 1000.0 g. (1) Dilute HCl so that 1.0 cc. is not quite
2. Glucose (Commercial) 50.0 g. neutralized by 1.0 cc. of silicate solu-

3. KNO3 5.0 g. tion made by dissolving 24.0 g. K2Si03
4. KH2PO4 1.0 g. and 8.4 g. NajSiOs in 500.0 g. distilled
5. AIgS04 0.5 g. water. Phenolphthalein as indicator.

Preparation (2)Add to HCl the following salts.
(1) Dissolve 2, 3, 4 and 5 in 1. MgSOi. 0.5 g.
(2) Adjustment of reaction not specified. CaO 0.01 g.

Sterilization: Method not given. Fe2(S04)3 0.01 g.
Use: Cultivation of Monilia sitophila MnS04 0.01 g.
(Mont) Sacc. Organism seems to grow HNO3 (amount not specified).
best in damp atmosphere. Also grows (3) Standardize (2) against silicate solu-
well on rice, arachis seeds, bread, roots tion so that 1.0 cc. is equivalent to
of Daucus carota, milk, etc. 1.0 cc. using methyl orange as
Reference: Went (1901 p. 546). indicator.
same way as HCl omitting the salts.
340. Charpentier's Glucose Nitrate Solution
(5) Standardize H3PO4 in similar manner
Constituents as HCl omitting the salts and using
1. Water 1000.0 cc. phenolphthalein as indicator.
2. MgS04 1.0 g. (6)Mix the acids in the following ratio:
3. K2HPO4 2.0 g. HCl 153.5 cc.
4. KNO3 2.0 g. H2SO4 77.0 cc.
5. CaNOa 0.05 g. H,P04 116.0 cc.
6. FeS04 trace (7) Mix equal quantities of N/0.6205
7. Glucose 10.0 g. KOH and N/0.2578 NaOH.
Preparation: (8) 1.0 cc. of (7) should neutralize 1.0
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. cc. of (6) using phenolphthalein as
(2) Heat at 120C. indicator.
(3) Filter. (9) Draw acid and base solution in
Sterilization: Sterilize at 120C. separate burettes and allow to stand
Use: To determine nitrate reduction by several hours to sterilize.
Algae, Cystococcus humicola. The (10) Add enough sterile glucose solution
author reported that the total nitrogen to a mixture of equal parts of (6)
content is raised after 13 days incubation. and (7) to give 10 g. of glucose per
Variant: The author omitted the 2.0 g. liter solution.
KNO3 and added 1.0 g. of Ca(N03)2 in- Sterilization: Method not given.
stead of only 0.05 g. Use: A general synthetic medium.
Reference: Charpentier (1903 p. 327). Reference: Doryland (1916 pp. 146-148).
342. Bokorny's Sucrose Nitrate Solution 1.0 or 2.0 g. of NH4NO3 to 100.0 cc.
of solution.
Constituents:
Reference: Stoklasa and Vitek (1905
1. Water 1000.0 cc.
p. 198).
2. KNO3 (0.4%) 4.0 g.
3. Sucrose (10.0%) 100.0 g. 344. Czapek's Sucrose Nitrate Solution
4. Phosphoric acid (PaOs)
Constituents:
(0.13%) 1.3 g.
Preparation: (1) Dissolve 2, 3, 4 in 1.
2. NaNOs 2.0 g.
3. K2HPO4 1.0 g.
Use: Cultivation of molds.
4. KCl 0.5 g.
Variants: Bottger used 0.1% K2HPO4 in-
5. MgS04 0.5 g.
stead of phosphoric acid and used 0.2,
6. FeS04 0.01 g.
0.5 or 1.0% KNO3 to determine the
7. Sucrose 30.0 g.
toxicity of nitrates for yeast. He re-
Preparation
ported that nitrate presence had little
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
effect on loss of weight, measured in
(2) Place in 100.0 cc. portions in flasks.
grams.
Plugg.
Reference: Bokorny (1917 p. 316), Bottger
(3) Adjustment of reaction, not given.
(1921 p. 224).
Sterilization: Sterilize at 15 pounds for 15
minutes.
343. Stoklasa and Vitek's Sucrose Nitrate
Use: Cultivation of soil forms.
Solution
Variants: Tanner specified the use of 0.5 g.
Constituents: MgS04-7H20.
1. Water References: Waksman (1918 p. 479), Tan-
2. K2HPO4 1.25 g. ner (1919 p. 66).
3. K2SO4 0.2 g.
4. CaClj 0.05 g. 345. Gerlach and Vogel's Cellulose Nitrate
MgCl2 0.05 g. Solution
5.
6. NajCOa 0.1 g. Constituents
7. reP04 0.05N 1. Distilled water 1000.0 cc.
8. Saccharose 5.0 g. 2. NaNOs 5.0 g.
9. NaNOs 2.0 g. 3. Potassium phosphate 0.5 g.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1 liter 5. NaCl 0.5 g.

of water. 6. NaHCOs 0.5 g.
(2) Add 8 and 9 to 50.0 cc. of (1). 7. FeS04 some
(3) Make (2) up to 500.0 cc. 8. Straw 25.0 g.
Use: To study nitrate reduction by Clos- (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
tridium gelalinosum, Bad. Hartlebi. (2) Distribute in 200.0 cc. lots.
The authors reported that when more (3) To each flask add 5.0 g. straw.
saccharose was added more nitrate was (4) Adjustment of reaction not given.
left unchanged. Sterilization: Sterilize in the steamer.
Variants Use: Cultivation of albumin formers from
(a) The authors suggested the addition soil and stable manure. The author
of 2.0 g. of CaCOs with 5.0 or 17.1 g. found that if the medium was inoculated
sucrose and 2.0 or 4.25 g. NaNOa per with albumin formers alone all the
50.0 cc. solution. nitrate nitrogen was changed to albu-
(b) The authors suggested that 5.0 or minous nitrogen. When denitrifying bac-
17.1 g. sucrosebe added with 2.0 or teria were inoculated with the albumin
4.25 g. NaNOs per 50.0 cc. solution. formers some of the nitrogen was lost.
(c) The authors added 2.0 g. sucrose and Reference Gerlach and Vogel (1901
: p. 619).
346. Dubos' Cellulose Sodium Nitrate 3. KNO3 10.0 g.

Solution 4. K2HPO4 0.5 g.
Preparation
Constituents:
(2) Inoculate with soil.
2. NaNOa 0.5 g.
Incubate under anaerobic conditions.
(3)
3. K2HPO4 1.0 g.
4. MgS04 0.5 g.
Use: To denitrification by soil
study
5. KCl 0.5 g.
forms, and Bacillus pyocyaneus. The
6. FeS04 0.01 g.
author reported that the medium became
7. Filter paper
blue green due to Bacillus pyocyaneus.
Preparation
Variants: Tanner used 5.0 cc. of ethyl
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
alcohol and specified the use of tap water.
(2) Tube in 5 cc. quantities in tubes con-
References Beijerinck and Minkman
: (1910
taining a strip of filter paper. The
p. 35), Tanner (1919 p. 67).
paper is partially immersed in water.
Sterilization: Not specified. 350. Bokomy's Alcohol Nitrate Solution
Use: Isolation of aerobic bacteria decom-
Constituents:
posing cellulose.
1. Water
Reference: Dubos (1927 p. 52).
2. Methyl alcohol
3. KH2PO4
347. Krainsky's Starch Nitrate Solution 4. MgS04
5. Ca(N03)2
Constituents:
Preparation
1. Water 1000.0 cc.
(1) Prepare a mineral solution by dissolv-
2. Starch (1.0%) 10.0 g.
KNO3 3.0 g.
0.05% KH2PO4, 0.025% bitter
ing
3. (0.3%)
and 0.04% Ca(N03)2 in water.
salts
4. K2HPO4 (0.05%) 0.5 g.
(2) Prepare solutions with 0.1% (1) and

1.0% methyl alcohol.
Use: Enrichment of Actinomycetes coeli-
Use: Cultivation of fungus utilizing methyl
color, Actinomycetes diastaticus.
alcohol as carbon source.
Reference: Krainsky (1914 p. 658).
Variants: The author used 10.0, 5.0, 2.0,
0.5, 0.005 or 0.0025% methyl alcohol
348. Lohnis' Cellulose Nitrate Solution
instead of 1.0%.
Constituents: Reference: Bokorny (1911 p. 178).

351. Kiihne's Glycerol Nitrate Solution
2. Cellulose 20.0 g.
(Proskauer and Beck)
3. KNO3 2.5 g.
Constituents:
4. K2HPO4 0.5 g.
and 1. Water 1000.0 cc.
Preparation: (1) Dissolve 3, 4 20.0 g.
2. Ca(N03)2 2.0 g.
of cellulose (filter paper or cotton wool)
in
3. KCl 0.5 g.
1.

4. MgS04 0.5 g.
Use: To study denitrification.

5. KH2PO4 0.5 g.
6. Glycerol 30.0 g.
Reference: Lohnis (1913 p. HI).
in 1.
349. Beijerinck and Minkman's Alcohol
Nitrate Solution
Use: Cultivation of tubercle bacilli.
Constituents Growth was very scant.
1. Water 1000.0 cc. Reference: Proskauer and Beck (1894
2. Ethyl alcohol. 5.0 g. p. 131).
352. Weissenberg's Glycerol Nitrate Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 8

Solution in 1.
Constituents
Use: To study action of Azotobacter and
Radiobacter on nitrates. Author found
2. Sodium phosphate (neu-
that nitrates were reduced.
tral) 2.5 g.
3. Glycerol 40.0 g.
4. NaCl 5.0 g.
5. NH4NO3 3.75 g. 355. Doryland's Acetic Acid Nitrate Solution
Preparation
Constituents:
(2) Place 0.75 g. NH4NO3 in 175.0 cc.
Erlenmeyer flasks.
2. HCl dilute
Fill each flask containing NH4NO3 3. MgS04 0.5 g.
(3)
with
4. CaO 0.01 g.
(1).
5. re2(S04)3 0.01 g.
Sterilization: Sterilize carefully (method
not given).
6. MnS04 0.01 g.
Use: To study denitrification by B. pyo-

7. HNO3 (amount not given)
8. H2SO4
cyaneus. The author found that ammo-
nia was produced.
9. CH3COOH
10. H3PO4
Reference: Weissenberg (1897 p. 284).
11. N/0.2578 NaOH
12. N/0.6205 KOH
353. Lantzsch's Glycerol Nitrate Solution
Preparation
Constituents: (1) Dilute the HCl so that 1.0 cc. is not
1. Water 1000.0 cc. quite neutralized by 1.0 cc. silicate
2. KNO3 0.5 to 1.5 g. solution. Prepared by dissolving
3. Glycerin 20.0 g. 24.0 g. KjSiOa and Na2Si03 in
8.4 g.
4. Malic acid 7.0 g 500.0 cc. distilled water (phenol-
5. NaoHPOi 0.5 g phthalein).
6. NaCl 0.5 g (2) Add the following salts to the HCl:
7. NasCOa 0.5 g MgS04 0.5 g.
8. MgS04 0.1 g CaO 0.01 g.
Preparation: (1) Dissolve 2, 3, 4 (neutralize Fe2(S04)3 0.01 g.
acid with soda) 5, 6, 7 and 8 in 1. MnS04 0.01 g.
Sterilization: Not specified. HNO3 (amount not given)
Use: To determine nitrate reduction by (3) Standardize (2) so 1.0 cc. equivalent
fluorescens group. Acidify the culture to 1.0 cc. silicate solution, using
with 5.0% H2SO4 and add a strong zinc methyl orange as indicator.
iodide solution. A blue color indicates (4) Standardize a solution of H2SO4 in
nitrate formation or presence. Test for the same way as HCl omitting the
NH3 with Nessler's reagent. salts.
Reference: Lantzsch (1921 p. 82). (5) Standardize H3PO4 and CH3COOH
solution in the same way as HCl
354. Stoklasa's Mannitol Nitrate Solution
omitting the salts and using phenol-
Constituents: phthalein as indicator.
1. Distilled water 1000.0 cc. (6) Mix the acids in the following ratio:
2. Mannite 20.0 g HCl 153.5 cc.
3. NaNOa 2.0 g CH3COOH 153.5 cc.
4. K2HPO4 0.5 g H2SO4 77.0 cc.
5. K2SO4 0.2 g H3PO4 116.0 cc.
6. CaCl2 0.05 g (7) Mi.x equal quantities of N/0.6205
7. MgCl. 0.05 g KOH and N/0.2578 NaOH solutions.
8. FePOi 0.05 g (8) 1.0 cc. of (7) should neutralize 1.0 cc.
(6) using phenolphthalein as indi- Sterilization: Method not specified.

cator. Use: To study denitrification.
(9) Acid and base solutions are to be Reference: Fercival (1920 p. 167).
drawn into separately plugged bu-
rettes and allowed to stand several 358. Gottheil's Nitrate Solution No. VII
hours to sterilize.
Constituents:
(10) Mix equal quantities of (6) and (7)
when ready for use.
Use: As a general synthetic medium. 3. CaCla 0.1 g.
Reference: Doryland (1916 pp. 146-148). 4. MgS04 0.3 g.

5. NaCl 0.1 g.
6. Iron trace
356. Giltay and Aberson's Citrate Nitrate
Solution (Murray)
7. KNO3 10.0 g.
8. Sucrose 5.0 g.
Constituents 9. Glycerol 10.0 g.
1. Water 1000.0 cc. Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8
2. KNO3 2.0 g. and 9 in 1.
3. MgSO^ 2.0 g. Sterilization: Not specified.
4. Citric acid 5.0 g. Use: General culture medium for organ-
5. K2HPO4 2.0 g. isms found in the soil, on roots and on
6. CaClz 0.20 g. rhizomes.
7. NasCOa 4.25 g. Reference: Gottheil (1901 p. 432).
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
in 1.
359. Burri and Stutzer's Nitrate Solution
Constituents:
Use: To study denitrification under aerobic
and anaerobic conditions. 1. Water 1000.0 cc.
Variants 2. KNO3 2.0 g.
(a) Fred used 0.2 g. K2HPO4 instead of

3. Glucose 2.0 g.
2.0 g. K2HPO4. 4. MgS04 2.0 g!
(b) Fred used 2.0 g. NaNOs instead of

5. Citric acid 50 g.
2.0 g. KNO3. 6. Potassium phosphate 2.0 g.
Christensen omitted the Na2C03, 7. CaCl2 0.2 g.

(c)
used 3.0 g. of NaNOs instead of 8. FeCl, 2 drops
Preparation
KNO3, used 0.5 g. sodium citrate
(1) Dissolve 2 and 3 in part of 1.
instead of citric acid and added a
trace of iron chloride.
(d) Harvey and Fercival omitted the mainder of 1.
(3) Boil (2) over a flame and neutralize

Na2C03, added a trace of FeCls and
used 2.0 of Na2N03 instead of with soda.
g.
2.0 g. KNO3. (4) Mix (1) and (3) and make up to 1 liter.
Sterilization: Sterilize in the steamer.
References: Murray (1916 p. 611), Fred
(1911-12 p. 422), Christensen (1913 p. 421), Use: To study nitrate reduction by B.
denitrificans
Harvey (1921-22 p. 108), Fercival (1920
Variants
p. 168).
(a) Jensen used 2.0 g. NaNOj instead of
357. Percival's Tartrate Nitrate Solution
2.0 g. KNO3, specified
the use of
K2HFO4 and omitted the FeCls.
Constituents (b) V. Bazarewski used either 2.0 g. of
1. Water 1000.0 cc. NaNO, or KNO3 and specified the
2. Calcium tartrate 20.0 g. use of KH2FO4.
3. KNO3 20.0 g. Reference: Burri and Stutzer (1895 p. 393),
4. K2HPO4 0.5 g. Jensen (1897 p. 623), v. Bazarewski
Preparation: (1) Dissolve 2, 3 and 4 in 1. (1905 p. 4).
360. Kuntze's Nitrate Solution Use: To study nitrate reduction.

Constituents:
p. 104).
2. Salt peter 2.0 g. 362. Giltay's Sucrose Nitrate Solution
3. Glucose 2.0 g. (Fred)
4. MgS04 2.0 g.
Constituents:
5. Citric acid 5.0 g.
6. Potassium sulphate 2.0 g.
2. NaNOa 2.0 g
7. CaCl2 0.2 g.
3. MgS04 2.0 g
8. Iron chloride 2 drops
4. K2HPO4 2.0 g
Preparation
5. CaCla 0.2 g
6. Citric acid 5.0 g
7. NaoCOa 4.25 g
mainder of 1.
8. Sucrose 2.0 g
(3) During the heating of (2) neutralize
Preparation: (1) Dissolve 2, 3, 4, 5, 6,
with soda.
7 and 8 in 1.
(4) Mi.x (3) and (1).
C5) Distribute into 100.0 cc. lots in com-
Use: To study nitrate reduction by B.
bustion flasks.
fluorescens liquefaciens, B. pyocyaneus,
(6) Inoculate with 5.0 g. manure.
B. Hartlebi (H. Jensen), B. denitrificans
Reference: Fred (1911-12 p. 422).
Use: To study morphology and physiology
of denitrifying organisms, Bacillus deni- 363. Behren's Cellulose Nitrate Solution
trificnns agilis (Ampola and Garino) and
Constituents:
Bacillus oxalaticus (Zopf).
1. Water 1000.0 cc.
Variants: Giltner gives the following solu-
2. NH4NO3 10.0 g
tion:
3. Sucrose 2.0 g
1. Distilled water 1000.0 g.
4. Potassium phosphate 5.0 g
2. KH2PO4 2.0 g.
5. MgS04 1.0 g
3. MgSOi 2.0 g.
6. Lactic acid 2.0 g
4. KNO3 1.0 g.
7. Swedish filter paper
5. CaCh 0.2 g.
Preparation
6. FeoCU solution 2.0 drops
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Filter.
8. Glucose 10.0 g.
References: Kuntze (1904 p. 2), Giltner
(4) Add pieces of purified Swedish filter
(1921 p. 376).
paper to each flask.
361. Giltay and Aberson's Nitrate Solution Sterilization: Not specified.

(Stoklasa and Vitek) Use: To study utilization of cellulose by
Constituents Botrylis vulgaris, Pencillium glaucum
1. Water 1000.0 cc. and Penicillium luteum. The author

2. NaNOi 2.0 g. reported that Botrylis vulgaris dissolved
3. K2HPO4 2.0 g. the cellulose.
4. CaCU 0.2 g. Reference: Behrens (1898 p. 549).
5. Glucose 2.0 g.
364. Maassen's Malic Acid Nitrate Solution
(Stoklasa and Vitek)
7. Iron chloride few drops
(1) Dissolve 2, 3, 4 and 5 in 1. 1. Water 1000.0 cc.
(2) Neutralize 5.0 g. citric acid with soda. 2. NaCl 0.5 g.
(3) Add (2) and several drops of iron 3. Na.COa 0.5 g.

chloride to (1). 4. MgS04 0.1 g.
Sterilization: Not specified. 5. Malic acid 7.0 g.
6. Glycerol 20.0 g. Went's Basal Leucine Solution 372

7. NaNOa 2.5 g. Cb. Amino nitrogen supplied as aspartic
Preparation acid or asparagin.
(1) Dissolve 2, 3 and 4 in 1. Di. Containing no other added organic
(2) Neutralize 7.0 g. malic acid with carbon.
NajCOa. van Delden's Basal Asparagin Salt
(3) Add (2), 6 and 7 to (1). Solution 373
Sterilization: Not specified. Uschinsky's Basal Asparagin Solution
Use: To study nitrate reduction. (Kendall, Day and Walker) 374
Reference: Stoklasa and Vitek (1905 Kita's Basal Asparagin Solution 375
p. 104). Proskauer and Beck's Basal Aspara-
gin Solution 376
365. Maassen's Stearate Nitrate Solution
Hefferan's Basal Asparagin Solution. 377
(Vierling)
Zikes' Basal Asparagin Solution 378
Constituents: Miehe's Basal Asparagin Solution. 379 . .
1. Water 1000.0 cc. Gottheil's Basal Asparagin Solution. 380

2. Glycerin 20.0 g. Went's Basal Asparagin Solution 381
3. KNO, 2.5 g. Sullivan's Basal Asparagin Solution. 382
4. Potassium stearate 1-0 g. D2. Containing additional organic carbon.
5. MgS04 1.0 g. El. Only one type of organic carbon added.
6. K2HPO4 0.5 g. Fi. Carbohydrates, only, added.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 Munter's Basal Glucose Asparagin
in 1. Solution 383
Sterilization: Not specified. Ono's Basal Sucrose Asparagin Solu-
Use: To study the reduction of nitrate by tion 384
Mycobacteria. Author reported that Bokorny's Basal Asparagin Solution. 385
little or no ammonia was produced. Higgins' Basal Carbohydrate Aspara-
Reference: Vierling (1920 p. 202). gin Solution 386
F2. Alcohols only added.
SUBGROUP I-C. SECTION 5
Peklo's Basal Asparagin Solution... 387
Liquid media or basal solutions with con- Sullivan's Basal Glycerol Asparagin
stituents ofknown chemical composition; Solution 388
organic nitrogen present as amino acids; Waksman's Basal Glycerol Asparagin
other forms of organic nitrogen may also Solution 389
be present. F3. Acids or their salts, only, added.
Ai.* Only one amino acid present. Tanner's Basal Lactate Asparagin
Bi.t Incomplete or basal solutions, used Solution 390
with the addition of a variety of other Hiss' Basal Litmus Asparagin Solu-
nutrients. tion 391
Ci. Amino nitrogen supplied as glycocoll. Harvey's Basal Lactate Asparagin
Went's Basal Glycocoll Solution 366 Solution 392
Wherry's Basal Glycocoll Solution. . . 367 Capaldi and Proskauer' s Basal Ci-
Waksman's Basal Glycerol Glycocoll trate Asparagin Solution 393
Solution 368 Mendel's Basal Citrate Asparagin
C2. Amino nitrogen supplied as alanine. Solution 394
Went's Basal Alanin Solution 369 Maassen's Basal Malic Acid Aspara-
C3. Amino nitrogen supplied as tyrosine. gin Solution (Smith) 395
Went's Basal Tyrosine Solution 370 E2. More than one type of organic carbon
C4. Amino nitrogen supplied as leucine. added.
Waksman's Basal Glycerol Leucine Lockemann's Basal Citrate Glycerol
Solution 371 Solution 396
Mendel's Basal Citrate Glycerol
'
See page 104 for A2. Solution 397
See bottom of next column for B2. B2. Complete media.
Ci.* Organic nitrogen supplied only as Henneberg's Asparagin Salt Solution 419 .
amino acids or their salts. Arnaud and Charrin's Asparagin Salt

Di. Amino nitrogen supplied as glycocoU. Solution (Roux and Rochaix) 420
Beijerinck and Minkman's Nitrate E2. Containing additional organic carbon.
GlycocoU Solution 398 Fi.* Only one type of organic carbon added.
Bokorny's Sucrose GlycocoU Solu- Gi. Additional carbon supplied as carbo-
tion 399 hydrates.
Hadley's Glycerol GlycocoU Solution. 400 Hi. Monosaccharides added.
Di. Amino nitrogen supplied as alanine. 1 1. Containing salts of monovalent cations
Beijerinck and Minkman's Nitrate only.
Alanine Solution 401 Saltet's Glucose Asparagin Solution. 421
D3. Amino nitrogen supplied as tyrosine. Saltet's Thiosulphate Asparagin
Berthelot's Tyrosine Salt Solution.. 402 Solution 422
Vansteenberge's Glucose Tyrosine Blanchetiere's Glucose Asparagin
Solution 403 Solution 423
Bokorny's Sucrose Tyrosine Solution. 404 12. Containing salts of monovalent and
Waksman's Glycerol Tyrosine Solu- other cations.
tion 405 Ji. Calcium salts added.
D^. Amino nitrogen supplied as cystine. Capaldi and Proskauer's Glucose As-
Myers' Cystine Solution 406 paragin Solution 424
Heap and Cadness' Cystine Solution. 407 Gottheil's Glucose Asparagin Solu-
D5. Amino nitrogen supplied as trypto- tion No. X 425
phane. J2. Calcium salts not added.
Rogers, Clark and Evans' Trypto- Capaldi and Proskauer's Glucose
phane Solution 408 Asparagin Solution No. II 426
Frieber's Glucose Tryptophane Solu- Stutzer's Glucose Asparagin Solution. 427
tion 409 Saltet's Magnesium Sulphate Aspara-
De. Amino nitrogen supplied as leucine. gin Solution 428
Bokorny's Sucrose Leucine Solution. 410 Samkow's Glucose Asparagin Solu-
D7. Amino nitrogen supplied as asparagin tion 429
or aspartic acid. Sullivan's Glucose Asparagin Solu-
Ei.f Containing no other organic carbon. tion 430
Fi. Containing only salts of monovalent Boehncke's Glucose Asparagin Solu-
cations. tion 431
Gi. Ammonium salts added. H2. Disaccharides added.
Dolt's Ammonium Asparagin Solu- Zikes' Sucrose Asparagin Solution.. 432
tion No. 1 411 Korf's Sucrose Asparagin Solution.. 433
Kendall, Walker and Day's Ammo- Zikes' Magnesium Sucrose Asparagin
nium Asparagin Solution 412 Solution 434
G2. Ammonium salts not added. Chrzaszcz's Sucrose Asparagin Solu-
Dolt's Asparagin Solution No. 2. 413
. . . tion 435
Beijerinck and Minkman's Nitrate Gj. Additional carbon supplied as alcohols.
Asparagin Solution 414 Sullivan's Glycerol Asparagin Solu-
Blanchetiere's Asparagin Salt Solu- tion 436
tion 415 Schweinitz and Dorset's Glycerol
Park, Williams and Krumwiede's Asparagin Solution (Goris) 437
Asparagin Salt Solution 416 Waksman and Joffe's Glycerol As-
Fj. Containing salts of monovalent and paragin Solution 438
other cations. Hadley's Glycerol Asparagin Solution
Bokorny's Asparagin Salt Solution. 417 . Nos. Ill and IV 439
Uschinsky's Asparagin Salt Solution Peklo's Mannitol Asparagin Solution. 440
(Giltner) 418 Capaldi and Proskauer's Mannitol
Asparagin Solution 1 441
*See next page for Cj.
tSee next column for E2. *See next page for F2
Gg. Additional carbon supplied as organic Clark and Lubs' Solution 461
acids or their salts. Remy and Sugg's Citrate Asparagin
Hi. Acetic acid or its salts added. Solution (Proskauer and Beck).. 462 . .
Ellrodt's Acetate Asparagin Solu- G3. Alcohols and organic acids added.
tion 441a (Carbohydrates may or may not be
H2. Lactic acid or its salts added. present.)
Ii. Ammonium salts added. Uschinsky's Glycerol Asparaginate
Voges' Lactate Asparaginate Solu- Solution 463
tion 441b Sullivan's Glycerol Nitrate Aspara-
Frankel and Voges' Salt Asparagin gin Solution 464
Solution (Besson) 441c Hadley's Glycerol Asparagin Solu-
Frankel' s Lactate Asparagin Solu- tion No. V 564
tion (Tanner) 442 Lowenstein's Glycerol Asparagin
Uschinsky's Lactate Asparaginate Solution 466
Solution (Smith) 443 Lockemann's Citrate Glycerol As-
Melick's Lactate Asparagin Solution. 444 paragin Solution (Lowenstein) .... 467
van Delden's Lactate Ammonia As- Frouin's Glycerol Asparagin Solu-
paragin Solution 445 tion (Bezancon) 468
I2. Ammonium salts not added. C2. Organic nitrogen present in addition to
Beijerinck's Lactate Asparagin Solu- amino nitrogen.
tion 446 Di. Amino nitrogen present as glycocoll.
van Delden's Lactate Asparagin Solu- Hadley's Urea Glycocoll Solution... 469
tion 447 D2. Amino nitrogen present as aspartic
Schroeder and Junger's Lactate As- acid or asparagin.
paragin Solution 448 Beijerinck's Urea Asparagin Solu-
Remy's Organic Acid Asparagin tion 470
Solution 449 Beijerinck's Urea Ammonia Car-
H3. Citric acid or its salts added. bonate Solution 471
Kuntze's Citrate Asparagin Solution. 450 Sears' Uric Acid Asparagin Solution. 472
Giltay and Aberson's Citrate Aspara- Kappen's Cyanamide Asparagin Solu-
gin Solution (Tanner) 451 tion 473
Maassen's Basal Citrate Asparagin A2. More than one amino acid present.
Solution (Lohnis) 452 Bi. Incomplete or basal solutions employed
H4. Malic acid or
its salts added. with the addition of other materials.
Maassen's Malate Asparagin Solu- Davis and Ferry's Basal Cystine
tion 453 Tryptophane Solution 474
Beijerinck's Malate Asparagin Solu- B2. Complete media.
tion 454 Ci. Containing glycocoll.
F2. More than one type of additional Berthelot's Amino Acid Solution 475
organic carbon employed. Armand-Delille, et al., Glycocoll
Gi. Carbohydrates and alcohols, only, Arginine Solution (Synthetic Me-
added. dium 164) 476
Zikes' Alcohol Asparagin Solution . . 455 Armand-Delille, et al., Synthetic
Gosio's Glycerol Aspartic Acid Solu- Solution 104 477
tion 456 Armand-Delille, et al., Glycocoll As-
Gartner's Glycerol Asparagin Solu- partic Acid Solution (Synthetic
tion 457 Medium 118) 478
Borel de coulon, Boez and Quimaud's C2. Not containing glycocoll.
Glycerol Asparagin Solution 458 Zipfel's Tryptophane Asparagin Solu-
G2. Carbohydrates and organic acids, only, tion 479
added.
366. Went's Basal Glycocoll Solution
Muller, Thurgan and Osterwalder's
Malate Asparagin Solution 459 Constituents:
Maassen's Glucose Asparagin Solu- 1. Water 1000.0 cc.
tion 460 2. Glycocoll (0.66%) 6.6 g.
Preparation
(1) Prepare 5.0% solutions of one of the
materials listed in added nutrients.
(2) Flask in 25.0 cc. quantities.
(3) Prepare a 0.66% solution of glycocoll.
(4) Add 25.0 cc. of (3) to each flask of (2).
Use: To study nutrients for Monilia sito-
phila (Mont.) Sacc. The materials
ranked as follows as suitable additional
carbon sources: maltose, glucose, lactose,
sucrose and glycerol.
Added nutrients: The author used the
following additional carbon sources:
maltose glucose
lactose glycerol
sucrose
tion without additional materials.
Reference: Went (1901 p. 593).
367 Wherry's Basal Glycocoll Solution
Constituents:
2. NaCl
3. KCl
4. CaClj
5. MgSOi
6. Glycocoll
7. (NH4)2HP04
370. Went's Basal Tyrosine Solution Preparation

Constituents (1) Prepare 5.0% solutions of one of the
1. Water 1000.0 cc.
materials listed in added nutrients.
2. Tyrosine (0.66%) 6.6 g. (2) Flask in 25.0 cc. quantities.
Preparation (3) Prepare a 0.66% solution of leucine.
(1) Prepare 5.0% solutions of one of the (4) Add 25.0
cc. of (3) to each flask of (2).
materials listed in added nutrients. Sterilization: Method not given.
Flask in 25.0 cc. quantities. Use: To study the nutrients for Monilia
(2)
(3) Prepare a 0.66% tyrosine solution. sitophila (Mont.) Sacc. The materials
ranked as follows as suitable carbon
(4) Add 25.0 cc. of (3) to each flask of (2).
Sterilization: Method not given. sources with leucine as nitrogen source:
Use: To study nutrients for Monilia sito- glucose and maltose, sucrose, glycerol,
lactose.
phila (Mont.) Sacc. The materials
ranked as follows as suitable carbon Added nutrients: The author used the
sources with tyrosine as a nitrogen source following additional carbon sources:
maltose, glucose, sucrose, lactose, glyc- maltose glucose
erol. lactose glycerol
Added nutrients: The author used the sucrose
following additional carbon sources: Variants: The author used the basic solu-
maltose glucose tion without additional materials.
lactose glycerol Reference: Went (1901 p. 593).
sucrose
Variants: The author used the basic solu- 373. van Delden's Basal Asparagin Salt
tion without additional materials. Solution
Constituents
371. Waksman's Basal Glycerol Leucine 1. Water, tap 1000.0 cc,
Solution 2. K2HPO4 0.5 g.
Constituents: 3. Asparagin 5.0 g.
2. Glycerol 30.0 g. (1) Dissolve 2 and 3 in 1.
3. K2HPO4 1.0 g. (2) Additional nutrients are added as
4. KCl 0.5 g. indicated below.
5. MgS04 0.5 g. Sterilization: Not specified.
6. reS04 0.01 g. Use: To study sulphate reduction by
7. Leucine 5.0 g. Microspira desidfuricans.
Preparation Added nutrients and variants The author :
(1) Dissolve 2, 3, 4, 5, 6, 7 and 2.0 g. of employed the following solutions:

one of the added nutrients in 1. (a) Added 2.0 g. MgS04-7H20.
(2) Tube in 10-12 cc. lots. (b) 5.0 g. potassium succinate, 5.0 g.
(3) Adjustment of reaction not specified. gypsum and 1.0 g. of asparagin.

Sterilization: Sterilize at 15 pounds for 15 (c) 5.0 g. potassium citrate, 5.0 g. gyp-
minutes. sum and 1.0 g. of asparagin.
Use: To study metabolism of actinomy- (d) Specified the use of sea water in the
cetes. preparation of the basic solution,
Added nutrients: The author added 2.0 g. used 1.0 g. of asparagin and added
of one of the following: 5.0 g. of sodium lactate or 2.0 g.
NaXOs (NH 4)280 4 glucose.
NaNOs (NH4)2C03 (e) 1.0 g. asparagin in the basic solution
Reference: Waksman (1920 p. 3). and added 30.0 g. NaCl, and 8.0 g.
MgS04-7H20. To this solution he
372. Went's Basal Leucine Solution added 0.0, 5.0 or 10.0 g. of sodium
Constituents lactate.
1. Water 1000.0 cc. (f) Added 30.0 g. NaCl and 12.0 g. of
2. Leucine (0.66%) 6.6 g. gypsum to the basic solution.
(g) Added 10.0 g. glucose, 30.0 g. NaCl References: Kendall, Day and Walker
and 8.0 g. MgSOi-THaO to the basic (1914 p. 428), Kendall, Walker and Day
solution. (1914 p. 355).
(h) Used 2.5 g. asparagin in the basic
375. Kita's Basal Asparagin Solution
solution and added 30.0 g. NaCl,
8.0 g. MgS04-7H20 and 5.0 g. sodium Constituents:
lactate. 1. Water 1000.0 cc.
(i) Used 1.0 g. asparagin in the basic 2. MgS04 2.5 g.
solution, added 8.0 g. MgS04-7H20, 3. KH2PO4 5.0 g.
30.0 g. NaCl and 5.0 ^g. of sodium 4. FeCh solution drops
lactate or potassium succinate. 5. Asparagin 5.0 g.
(j) Used 1.0 g. of asparagin in the basic Preparation
solution and added 5.0 g. sodium (1) Dissolve 2, 3, 4 and 5 in 1.
lactate, 75.0 g. NaCl and 8.0 g. (2) Add 5.0% of any carbohydrate.
MgS04-7H20. Sterilization: Not specified.
Reference: van Delden (1903-04 p. 85, 105). Use: Cultivation of Japanese molds, Asper-
gillus Okazaki, Aspergillus candidus,
374. Uschinsky's Basal Asparagin Solution
Aspergillus albus, Aspergillus tamarii,
(Kendall, Day and Walker) Pseudorhizopus, Aspergillus glaucum.
Constituents: Added nutrients: Add 5.0% of any carbo-
1. Distilled water 1000.0 cc. hydrate.
2. Asparagin 4.0 g. Variants
3. NasHPOi 2.0 g. (a) Buromsky used the following solution
4. NaCl 5.0 g. to study effect of organic acids on
Preparation yeast, Saccharomyces ellipsoid, Sac-
(1) Dissolve 2, 3, 4 and one of the ma 4- charomyces pastorianus.
terials listed under added nutrients 1. Water 1000.0 g.
in 1. 2. MgSOi 0.5 g.
(2) Distribute in 100.0 cc. lots. 3. KH2PO4 1.0 g.
(3) Adjustment of reaction not given. 4. Asparagin 5.0 g.
Sterilization: Method not given. The solution was prepared by
Use: To study metabolism by the tubercle (1) Dissolve 2, 3 and 4 in 1.
bacilli. Authors used alizarin, neutral (2) Distribute in 100.0 cc. lots in 500.0
red and phenolphthalein to study changes cc. Erlenmeyer flasks.
in reaction; Ziehl-Neelsen stain for (3) Add one of the listed materials
staining. below as additional carbon source.
Added nutrients: The following organic (4) Sterilize on 3 successive days in a
materials were added: Koch steamer, 30 minutes the first
glucose 10.0 g. day, and 20 minutes on each the
mannitol 10.0 g. 2nd and 3rd day, (a slight turbidity
glycerol 30.0 g. develops when glycerin or mannite
The following materials were added to is added).
study lipase production: different es- The following nutrients were added:
ters were incubated for 24 hours with quinic acid 0.5%
clear bacteria free culture broth, and citric acid 1-0%
amount of acid produced, measured in tartaric acid 1-0%
terms of n/50 NaOH, determined lipase succinic acid 1-0%
production, glycerol 1.0%
ethyl alcohol and (NH4)2HP04 amounts mannitol 1 .0%
not given, (b) von Bronsart used 0.25% KH2PO4

glycerol and (NH4)2HP04 amounts not and 5.0% asparagin instead of 0.1%
given, KH2PO4 and 0.5% asparagin. He
glucose and (NH4)2HP04 amounts not added one of the following carbon
given. sources (amount not specified).
starch levulose (g) Glycerol 1.0%

dextrin glucose citric acid 0.1%
sucrose alanin 0.1%
He used the solution for the cultivation (h) citric acid 1-5%
of Xylaria, Xylaria hypoxylon. One of the following:
References: Kita (1913 p. 434), Buromsky alanin 0.1%
(1914 p. 532), von Bronsart (1919 p. 59). fumaric acid 0.075%
aconitic acid 0.075%
376. Proskauer and Beck's Basal Asparagin citraconic acid 0.075%
Solution itaconic acid 0.075%
Constituents: (i) citric acid 1.0%
1. Water 1000.0 cc.
glyceric acid 1-0%
or
2. Potassium phosphate
Biuret 0.075%
(0.5%) 5.0g.
Variants
3. MgSO4(0.25%) 2.5 g.
4. Asparagin (0.5%) 5.0 g.
(a) The authors used 0.3% potassium
Preparation phosphate and added 0.25% mag-
Dissolve 3 and 4 in 1.
nesium citrate and 1.5% citric acid.
(1) 2,
Add one of the combinations listed

(b) The authors used 0.06% MgS04 and
(2)
under added nutrients. added 0.25% magnesium citrate and
1.5% citric acid.
Use: To study the constituents essential (c) The authors used 0.1, 0.2, 0.3, 0.4,
0.5 or 0.6% asparagin without addi-
for the growth of tubercle bacilli.
tions.
Added nutrients The authors added one of
:
Reference: Proskauer and Beck (1894

the following combinations:
p. 139).
(a) citric acid 0.075%
glycerol 4.0%
glucose
377. Hefferan's Basal Asparagin Solution
1.0%
K2SO4 0.25% Constituents
NaCl 0.15% 1. Redistilled water 1000.0 cc.
(b) citric acid 0.75% 2. Asparagin 2.0 g.
glycerol 4.0% Preparation
K2SO4 0.25% (1) Dissolve 2 in redistilled water.
NaCl 0.15% (2) Reaction is neutral to phenol-
(c) Glycerol 4.0% phthalein.
glucose 1.0 or 0.0% (3) Distribute in Jena flasks that have
K2SO4 0.25% been cleaned in acid and rinsed re-
NaCl 0.15% peatedly in distilled water.
(d) Glycerol 1.5% (4) Add one of the combinations given
K2SO4 0.25% below.
NaCl 0.15% Sterilization: Not specified.
(e) Glycerol 1.0% Use: To study pigment production by red
K2SO4 0.25% pigment forming bacteria, B. prodigiosus,
NaCl 0.15% B. ruberbalticus, B. kiliensis, B. ruber-
(f) Glycerol 1.0% miquel, B. rutilus, B. amyloruber. Pig-
One of the following: ment production generally poor.
citric acid 1-0% Added nutrients One of the
: following ma-
lactic acid 0.1% terials or combinations may be added;
tartaric acid 0.75% (a) 1.0 g. MgS04 and 1.0 g. K2HPO4.
malic acid 0.75% (b) 1.0 g. K2HPO4.
succinic acid 0.75% (c) 1.0 g. MgS04.
aspartic acid 0.75% (d) 1.0 g. MgS04, 1.0 g. K2HPO4 and
alanin 0.75% 20.0 g. glycerol.
(e) 1.0 g. MgS04, 1.0 g. K2HPO4 and 380. Gottheil's Basal Asparagin Solution
10.0 g. glucose.
Constituents:
(f) 1.0 g. MgS04, 1.0 g. K2HPO4 and
10.0 g. lactose.
(g) 1.0 g. MgS04, 1.0 g. K.HPO^ and
3. CaClj 0.1 g.
10.0 g. sucrose.
Reference: Hefferan (1903-4 p. 522).
4. MgS04 0.3 g.
5. NaCl 0.1 g.
6. Ifon trace
378. Zikes Basal Asparagin Solution
7. Asparagin 10.0 g.
1. Water 1000.0 cc. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
2. Asparagin 2.5 g. (2) Add one of the added nutrients in
3. K2HPO4 1.0 g. the amount indicated.
Preparation Use: Cultivation of organisms found in the
(1) Dissolve 2, 3 and 4 in 1. soil, on roots and on rhizomes.
(2) Add 10.0% one of the carbon

of Added nutrients One : of the following com-
sources listed under added nutrients. binations were added:
Sterilization: Not specified. (a) glycerol 10.0 g.
Use: Volutin production by yeast, Oidium sucrose 5.0 g.
lactis. (b) sucrose 5.0 g.
Added nutrients: The author used 10.0% (c) galactose 3.0 g.
of one of the following carbon sources: lactose 3.0 g.
glucose lactose (d) galactose 3.0 g.
fructose raffinose glycerol 3.0 g.
galactose dextrin Reference: Gottheil (1901 p. 432).
maltose inulin
sucrose 381. Went's Basal Asparagin Solution
Constituents
379. Miehe's Basal Asparagin Solution

1. Water 1000.0 cc.
2. Asparagin (0.66%) 6.6 g.
1. Water 1000.0 cc. (1) Prepare 5.0% solutions of one of the
2. K2HPO4 (0.1%) 1.0 g. materials listed in added nutrients.
3. MgS04 (0.02%) 0.2 g. (2) Flask in 25.0 cc. quantities.
4. CaCl2 (0.01%) 0.1 g. (3) Prepare a 0.66% solution of asparagin.
5. Asparagin (0.5%) 5.0 g. (4) Add25.0 cc. of (3) to each flask of (2).
(1) Dissolve 2, 3, 4 and 5 in 1. Use: To study the nutrients for Monilia
(2) Add 4.0% of one of the added nu- sitophila (Mont.) Sacc. The materials
trients. ranked as follows as suitable carbon
Sterilization: Not specified. sources, with nitrogen supplied as aspara-
Use: To study carbon sources suitable for gin: sucrose, glycerol, glucose, maltose
tubercle bacilli. and lactose.
Added variants: The following materials Added nutrients: The author used the
were added: following additional carbon sources:
glycerol 4.0% maltose glucose
dextrin 4.0% lactose glycerol
glucose 4.0% sucrose
maltose 4.0% Variants: The author used the basic solu-
xylose 4.0% tion without additional materials.
Reference: Miehe (1908-9 p. 137). Reference: Went (1901 p. 593).
no CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
382. Sullivan's Basal Asparagin Solution (2) Neutralize with NasCOj.

Constituents: (3) Distribute in 50.0 cc. lots in small
1. Water 1000.0 cc. Erlenmeyer flasks.
2. Asparagin 10.0 g. (4) Add one of the added nutrients.
(1) Dissolve 2 in 1. Use: Cultivation of Actinomyces odorifer,
(2) Add one of the materials listed under Act. chromogenes, Act. albus I and II,
added nutrients. Act. S. a, b, andc. The author reported
Sterilization: Not specified. that the actinomycetes preferred a
Use: To study pigment formation. The neutral medium. They tolerated a very
author reported that the blue-green small amount of acid however. No
pyocyanin was formed whenever there growth in highly acid solutions.
was present a suitable combination of Added nutrients: Add one of the following
carbon, hydrogen, oxygen, and nitrogen to each flask:
and some salts to aid synthesis. acetic acid 0.01% or 0.1%.
Its
production was independent of either a succinic acid 0.004%, 0.04% or 0.1%.
phosphate or a sulphate. malic acid 0.004%, 0.01% or 0.1%.
Added nutrients: aspartic acid 0.01%, 0.05%, 0.1%, 0.2%
or 0.4%.
fMgS04 2.0 g.
(a) K2HPO4 1.0 g.
[NasS 0.2 g. tion without any additions.
(b) MgS04 0.2 g. Reference: Mlinter (1913 p. 378).
. fK,HP04 l.Og.
^''^ 384. Ono's Basal Sucrose Asparagin
\Na2SO4 l.Og.
Solution
, [MgSO^ 0.2 g.
^""^ Constituents
IK2HPO4 l.Og.
1. Water 1000.0 g.
. . fMgS04 0.01 to l.Og.
^*''' 2. Sucrose 85.0 g.
\K2HPO4 l.Og.
fMgS04 1.0 to 16.0 g. 4. MgS04
.J. 3.0 g.
[peptone 10.0 g.
5. KH2PO4 5.0 g.
fMgS04 0.2 g. 6. FeS04 trace
^^^ \K2HPO4 0.01 to 5.0 g. Preparation
[MgS04 0.2 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
(h) K2HPO4 l.Og. (2) Distribute in 30.0 cc. lots.
[peptone 5.0 g. Sterilization: Sterilize in the steamer.
Variants: The author used 5.0 g. asparagin Use: To study the effect of small amounts
and added 0.2 g. MgS04, 1.0 g. K2HPO4, of chemicals on the growth of Aspergillus
and 4.0 g. ammonium lactate. niger. The author reported that small
Reference: Sullivan (1905-06 pp. 127-140). amounts of CUSO4 tended to increase
growth while concentrations higher than
383. Miinter's Basal Glucose Asparagin
1/250 molar CUSO4 tended to hinder
Solution
growth.
Constituents: Variants and added nutrients:
1- Water 1000.0 cc. (a) The author added CUSO4 solution so
2. NaCl 0.5 g. that the medium was from 1/32,000 to
3. MgS04 0.5 g. 1/250 molar CUSO4.
4. CaCl2 Olg (b) Henneberg used 0.3% asparagin, 0.2%
5. K2HPO4 1.5 g. MgS04, 5.0% sucrose and omitted the
6. NH4NO3 l.Og. FeS04. To this solution he added
7. Asparagin 0.5 g. the following materials
8. Dextrose 9-0 g. NaCl 0.1%
Preparation CaCl2 0.1%
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. K2SO4 0.1%
Iron sulphate 0.01% KBr were toxic for yeast. Na2C03

Calcium lactate 10% inhibited growth. NaF inhibited
gypsum 0.1% growth of yeast. A heavier growth of
K2CO, 0.5% yeast appeared on medium contain-
soda 0.1 to 0.6% ing only 0.5% Na2S203.
MgCOa 0.1 to 1.0% (f) Bokorny used a medium containing
(c) Henneberg used 3.0 to 6.0% aspara- 1.0% sucrose, 0.1% asparagin, 0.1%
gin, 10.0 to 15.0% sucrose, 0.2% KH2PO4, 0.05% MgS04 and a trace
MgSO* and omitted the FeS04 from of iron chloride. He
reported that
the basic solution. To this solution yeast would grow more luxuriantly
he added one of the following: if the medium contained 0.05% CaCl2.
dextrin 1.0% Calcium was thought to be a necessity

ammonium tartrate 0.3% for growth. Growth occurred in the
peptone 0.5% solution without calcium due to the
potassium phosphate 0.5% probable presence of calcium in the
K2HPO4 0.5% sucrose or asparagin as an impurity.
[peptone 0.5% (g) Bokorny reported that spirogyra
ICaClj 0.1% grew much more luxuriant and more
fCaClj 0.1% characteristic in the following me-
\Chalk dium when 0.05% CaCl2 was added
(d) Henneberg used 0.3% asparagin, 1. Water 1000.0 g.
15.0% sucrose, 0.2% MgS04 and 2. Sucrose 10.0 g.
specified the use of either KH2PO4 3. Asparagin 2.5 g.
or K2HPO4. To this solution he sug- 4. KH2PO4 0.5 g.

gested that one of the following might 5. MgS04 0.5 g.
be added 6. CaCls 0.0 or 0.5 g.
gypsum 2.0% (h) Zikes used the following solution to
potassium phosphate 2.0% study perithecium formation by
CaCla 0.6% Aspergillus oryzae:
potassium lactate 2.0% 1. Water 1000.0 g.
K2CO3 2.0% 2. Asparagin 10.0 g.
soda 0.6% 3. K2HPO4 5.0 g.
K2SO4 0.6% 4. MgS04 2.5 g.
(e) Bokorny used a medium containing Sucrose

5. 75.0 g.
(i) Zikes used the following solution to
0.5% asparagin, 0.03% MgS04, a
trace of CaCl2, 10.0% sucrose and study volutin production by a num-
added one of the following: ber of wine and other yeasts:
K2HPO4 0.1, 0.5, 1.0, 2.0 or 4.0% 1. Water, distilled 1000.0 cc.
KH2PO4 0.1, 0.5, 1.0, 2.0 or 4.0% 2. Sucrose 100.0 g.
NajHPOi 0.1, 0.5, 1.0, 2.0 or 4.0% 3. Asparagin 2.5 g.
KH2PO4 0.1% with NaCl 0.5, 1.0, 2.0 or 4.K2HPO4 1.0 g.
4.0% 5.MgS04 0.3 g.

KH2PO4 0.1% with KI 0.01, 0.05, 0.1 or pH = 5.0706, CH = 8.50 X 10-
0.5% References: Ono (1902 p. 155), Henneberg

KH2PO4 0.1% with KBr 0.01, 0.05, 0.1 (1907pp. 40-45), (1909 pp. 104, 105),
or 0.5% Bokorny (1912 pp. 119, 120, 135, 138, 144,
KH2PO4 0.1% with NasCOs 0.5 or 1.0% 147), Zikes (1922 pp. 29, 340).
KH2PO4 0.1% with NaF 0.05, 0.1 or
385. Bokorny's Basal Asparagin Solution
0.5%
KH2PO4 0.1% with Na2S203 0.5 or 1.0% Constituents:
He reported that 4.0% K2HPO4, 1. Water 1000.0 cc.
KH2PO4 or Na2HP04 did not inhibit 2. Sucrose 100.0 g.
the growth of yeast, 2 and 4.0% NaCl 3. Asparagin (0.5%) 5.0 g,
inhibited growth. Generally KI and 4. KH2PO4 (0.1%) 1.0 g.
5. MgSOi (0.03%) 0.3 g. 387. Peklo's Basal Asparagin Solution

6. CaCl2 trace Constituents:
Preparation 1. Distilled water 1000.0 cc.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. 2. KH2PO4 10.0 g.
(2) Add one of the salts given in added 3. CaClj l.Og.
nutrients. 4. MgS04 3.0 g.
Sterilization: Not specified. 5. NaCl l.Og.
Use: To study the effect of metallic salts 6. Fe2Cl6 (Merck siccum) trace
on development of yeast. 7. Mannitol 20.0 g.
Added nutrients: The following salts were 8. Asparagin 4.0 g.
added: Preparation
CaS04 0.1 or 0.02 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
MnS04 1.0, 3.0or 5.0 g. (2) Add one of the added nutrients.
KMn04 0.1, 0.05 or 0.01 g. Sterilization: Generally sterilize all
solu-
NiS04 0.02, 0.03 or 01 g. tions for 30 minutes (method not given).
KzCrOi (red 1/3 neutral) 0.1, 0.05 or 0.5 g. Use: Cultivation of plant actinomyces.
K2Cr04 (yellow) 0.1 or 0.001 g. Added nutrients with modifications: The
Co(NO3)3 0.1, 0.02 or 0.2 g. following combinations were added:
ZnCls 0.2, 0.1, 0.05, 0.025 or 0.01 g. (a) The author used KH2PO4, 0.1
1.0 g. g.
ZnSO4 0.1, 0.5 or 1.0 g. CaCl2, 0.3 g. MgS04 and 0.1 g.
perosmic acid Os04 0.05, 0.01, 0.005, 0.5 NaCl and added 20.0 g. mannitol
or 0.1 g. and 4.0 g. asparagin. The reaction
Author omitted the trace of CaCl2. was acid before sterilization. May
Reference: Bokorny (1921 pp. 153-182). be neutralized by the addition of
Na2C03.
386. Higgins' Basal Carbohydrate Asparagin (b) The author used 7.5 g. KH2PO4, and
Solution added 20.0 g. of mannitol and 4.0 g.
asparagin. The reaction was slightly
Constituents
acid before sterilization. May be
1. Water 1000.0 cc.
neutralized by the addition of
2. Asparagin 4.0 g.
NaaCOs.
3. Lactose 2.5 g.
(c) Add 100.0 cc. of the basal solution to
4. Glucose 2.5 g.
900.0 cc. of distilled water, and then
Preparation
add 20.0 g. mannitol and 4.0 g.
asparagin. The reaction was weakly
(2) Add one of the combinations given
acid. The author specified that the
under added nutrients.
solution might be neutralized by the
(3) Adjust to neutral to phenolphthalein.
addition of Na2C03.
(d) Mi.x 250.0 cc. of the basal solution and
Use: Cultivation of organisms causing
add 750.0 cc. of distilled water, and
cholera and Picaton cattle disease.
add 20.0 g. mannitol and 4.0 g.
Added nutrients: The following combina- asparagin. The action was distinctly
tions were added:
acid.
(a) Potassium phosphate 2.0 g
Mi.x 100.0 cc. of the basal solution
(e)
KNO3 4.0 g
with 900.0 cc. of distilled water. Add
(b) Ammonium succinate 4.0 g 30.0 g. mannitol and 4.0 g. asparagin.
potassium phosphate 2.0 g The reaction was weakly acid.
K2SO4 1.0g (f) Mix 250.0 cc. of the basal solution
NH4CI 1.5 g
with 750.0 cc. of distilled water. Add
(c) Ammonium phosphate 2.5 g 30.0 g. mannitol and 4.0 g. asparagin.
KNO3 2.5 g The reaction was distinctly acid.
KCl 1.5 g The author used 5.0 g. KH2PO4 and
(g)
(NH4)2S04 l.Og 0.5 g. CaCU and added 5.0 g. K2HPO4
Reference: Higgins (1898 pp. 666-667). to the basal solution. To this solu-
tion 20.0 g. mannitol and 4.0 g. aspara- (c) Added 1.0 g. KXO,.
gin were added. Reaction was dis- (d) Used 20.0 g. glycerol, 3.0 g. asparagin
tinctly acid. Filter the precipitate and added 1.0 g. XaCl.
after the first sterilization. Sterilize (e) Omitted the K2HPO4, used 10 to
for 90minutes all together, 20.0 g. glycerol, 10.0 g. asparagin,
(h) The author used 5.0 g. KH2PO4 and and added 0.2 to 1.0 g. KNO3 and
0.5 g. CaCl2 and added 5.0 g. K2HPO4 0.2 to 1.0 g. XaCl.
to the basal solution. 100.0 cc. of (f) Used 10 to 20.0 g. glycerol, 10.0 g.
this solution was diluted with 900.0 asparagin and added 0.2 to 1.0 g.
cc. of water, and 20.0 g. mannitol and HBr.
4.0 g. asparagin were added. Reac- Used 20.0 g. asparagin, 20.0 g. glycerol
(g)
tion was weakly acid. Filter the and added 0.2 to 1.0 g. KI.
precipitate after the first steriliza- (h) Used 10.0 g. asparagin, 20.0 g.
tion. Sterilize for 90 minutes all glj'cerol and made no additions, or
together, added 0.2 to 1.0 g. KCl, 0.2 to 1.0 g.
(i) The author used KH.PO4, and
5.0 g. K2SO4, or 0.2 to 1.0 g. NaoS04.
0.5 g. CaCl2, MgSOi and
omitted the Reference: Sullivan (1905-06 p. 127-140).
NaCl from the basal solution and
added 5.0 g. K2HPO4 and 3.0 g. 389. Waksman's Basal Glycerol Asparagin
MgS04. To 200.0 cc. of this solution Solution
added 800.0 cc. distilled water, 20.0 g.
Constituents:
mannitol and 4.0 g. asparagin. The
1. Water 1000.0 cc.
reaction is weakly acid. Sterilize
2. Glycerol 30.0 g
for one hour.
Reference: Peklo (1910
3. K2HPO4 1.0 g
p. 470).
4. KCl 0.5 g
388. Sullivan's Basal Glycerol Asparagin 5. MgS04 0.5 g
Solution 6. FeS04 0.01 g
Constituents:
7. Asparagin 5.0 g,
Preparation
1. Water 1000.0 cc.
2. Glycerol (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
10.0 g.
3. Asparagin 2.0 g.
trients.
4. K2HPO4 1.0 g.
Preparation (3) Tube in 10-12 cc. lots.
Dissolve 3 and 4 in
(1) 2, 1.
(2) Add the various materials as indi-

Sterilization: Sterilize at 15 pounds for
15 minutes.
cated under added nutrients.
Use: To study metabolism of Actinomy-
cetes.
Use: To study pigment formation. The
author reported that the blue-green Added nutrients: The author added 2.0 g.
pyoc3'anin was formed whenever there
was present suitable combination of car- NaNOa (NH4)2S04
bon hydrogen, oxygen and nitrogen and NaXOa (XH 4)200 3
some salts to aid synthesis. For the Reference: Waksman (1920 p. 3).
formation of the fluorescent pigment,

the presence of both sulphur and phos- 390. Tanner's Basal Lactate Asparagin
phorus was essential. Solution

Added nutrients and variants: Constituents:
(a) The author added 0.2 g. of MgSOi to 1. Distilled water 1000.0 cc.
the basal solution. Most chromo- 2. XaCl 5.0 g.
genic bacteria produced a pigment on 3. CaH4(P04)2-H20 2.0 g.
this medium. 4. Ammonium lactate 6.0 g.
(b) Substituted 5.0 g. of asparagin for 5. XaOH (normal) 20.0 cc.
2.0 g. 6. Asparagin 4.0 g.
Preparation 3. Na2S04 2.0 g.
(1) Dissolve 2, 3, 4, and 6 in 1.

5 4. MgS04 0.2 g.
Add one of the listed added nutrients. 5. Asparagin 5.0 g.

(2)
Tube. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
(3)
Sterilization: Sterilize in the Arnold Sterilization: Not specified.
Sterilizer. Use: To ascertain production of indol test.

Use: To study metabolism of sulphur. Added nutrients: Author added 30.0 g. of
Production of H2S by the fluorescent peptone, or 0.1, 0.3, or 0.5 g. of 1-1000
group, colon-typhoid group and others. tryptophane.
H2S production may be detected by the Variants: The basic solution was used as
blackening of lead acetate paper. culture medium without further addi-
Added nutrients: One of the following tions.
materials was employed: Reference: Harvey (1921-22 p. 102).
Cystine (prepared from wool, Folin's

393. Capaldi and Proskauer's Basal Citrate
method) 60.0 g.
Asparagin Solution
2-thiohydantoin 1.0 cc. to each 5.0 cc. of
medium. Constituents:
100.0 g. 1. Water 1000.0 cc.
Thiourea
2. Asparagin 2.0 g.
Taurine, amount not given.
Na2S203-5H20 3.0 g. 3. MgS04 2.0 g.
Na2S03 30.0 g.
Reference: Tanner (1917 p. 586). 5. KH2PO4 2.0 g.
6. CaCl2 0.2 g.
391. Hiss' Basal Litmus Asparagin Solution Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Constituents:
Distilled water 1000.0 cc. (2) Add NaOH until the reaction is
1.
slightly alkaline.
2. K2HPO4 2.0 g.
(3) Add 0.2% of one of the carbon sources
3. MgS04 0-4 g-
listed under added nutrients.
4. NaCl 5.0 g.
4.0 g.
5. Asparagin
Use: To study fermentation by members
6. Ammonium lactate 5.0 cc.
The authors
15.0 cc. of the colon-typhoid group.
7. Litmus (5.0% soln.)
reported no growth of Bacterium hjphi.
Preparation :
B. colt produced gas from glucose, levu-

(1) Dissolve 2, 3, 4, 5 and 6 in 1.
lose, lactose and mannose.
(2) Add 6.0 cc. of normal NaOH. of the follow-
Added nutrients: 0.2% of one
(3) Add 15.0 cc. of 5.0% litmus solution.
ing was added:
(4) Add 1.0% of one of the carbon sources maltose
glucose
given in added nutrients.
levulose raffinose
(5) The medium is clear.
lactose maltose
Sterilization: Method not given. .
sucrose mannose
Use: To study fermentation by the dysen-
Reference: Capaldi and Proskauer (1896
tery group. Acid production turns the
p. 454).
medium from purplish blue to red.
Added nutrients: The author added 1.0% 394. Mendel's Basal Citrate Asparagin
of one of the following: Solution
glucose de.xtrin
maltose mannitol Constituents:
sucrose
Reference: Hiss (1904 p. 32). 2. KNO3 2.0 g.
3. Asparagin 5.0 g.
392. Harvey's Basal Lactate Asparagin 4. MgS04 2.0 g.
Solution 5. Citric acid 5.0 g.
Constituents 6. K2HPO4 2.0 g.
1000.0 cc. CaCl2 0.2 g.

1. Distilled water 7.
2. Ammonium lactate 5.0 g. 8. Iron chloride

Preparation 396. Lockemann's Basal Citrate Glycerol

(1) Dissolve 2 and 3 in part of 1. Solution
(2) Dissolve 4, 5, 6, 7 and 8 in the rest of 1.
Constituents:
(3) Neutralize (2) with KOH
during the
1- Water
heating. lOOO.O cc.
Mix and
2. MgHP04-7H20 4.93 g.
(4) (3) (1).
Sterilization: Sterilize for 20 minutes on
3. H3PO4 (1/3 molar) 20.0 cc.
4. Citric acid (3 normal) 10.0 cc.
each of two successive days. On the
third day
add 1.0% of one of the added
6.Glycerol 20.0 g.
nutrients and sterilize for 15 minutes.
Preparation
Use: To study decomposition of sugars by
Bacterium coli and \Bacterium Fitzi- (1) Dissolve 2, 3, 4, 5 and 6 in 1.
anus. The author reported the following (2) Add one of the combinations given
reactions:
under added nutrients. The reaction
of the various solutions will also be
Variants: The author used 10.0 g. asparagin
instead of 5.0 g. in the basic solution. given there.
Added nutrients: The author employed Sterilization: Not specified.
1.0% of one of the following as carbon
sources:
Added nutrients and modifications: The
author added the following materials:
glucose lactose
maltose sucrose
(a) K2HPO4 0.35 g. and 10.0 cc. normal
Reference: Mendel (1911 H2SO4. The acid titer was +4.7.

p. 297).
Luxuriant growth was reported.
395. Maassen's Basal Malic Acid Asparagin (No indicator specified.)
Solution (Smith) (b) Used 5.0 g. asparagin, 12.0 cc. citric
Constituents: acid, 25.0 cc. H3PO4, 4.0 g. MgHP04--
1. Distilled water 1000.0 cc. 7H2O and added 0.25 g. of NaH2P04-
2. Malic acid 7.0 g. 12 H2O, and 0.25 g. of KH2PO4. The
3. Asparagin 10.0 g. acid titer was +4.0. Growth was
4. Sodium phosphate reported as being quite good.
(secondary) 5.0 g (c) Used 5.0 g. asparagin 12.0 cc. citric
5. MgS04 2.5 g. acid, 25.0 cc. of H3PO4, 3.75 g.
6. NaOH 2.5 g. MgHP04-7H20 and added 0.25 g.
7. CaCl2 0.01 g. CaHP04, 0.25 g. KH2PO4 and 0.25 g.
Preparation NaH2P04. The reaction was +4.2.
(1) Dissolve 2 in 1000.0 cc. of distilled Growth was reported as slight.
water. (d) Added 0.72 g. NaH2P04, and 10.0 cc.
(2) Neutralize exactly to litmus, using normal H2SO4 to the basic solution.
7.0% KOH. The acid titer was +4.7. Growth
(3) Make up to 1000.0 cc. was reported as very slight.
(4) Dissolve and 6 in (3).
4, 5 (e) Added 10.0 cc. of normal H2SO4 and
(5) Add CaCl2 to (4).
0.01 g. 0.35 g. K2HPO4 to the basic solution.
(6) Dissolve one of the added nutrients The acid titer was +4.6. Growth
in (5). reported as luxuriant.
Sterilization: Not specified. (f) Added 10.0 cc. normal H2SO4, 0.18 g.
Use: As a general culture medium. K2HPO4 and 0.36 g. KH2PO4 to the
Variants: Kolle and Wasserman used 0.4 g. basic solution. The acid titer was
MgS04 in the basal solution and 2.5 g. +4.2. Growth reported as being
of pure crystalline soda instead of luxuriant.
NaOH. He specified the use of 0.5 to Reference: Lockemann (1919 p. 421).
1.0% of any desired carbohydrate, alco-
hol, etc. as added nutrients. 397. Mendel's Basal Citrate Glycerol
Added nutrients: Smith added any desired Solution
carbohydrate, alcohol, etc. Constituents
Reference: Smith (1905 p. 198), Kolle and 1. Distilled water 1000.0 cc.
Wasserman (1912 p. 394). 2. Asparagin 5.0 g.
3. Citric acid 0.75 g. 7. K2HPO4 2.3 g.
Glycerin 10.0 g. 8. NaCl 6.0 g.

4.
5. Potassium biphosphate 5.0 g. Preparation: (1) Dissolve 2, 3, 4, 5, 6,
8 in sufficient water to make 1000.0 cc.

7 and
6. MgS04 2.5 g.
(NHO2SO4 5-0 g. SterUization: Not specified.

7.
Preparation Use: Cultivation of Bacillus diphtheriae
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. and study of toxin production.
Adjustment of reaction not specified. Variants: The author omitted the FePO*
(2)
Sterilization: Sterilize for 30 minutes on and added 0.8 g. CaCU.
each of two successive days. On the Reference: Hadley (1907 p. 101).
third day add 1.0% of one of the added 401. Beijerinck and Minkman's Nitrate
nutrients and sterilize for 15 minutes. Alanine Solution
Use: To study decomposition of sugars by
Bacterium coli and Bacterium Fitzianus. Constituents:
Water 1000.0 cc.
Added nutrients: The author added 1.0% 1.
2. Alanine (0.5%) 5.0 g.

lactose 3. KNO3 (1.0%) 10.0 g.
glucose
maltose sucrose Preparation
Reference: Mendel (1911 p. 297). (1) Dissolve 2 and 3 in 1.

398. Beijerinck and Minkman's Nitrate SteriUzation: Not specified.
GlycocoU Solution Use: To study denitrification by soU forms,
Constituents Bacillus nitroxus.
1000.0 cc. Reference: Beijerinck and Minkman (1910
1. Water
2. GlycocoU (2.5%) 25.0 g. p. 37).
3. NaNOs (1.0%) 10.0 g.

402. Berthelot's Tyrosine Salt Solution
Sterilization: Not specified. Constituents
Water 1000.0 cc.
Use: To study denitrification by soil forms, 1.
{Bacillus nitroxus). The medium was 2. K,S04 0.2 g.
inoculated with soil. 3. MgS04 0.2 g.
Reference: Beijerinck and Minkman (1910 4. K2HPO4 0.5 g.
p. 37).
5. KNO3 0.25 g.
6. CaCl2 0.02 g.
399. Bokorny's Sucrose GlycocoU Solution Tyrosine 2.0 g.
7.

Constituents:
Water 1000.0 cc. in 1.
1.
2. GlycocoU 2.5 g. Sterilization: Not specified.
3. Sucrose 50.0 g. Use: Enrichment and isolation BaciWus

phenologenes. Bacillus phenologenes is
4. KH2PO4 2.0 g.
MgS04 1-0 g- enriched in this medium and then plated
5.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. on the same medium solidified with
SterUization: Not specified. agar.
Use: Cultivation of Yeast. Reference: Berthelot (1913 p. 19).

403. Vansteenberge's Glucose Tyrosine
Solution
400. Hadley's Glycerol GlycocoU Solution
Water 1000.0 cc.
1. Distilled water 1000.0 cc. 1.
Glucose 20.0 g.
2. Ammonium lactate 7.5 g. 2.
3. Tyrosine 0.2 g.
3. FeP04 0.8 g.
4. Glycerin 30.0 g. 4. K2HPO4 0.5 g.
5. GlycocoU 10 g. 5. MgS04 2.0 g.

6. MgS04 3.2 g.
117
sterilization: Not specified. (b) Inoculate and incubate.

Use: Cultivation of B. coli. (c)After a few days litmus is turned blue
Reference: Vansteenberge (1917 p. 609). again and more HCl is added.
(d) Suspend a sterile piece of lead acetate
404. Bokorny's Sucrose Tyrosine Solution
soaked filter paper in each tube to
Constituents:
H2S production.
test or
1- Water 1000.0 cc. Reference: Myers (1918 p. 250).
2. Tyrosine 2.5 g.
3. Sucrose 50 g
407. Heap and Cadness' Cystine Solution
4. KH2PO4 2 0g' Constituents:
5. MgS04 1.0 g. 1. Distilled water 1000 cc
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 2. NaCl 5.0g.'
Sterilization: Not given. 3. Na2HP04 4.2 g.
Use: Cultivation of yeast. 4. Sodium citrate 6.0 g.
Reference: Bokorny (1917 p. 340). 5. Cystine 0.2 g.
405. Waksman's Glycerol Tyrosine Solution Sterilization: Not specified.
Constituents: Use: To study hydrogen sulphide produc-
1- Water 1000.0 tion by B. aertnjcke. The authors re-
cc.
2. K2HPO4 10 ported that the addition of glucose to
3. MgS04 0.5
thismedium gave earlier and accelerated
I.
4. KCI 0.5 g.
hydrogen sulphide production, but pro-
5. FeS04 0.01 g duction ceases after 24 hours.
6.Tyrosine Reference: Heap and Cadness (1924-25
1.0 g
7. Glycerol 3O.0 p. 86).
g
Preparation
408. Rogers, Clark and Evans' Tryptophane
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Solution
(2) Tube in 10-12 cc. lots.
Constituents
Sterilization: Sterilize at 15 pounds for 1- Water iqqo.O cc.
15 minutes.
2. Tryptophane 03g
Use: To show utilization of tyrosine by 3. K2HPO4 5.0 g.'
Actlnomycetes. Preparation
(1) Dissolve 2 and 3 in
1.
(2) Tube in 10.0 cc. lots.

406. Myers' Cystine Solution Sterilization: Not specified.
Use: To study production of indol. If
Constituents:
indol is present a violet color appears
when 1.0 cc. of a 2.0% alcoholic solution
2. Cystine 5.0 g.
of p-dimethylamidobenzaldehyde and
Preparation: (1) Dissolve the cystine in 1,
concentrated HCl are added (Zipfel's
using enough NajCOs to keep the cystine
method).
in solution.
Variants: Braun and Cahn-Bronner used
Sterilization: Sterilize by filtration, then
0.5% NaCl, 0.4% tryptophane, 0.2%
tube and test sterility.
K2HPO4 and added 0.7% normal NaHCO,
Use: To study H,S production. If H2S is
after the solution was neutralized to
formed, lead acetate paper is blackened.
litmus.
No growth appeared with organisms
References: Rogers, Clark and Evans
studied unless medium was acid. Some
(1914 p. 101), Braun and Cahn-Bronner
organisms produce H2S in acid medium.
Variant: The author gives the following
(1921 p. 199), Harvey (1921-22 p. 117).
variant 409. Frieber's Glucose Tryptophane
(a) Add a bit of sterile litmus paper to Solution
each tube and sterile 5.0% HCl until Constituents
the litmus is faintly red. The cys- 1- Water looO.O cc.
tin is precipitated at neutrality.
2. Tryptophane (0.03%) 0.3 g.
3. NaCl(0.5%) 5.0 g. 4. Na2HP04 10 g-
Potassium phosphate 5. NaCl 5.0 g.

4.
2.0 g. Preparation
(0.2%)
0.2 g. (1) Dissolve 2, 3, 4 and 5 in 1.
5. MgSO4(0.02%)
30.0 g. (2) Adjustment of reaction not given.
6. Glucose (3.0%)
7. CaCOa
Sterilization:Method not given.
Preparation
5 and 6 in 1. Use: To study lipolytic and proteolytic
(1) Dissolve 2, 3, 4,
activity by the tubercle bacilli. To
Distribute in flasks in 50.0 cc. lots. determine lipolytic action, suspend 1.0
(3)
each cc. of the bacteria free filtrate of
the
(4) Add 10.0%, sterile CaCOa to
culture in freshly boiled distilled water.
flask.
Sterilization: Not specified. Add 0.25 cc. of ethylbutyrate and 0.5
cc. of toluene and incubate at 37C.
The
Use: To study indol production. The
increase in acidity as measured in terms of
author reported that indol was not
formed in the presence of glucose. N/50 NaOH measures lipolytic activity.
Variants The authors used 4.0 g. asparagin
Reference: Frieber (1921-22 p. 268).
:
and omitted the Na2HP04.

410, Bokorny's Sucrose Leucine Solution Reference: Kendall, Walker and Day (1914
Constituents p. 463).
1. Water 1000.0 cc. 413. Dolt's Asparagin Solution No. 2

2. Leucine 2.5 g.
Constituents:
Sucrose 50.0 g. 1000.0 cc.
3.
1. Distilled water
4. KH2PO4 2.0 g.
2. Asparagin 10-0
5. MgS04 1-0 g-
3. Na2HP04 2.0 g.
Preparation: (1) Dissolve one of 2, 3, 4
Preparation
and 5 in 1.
Sterilization: Not specified. NaOH using phenol-
(2) Neutralize with
Use: To study the nutrient requirements phthalein as an indicator.
for growth of yeast. Sterilization: Method not given.
Reference: Bokorny (1917 p. 340). Use: Cultivation of colon group and organ-
isms found in water.
411. Dolt's Ammonium Asparagin Solution
Variants: Dolt added 5.0 g. NaCl and
No. I
neutralized the medium by the addition
Constituents of Na.CO.s
1. Distilled water 1000.0 cc. References: Dolt (1908 pp. 617, 620),
2. Asparagin 10.0 g. Tanner (1919 p. 65).
3. (NH4)2HP04 2.0 g.
414. Beijerinck and Minkman's Nitrate
Preparation
Asparagin Solution
phenolphthalein Constituents:
(2) Make neutral to
Water 1000.0 cc.
with NaOH. 1.
2. Asparagin (0.05%) 0.5 g.

Use: Cultivation of colon group and organ- 3. KNO3 (1.0%) 10.0 g.
Preparation
isms found in water.
1.
Reference: Dolt (1908 p. 620), Tanner
(1919 p. 65).
412. Kendall, Walker and Day's Ammo- Use: To study denitrification by soil forms,
nium Asparagin Solution Bacillus pyocyaneus.
Constituents Variants: Author employed the above me-

1. Redistilled water 1000.0 cc. dium with the addition of 5.0 g. K2HPO4.
2.0 g. Reference: Beijerinck and Minkman (1910
2. Asparagin
(NH4)2HP04 2.0 g. p. 37).
3.
415. Blanchetiere's Asparagin Salt Solution 418. Uschinsky's Asparagin Salt Solution
Constituents: (Giltner)
2. NaCl 5.0 g. 1. Distilled water 1000.0 cc.
3. Na2HP04 1.0 g. 2. Asparagin 34g
4. Potassium biphosphate 1-0 g. 3. NaCl 5.0 g.
5. Asparagin 3.0 g. 4. MgS04 0.2 g.
Preparation 5. CaCh 0.1 g.
(1) Dissolve 2, 3, 4 and 5 in enough water 6. KH2PO4 1.0 g.
to make 900.0 cc. 7. FeS04 trace
(2) Distribute in 450.0 cc. lots in liter Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
flasks. in 1.
Sterilization: Sterilize for 15 minutes at Sterilization: Not specified.
110 to 112C. Use: General culture medium.
Use: To study asparagin decomposition by Variants
Bacillus fluorescens liquefaciens (Flugge). (a) Gottheil did not specify the type of
Reference: Blanchetiere (1917 p. 294). phosphate used and used 0.3 g. MgS04
with 10.0 g. asparagin.
416. Park, Williams and Krumwiede's
(b) Linde used 0.01 g. Fe2Cl6 instead
Asparagin Salt Solution
of FeS04, used 0.3 g. MgS04-7H20,
Constituents: 0.1 g. NaCI, and did not specify the
1. Water 1000.0 cc. amount of asparagin added. H used
2. Asparagin 6.0 g. the solution with an alkaline reaction
3. Sodium phosphate (ortho).. 2.0 g. for the cultivation of Cladothrix.
4. NaCl 5.0 g. The addition of glucose, sucrose or
Preparation glycerol would have increased the
(1) Dissolve 2, 3 and 4 in 1. amount of growth.
(2) Ifnecessary make alkaline to litmus References: Giltner (1921 p. 369), Gottheil
by the addition of NaOH. (1901 p. 432), Linde (1913 p. 386).
(3) Tube in small quantities.
(4) Test for color production with. B. 419. Henneberg's Asparagin Salt Solution
pj/ocyaneus Constituents
Sterilization:Method not given. 1. Distilled water 1000.0 cc.
Use Culture medium for disinfection tests.
: 2. Asparagin (0.075 to
Reference: Park, Williams and Krum- 0.9%) 0.75 to 9.0 g.
wiede (1924 p. 122). 3. K2HPO4 (0.05 to
1-5%) 0.5 to 15.0 g.
417. Bokorny's Asparagin Salt Solution
4. MgS04 (0.02 to
Constituents 0.6%) 0.2 to 6.0 g.
1- Water 1000.0 cc. Preparation: (1) Dissolve 2, 3 and 4 in 1.
2. Asparagin (0.5%) 5.0 g. Sterilization: Not specified.
3. KH2PO4 (0.1%) 1.0 g. Use: Cultivation of yeast.
4. MgS04 (0.03%) 0.3 g. Variants
5. CaCIo trace (a) Bokorny used 25.0 g. asparagin, 5.0 g.
6. K2SO4 (0.1%) 1.0 g. KH2PO4 and 1.0 g. MgS04.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 (b) Evans used 5.0 g. asparagin, 1.0 g.
in 1.
MgS04 and 2.0 g. K2HPO4.
Sterilization: Not specified. (c) Heinemann and Tanner used 2.0 g.
Use: To study effect of metallic salts on asparagin, 1.0 g. MgS04 and 1.0 g.
yeast growth. The author reported that K2HPO4.
4.0% of K2SO4 did not inhibit the develop- (d) Tanner used 10.0 g. asparagin, 2.0 g.
ment of the yeasts. MgS04 and 1.0 g. KH2PO4 for pig-
Variants: The author used 0.5, 1.0, 2 or ment production by Ps. pyoctjaneus
4.0% K2SO4. and Ps. fluorescens liquefaciens.
Reference: Bokorny (1912 p. 122), (e) Boehncke used 2.0% asparagin, 0.5%
Preparation: Dissolve 5, 6, 7
NaCl and 0.05% MgS04 to determine (1) 2, 3, 4,
ammonia production by Proteus. and 8 in 1.
References: Henneberg (1907 pp. 40-45), Sterilization: Not specified.
(1909 p. Bokorny (1917 p. 338),

104), Use: To study thiosulphate reduction by
B. desulfuricans and B. coli. When
Evans (1916 p. 444), Heinemann (1922
p. 42), Tanner (1919 pp. 64, 66),
Boehncke ammonia and sodium nitroprusside were
(1911 p. 74).
added to either culture a negative reac-
tion was obtained.
420. Amaud and Charrin's Asparagin Salt Reference: Saltet (1900 p. 702).
Solution (Roux and Rochaix)
Constituents: 423. Blanchetiere's Glucose Asparagin

2. KH2PO4 01 g-
Constituents:
3. Na2HP04-12H20 0.1 g.
4. KHCO3 0.134 g.
2. NaCl 5.0 g.
5. CaCU 0.05 g.
3. Na2HP04 10 g.
6. MgS04-7H20 0.05 g.
4. K2HPO4 10 g.
7. Asparagin 5.0 g.
Asparagin 5.0 g.
5.
3.0 g.
6. Glucose
in 1.
Preparation
5 and 6 in enough
(1) Dissolve 2, 3, 4,
Use: Cultivation oi Bacillus pyocyaneus. water to make 1000.0 cc.
Reference: Roux and Rochaix (1911 p. 104). The reaction is alkaline to litmus
(2)
421. Saltet's Glucose Asparagin Solution before sterilization and neutral after
sterilization.
Constituents Sterilization: Autoclave at 112 for 40
minutes.
NaCl 12.0 g.
2.
Use: To study asparagin decomposition by
3. KCl 10 g.
Bacillus fluorescens liquefaciens (Flugge).
Na2HP04 0.40mg.
4.
Good growth when NH4CI is added but
5. Glucose 0.5 g.
produced.
no pigment is
6. Asparagin 1-0 g-
Variants: The author specified that 5.0 g.
7. Na2S04 varying amounts
NH4CI might be added.
Preparation:
Reference: Blanchetiere (1917 p. 301).
(1) Dissolve 2, 3, 4, 5 and 6
in 1.
(2) Add varying amounts of Na2S04 and Proskauer's Glucose

424. Capaldi
(amount not given) to (1). Asparagin Solution
Constituents
Use: To study sulphate reduction by B.
Water 1000.0 cc.
desulfuricans Sulphates were reduced.
.
1.
2.0 g.
Variants: The author substituted MgS04 2. Asparagin (0.2%)
for Na2S04.
3. NaCl (0.02%) 0.2 g.
Reference: Saltet (1900 p. 699). 4. MgS04 (0.01%) 0.1 g.
5. CaCl2 (0.02%) 0.2 g.

422. Saltet's Thiosulphate Asparagin 6. KH2PO4(0.2%) 2.0 g.
Solution 7. Dextrose (0.2%) 2.0 g.
Constituents:
1. Distilled water 1000.0 cc. in 1.
NaCl 12.0 g. Sterilization: Not specified.

2.
3. KCl 10 g. Use: Acid production by Bacterium coli

4. Na2HP04 30.0 mg. and typhoid bacillus.
5. Na2S04 1-0 g- Variants: Robertson and Davis used the
Na2S203 1-5 g- following solution to study the influence
6.
of vitamines on bacterial growth. All
7. Asparagin 1-0 g.
Glucose 1-5 g. materials were chemically pure. This

8.
medium did not give continued growth 3. Asparagin 20 g

of yeast. 4. KH2PO4 0.5 g.
1. water
Sterile distilled 1000.0 cc. 5. MgS04 0.25 g.
2. Asparagin (Merck) 3.4 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
3. CaCl2 0.1 g. Sterilization: Not specified.
4. Dextrose 20.0 g. Use: Cultivation of bacteroids from Pisum
5. MgS04 0.2 g. sativum.
6. K2HPO4 1.0 g. Variants
7. NaCl 5.0 g. (a) Hefferan used1.0 g. MgS04, 1.0 g.
References: Capaldi and Proskauer (1896 sodium phosphate and 20.0 g. of
p. 456), Robertson and Davis (1923 p. 154). glucose to cultivate B. rosaceus
metalloides. This organism forms
425. Gottheil's Glucose Asparagin Solution rosette-like groupings in liquid media.
No. X The addition of glucose aids the
Constituents growth of the organism.
(b) Kuntze used the following solution
2. Potassium phosphate 10 to study pigment production by
g.
3. CaCla Bacillus ruber indicus. He reported
1.0 g.
4. MgS04 that the organism produced pigment
0.3 g.
5. NaCl 0.1 g.
when grown in medium without
6. Iron trace
MgS04 for 6 days and then in medium
containing MgS04.
8. Glucose 1. Distilled water 1000.0 cc.
30.0 g.
Preparation: Dissolve and 2. d-glucose (Soxhlet) . 25.0 g.
(1) 2, 3, 4, 5, 6, 7
8 in 1.
4. K2HPO4 2.0 g.
Use: Cultivation of organisms found in the
5. MgS04 0.0 or 2.0 g.
soil, on roots and on rhizomes. (c) Kuntze used the following solution
Reference: Gottheil (1910 p. 432). to study pigment production by
Aspergillus niger and Bacillus prodi-
426. Capaldi and Proskauer's Glucose giosus.
Asparagin Solution No. II 1. Distilled water 1000.0 cc.

2. MgS04 1.0 to 2.0 g.
Constituents
3. K2HPO4 2.0 g.
1. Water 1000.0 g. 4. d-glucose 25.0 g.
2. Asparagin 2.0 g.
3. Glucose 2.0 g.
(d) Kuntze used the following solution to
4. NaCl 5.0 g. study pigment production by Bacil-
5. MgS04 2.0 g. lus prodigiosus. He reported that
6. Sodium phosphate 2.0 g.
when KH2PO4 was used pigment was
Preparation
produced:
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Add NaOH until the reaction is
slightly alkaline.
3. d-glucose 20.0 g.
4. KH2PO4 or K2HPO4 2.0 g.
Use: Acid production by Bacteriuyn coli
5. MgS04 2.0 g.
and typhoid bacilli.
(e) Zikes used a solution containing
100.0 g. glucose, 2.5 g. asparagin and
p. 456).
3.3 g. MgS04 per liter to determine
volutin production by Oidiurn lactis.
427. Stutzer's Glucose Asparagin Solution
He distributed the solution in 10.0 cc.
Constituents: quantities and added 0.25, 0.125,
1. Water 1000.0 g. 0.063, 0.032 or 0.016 g. K2HPO4 to
2. Glucose 10.0 g. each tube. He reported that de-
the K2HPO4 content de-

creasing Use: As a general culture medium, and
creased volutin production. also used to study pigment production.
References: Stutzer (1900 p. 901), Hefferan Reference: Sullivan (1905-06 p. 115).

(1902 p. 694), Kuntze (1907 pp. 301, 304,
431. Boehncke's Glucose Asparagin
307), Zikes (1922 p. 33). Solution
428. Saltet's Magnesium Sulphate Constituents:
Asparagin Solution 1. Water 1000.0 cc.
Constituents 2. Asparagin (5.0%).. . . 50.0 g.
1. Distilled water. 1000.0 cc 3. NaCl (0.5%) 5.0 g.
2. NaCl 12.0 g, 4. Potassium biphos-

3. KCl 1.0 g, phate (0.2%) 2.0 g.
4. NasHPO^ 0.03 g 5. MgS04 (0.05%) 0.5 g.
MgS04 10 g- 6. Dextrose (1.0 or

5.
NaoSOs 1-5 g- 2.0%) 10.0 or 20.0 g.
6.
Glucose 0-5 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6
7.
8. Asparagin 1-0 g. in 1.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, Sterilization: Not specified.
7 and 8 in 1. Use: To determine the formation of am-

Sterilization: Not specified. monia by Proteus types.
Use: To study sulphite reduction by B. Reference: Boehncke (1911 p. 103).
desulfuricans. No H2S reaction when

detect 432. Zikes' Sucrose Asparagin Solution
using sodium nitroprusside to
H2S. Constituents
Reference: Saltet (1900 p. 697). 1. Water 1000.0 cc.
2. K2HPO4 5.0 g.
429. Samkow's Glucose Asparagin Solution
3. K2SO4 2.5 g.
Constituents 4. Asparagin 10.0 g.
1. Distilled water. 1000.0 cc. 5. Sucrose 75.0 g.
2. Asparagin 10.0 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
3. Glucose 5.0 g. Sterilization: Not specified.

4. KH2PO4 2.0 g. Use: To study perithecium formation by
5. NasCOs 2.5 g. Aspergillus oryzae.
MgS04 0.4 g. Reference: Zikes (1922 p. 340).
433. Korffs' Sucrose Asparagin Solution
inl.
Use: To study pigment production by 1. Water
Bacillus prodigiosus. A red pigment was 2. KH2PO4 25.0 g.
produced. 3. MgS04 (crystalline) 8.5 g.
Reference: Samkow (1903 p. 306). 4. Asparagin 20.0 g.
5. Sugar candy
430. Sullivan's Glucose Asparagin Solution Preparation
Constituents (1) Dissolve 2, 3 and 4 in 1 liter of water.
1. Water 1000.0 g. (2) Dissolve 1000.0 g. of white sugar

2. Asparagin 10.0 g. candy in 500.0 cc. distilled water by
3. Dextrose 5.0 g. heating on the water bath.
4. MgS04 0-3 g. (3) Filter while hot in a hot water funnel
5. K2HPO4 10 g into a large porcelain dish.
6. KNO3 10 g- (4) Add 2 liters of warm absolute alcohol,

7. NaCl 10 g. stirring constantly.
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 (5) Pour the cool alcohol from the sugar
in 1.
and wash the sugar with absolute
Sterilization: Not specified. alcohol and then with ether.
(6) Dry at 60C. until all traces of damp- Use: Cultivation of chromogenic bacteria.
ness of alcohol or ether have dis- The author reported that the chromogenic
appeared. bacteria developed rapidly with good
(7) Prepare a 10.0% solution of (6) in color production on this medium.
distilled water. Variants: (1) Bezangon used 2.5 g. MgS04,
(8) Mix about 10.0% of (7) with 10.0% 1.5 g. potassium phosphate, 4.0 g. aspara-
of (1). gin and 15.0 g. glycerol. This solution
(9) Distribute in 150.0 cc. lots in fer- was used for the cultivation of tubercle
mentation flasks. bacilli.
Sterilization: Sterilize in streaming steam. Reference: Sullivan (1905-06 p. 116),
Use: To study fermentation by yeast. Bezangon (1920 p. 547).
Reference: Korff (1898 p. 532).
437. Schweinitz and Dorset's Glycerol
434. Zikes' Magnesium Sucrose Asparagin Asparagin Solution (Goris)
Solution
Constituents
Constituents 1. Water 1000.0 cc.
1. Water 1000.0 cc. 2. Glycerol 70.0 g.
2. Asparagin 10.0 g. 3. KH2PO4 1.0 g.
3. K2SO4 5.0 g. 4. Ammonium phosphate 10.0 g.
4. MgS04 2.5 g. 5. NaCl 10.0 g.
5. Saccharose 75.0 g. 6. Asparagin 2.0 g.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 7. MgS04 2.0 g.
Sterilization: Not specified. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
Use: To study perithecium formation by in 1.
Aspergillus oryzae. Sterilization: Not specified.

Reference: Zikes (1922 p. 342). Use: Cultivation of tubercle bacilli.
Reference: Goris (1920 p. 499).
435. Chrzaszcz's Sucrose Asparagin
Solution 438. Waksman and Joflfe's Glycerol Aspara-
gin Solution
Constituents:
2. Sucrose 100.0 g. 1. Water 1000.0 cc.
3. Mg(H2P04)2 2.0 g. 2. Glycerol 30.0 g.
4. Ca(H2P04)2 2.0 g. 3. MgS04 0.5 g.
5. K2SO4 2.0 g. 4. KCl 0.5 g.
6. Asparagin 5.0 g. 5. FeS04 0.01 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 6. Asparagin 5.0 g.
in 1. Preparation
Sterilization: Not specified. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Use: To study growth requirements for (2) Adjust medium to different pH from
yeasts. Author reported that yeast de- 4.4 to 8.7 with phosphates and
veloped very well in this medium. carbonates.
Reference: Chrzaszcz (1904 p. 149). (3) Tube in 10-12 cc. lots.
436. Sullivan's Glycerol Asparagin
15 minutes.
Solution
Use: To study effect of reaction (pH) on
Constituents: metabolism of actinomycetes.
1. Water 1000.0 cc. Reference: Waksman and Joffe (1923 p. 44).
439. Hadley's Glycerol Asparagin Solution
3. K2HPO4 1.0 g.
Nos. Ill and IV
4. MgS04 0.2 g.
5. Glycerol 20.0 g. Constituents
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Ammonium phosphate 10.0 g.
4. CaCl2 0.1 g.
5. Glycerin 40.0 g.
6. KNO3 0.3 g.
7. K2HPO4 0.3 g.
8. NaCl 6.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7
and 8 in 1.

Use: Cultivation of Bacillus diphtheriae
and study of toxin production.
Variants: The author used 0.3 g. MgS04
instead of 0.6 g. NaCl for solution No. 4. 1000.0
Reference: Hadley (1907 p. 101).
440. Peklo's Mannitol Asparagin Solution
Constituents
2. KH2PO4 15.0 g.
3. CaCU 10 g.
4. MgS04 3.0 g.
5. NaCl 1.0 g.
6. FeiCle (Merck siccum)

7. Mannite 20.0 g.
8. Asparagin 4.0 g.
Preparation
(1) Dissolve 2, 3, 4, 5 and 6 (amount of 6
not given) in 1.
(2) Neutralize with K2CO3.
(3) Partially correct the precipitate loss
by the addition of orthophosphoric
acid.
(4) Dissolve 7 and 8 in (3).
(5) Reaction is found to be neutral after

sterilization.
Variants
(a) Peklo prepared the following solu-
tion:
2. K2HPO4 5.0 g.
3. KH2PO4 4.5 g.
4. MgS04 3.0 g.
5. NaCl 10 g.
6. CaCh 1-0 g.
7. FesCle trace
He diluted 200.0 cc. of this solution
with 800.0 water and

cc. of distilled
added 20.0mannitol and 4.0 g.
g.
asparagin. This solution was steril-

ized twice for one hour. The final
reaction is weakly acid.
441b. Voges' Lactate Asparaginate Solution
Constituents
2. NaCl 8.0 g.
3. K2HPO4 2.0 g.
4. Ammonium lactate 6.0 g.
5. Sodium asparaginate 4.0 g.
in 1.

Use: Enrichment and isolation of cholera
bacilli from water. Add 800.0 cc. of the
water to be investigated to the medium.
Used also as a general culture medium. 1000.0 cc
Variants
(a) Sames used 5.0 g. NaCl, 4.0 g. aspara-
gin and 8.0 g. ammonium lactate.
He distributed the solution in 5.0 cc.
lots and added 0.0, 0.2, 0.25, 0.3,
0.35, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.1
cc. ofa 5.0% NaOH solution to each
tube. He reported that motile sar-
cine grew best when 0.25 to 0.3 cc. of
5.0% NaOH solution was added to
each 5.0 cc. medium.
(b) Heinemann used 2.0 g. Na2HP04
instead of K2HPO4, 5.0 g. NaCl,
6.3 g. ammonium lactate and 3.4 g.
asparagin.
(c) Kuntze used 2.0 g. KH0PO4 instead
of K2HPO4, 4.0 g. asparagin and 5.0 g.
NaCl. He used this medium to study
pigment production by Bacillus pro-
digiosus.
(d) Dolt used 5.0 g. NaCl, 2.0 g. Na2HP04
instead of K2HPO4 and 4.0 g. aspara-
gin. The solution was neutralized
by the addition of Na2C03.
(e) NicoUe, Raphael and Debaiens used
5.0 g. NaCl and 4.0 g. of asparagin.
(f) Magnusson used 5.0 g. NaCl. The
medium was used to study slime pro-
duction by Bacterium lactic viscosum
and streptococci.
(g) Tanner used 5.0 g. NaCl.
(h) Bezangon used from 5.0 to 7.0 g.
NaCl, 6.0 to 7.0 g. ammonium lactate
and 3.0 to 4.0 g. asparagin.
(i) Abbott, Dopter and Sacquepee, and
also Park, Williams and Krumuriede
used 4.0 g. asparagin, 5.0 g. NaCl,
and 2.0 g. Na2HP04. Abbott culti-
vated Pseudomonas aeruginosa, the
bacillus of green pus, on this medium.
443. Uschinsky's Lactate Asparaginate Preparation

Solution (Smith) (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
(2) Inoculate with sea water or sand

Constituents
from a beach.
1. Distilled water 1000.0 ce.
Use: Enrichment of sulphite and sulphate
3. Sodium asparaginate 2.5 g.
reducers from sea water, Microspira
4. Na2SO4-10H2O 2.5 g.
aestuarii.
5. NaCl 2.5 g.
Variants: Omit the NaoSOs if inoculation
6. K2HPO4 2.5 g.
is made with sea mud colored black with
7. CaClj 0.01 g.
H2S.
8. MgS04 0.01 g.
Reference: van Delden (1903-04 p. 93).
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7 and
Sin 1.
446. Beijerinck's Lactate Asparagin
Solution
Use: As a general culture medium.
References: Smith (1905 p. 197), Tanner Constituents
(1919 p. 69). 1. Water (ditch) 1000.0 cc.
2. Asparagin 2.5 g.
444. Melick's Lactate Asparagin Solu-tion 3. MgS04 2.5 g.
Constituents: 4. Potassium phosphate 2.0 g.
1. Redistilled water 1000.0 cc. 5. Iron lactate 1 -0 g.

3. Asparagin 3.4 g. (1) Dissolve 2, 3, 4 and 5 in ditch water
4. Na2HP04 2.0 g. ("Graben wasser").

5. MgS04 0.5 g. (2) Make alkaline with NaaCOa.
6. Ammonium lactate 6.3 g. Sterilization: Method not given.
Preparation Use: To study the reduction of sulphates.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Author reported strong and ammo- US
(2) Adjust to +0.2 acid by the addition of nium sulfide production after 48 hours
NaaCOs. at 30C.
Sterilization Reference: Beijerinck (1895 p. 107).
(1) Autoclave for 10 minutes at 10 pounds

447. van Delden's Lactate Asparagin
pressure.
Solution
(2) Tube and autoclave again at 10
pounds pressure for minutes.
10 Constituents
Discard all tubes showing a pre- 1. Water 1000.0 cc.
cipitate. 2. K2HPO4 0.5 g.
Use: Growth of organisms for preparation 3. Sodium lactate 5.0 g.
of bacterial antigens, in colon-typhoid 4. Asparagin 10 g.
group. 5. MgS04-7H20 1.0 g.
Reference: Melick (1922 p. 408). 6. FeS04 trace
445. van Delden's Lactate Ammonia
in 1.
Asparagin Solution
Constituents Use: Sulphate reduction by Spirillum
1. Water 1000.0 cc. (Microspira) desuljuricans Medium .
2. NaCl 30.0 g. turned dark due to H2S production.

3. K2HPO4 0.5 g. H2S may also be determined by titration
4. MgS04-7H20 2.5 g. (or more) with 0.01 N iodine solution.
5. Sodium lactate ... 5.0 to 10.0 g. Variants
6. Asparagin 10 g. (a) The author substituted 1.0 g. 'gypsum
7. Mohr's salt trace for MgS04.
(FeS04(NH4)2S04). (b) Tanner and Giltner specified the use
8. Na2S03 small quantity of tap water.
References: van Delden (1903-04 p. S3), 450. Kuntze's Citrate Asparagin Solution
Tanner (1919 p. 64), Giltner (1921 p. 377).
Constituents
448. Schroeder and Junger's Lactate
Asparagin Solution
2. KNO3 2.0 g.
3. Asparagin 1-0 g.
Constituents 4. MgS04 2.0 g.
1. Water 1000.0 cc. 5. Citric acid 5.0 g.
2. Asparagin 4.0 g. 6. KH2PO4 2.0 g.
3. Sodium lactate 6.0 g. 7. CaCl2 0.2 g.
4. Sodium phosphate (Ortho) 2.0 g. 8. Iron chloride several drops
Preparation (1) Dissolve 2 and 3 in part of 1.
(1) Powder 2, 3, and 5 separately.
4
(2) Dissolve the powdered salts sepa- mainder of 1.
rately in 50 to 100.0 cc. of water.
(3) Neutralize (2) with KOH during the
(3) Mix the solutions and add the re- heating.
mainder of the liter of water.
(4) Mix (3) and (1).
(4) Add NaOH to make slightly alkaline
(5) Distribute into 100.0 cc. lots in com-
to litmus. bustion flasks.
(5) Filter. Sterilization: Method not given.
(6) Tube. use: To study morphology and physiology
Sterilization: Sterilize on 2 consecutive of denitrifying organisms Bacillus deni-
days in the Arnold sterilizer. trificans agilis (Ampola and Garino) and
Use: Cultivation of Bacillus pyogenes and Bacillus oxalaticus (Zopf).
chromogenes. The author reported that Variants
a green band of pigment was produced (a) Wojtkiewicz used 3.0 g. KNO3, 7.6
after 24 hours. The band gradually in- potassium citrate instead of citric
creased until the whole tube was light acid, used 0.2 g. CaCla and specified
green. Non chromogenic organisms were a trace of FeClo.
inhibited. (b) Arnd and Harvey specified a trace of
Variants: Harvey used 2.0 g. of Na2HP04. FeCl2.
References: Schroeder and Junger (1912 References: Kuntze (1904 p. 3), Smith
p. 601), Harvey (1921-22 p. 102). (1905 p. 198), Wojtkiewicz (1914 p. 258),
Arnd (1916 p. 567), Harvey (1921-22
449. Remy's Organic Acid Asparagin p. 102).
Solution
and Aberson's Citrate
451. Giltay
Constituents:
Asparagin Solution (Tanner)
2. Asparagin 6.0 g. Constituents:
3. Oxalic acid 0.5 g. 1. Distilled water 1000.0 cc.
4. Lactic acid 0.15 g. 2. KNO3 2.0 g
5. Citric acid 0.15 g. 3. MgS04 2.0 g
6. Na^HPO^ 5.0 g. 4. Citric acid 5.0 g
7. MgS04 2.5 g. 5. K2HPO4 2.0 g
8. K2SO4 1.25 g. 6. CaCl2 0.2 g
9. NaCl 2.0 g. 7. NaaCOs 4.25 g
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8. Asparagin 1.0 g
8 and 9 in 1. Preparation
Sterilization: Not specified. (1) Dissolve 2 and 8 in a little distilled
Use: Isolation of typhoid bacilli. water.
Reference: Thoinot and Masselin (1902 (2) Dissolved 3, 4, 5, 6 and 7 in distilled
p. 337). water.
(3) Mix (1) and (2) and make up to a

liter.
(4) Tube in fermentation tubes.

Use: Cultivation of denitrifying bacteria.
Variants: Lohnis used 2.0 g. KH2PO4 in-
stead of K2HPO4, omitted the NajCOa
and added a trace of Fe2Cl6.
References: Tanner (1919 p. 67), Lohnis
(1913 p. 97).
452. Maassen's Basal Citrate Asparagin

Solution (Lohnis)
Constituents:
4. K2HPO4 2.0 g.
5. NasCOs (crystalline) 2.5 g.
6. MgS04 0.4 g.
7. CaCU 0.01 g.
Preparation
(1) Neutralize 7.0 g. citric acid with pure
KOH.
(2) Dissolve (1), 3, 4, 5, 6 and 7 in 1.
(3) Add from 1.5 to 4.0% of one of the

nutrients listed below to (2).
Sterilization: Sterilize on each of 3 suc-
cessive days.
Use: As a general synthetic medium.
Added nutrients: Lohnis added from 1.5
to 4.0% of one of the following materials:
sugars
alcohols
other sources of carbon
453. Maassen's Malate Asparagin Solution

Constituents
2. Malic acid
3. KOH
4. Asparagin
5. MgS04
6. Na2HP04
7. Soda
8. CaCla
Preparation
(1) Dissolve 7.0 g. malic acid in about
100.0 cc. distilled water.
(2) Neutralize (1) with KOH.
(3) Make volume to 1000.0 cc.
2. Sodium malat (natrium 457. Gartner's Glycerol Asparagin Solution

malate) 10.0 g.
Constituents:
3. Asparagin 5.0 g. 1. Water 1000.0 cc.
2. Asparagin 10 g.
5. NaCl 2.5 g.
3. Glycerol 2.5 g.
6. Mohr salt (NH4)2S04 1.0 g.
4. Dextrose 2.5 g.
(b) Beijerinck detected the production of 5. Potassium phosphate 1-0 g.
US and SO3 after 6 days cultivating Preparation
Spirillum destilfuricans in the follow-
ing solution:
(2) Flask in 30.0 cc. lots.
2. Gypsum (CaS04) 2.0 g.
Use: To study nitrite oxidation by nitrate
3. Sodium malat (natrium fungus. The author reported trace of
malate) 0.5 g. weeks incubation.
nitrite after 4
4. Asparagin 0.5 g.
Reference: Gartner (1898 p. 4).
6. Na.COs 458. Borrel, de Coulon, Boez and Quimaud's
Reference: Beijerinck (1895 pp. 55, 56,
Glycerol Asparagin Solution
107).
Constituents:
455. Zikes' Alcohol Asparagin Solution 1. Distilled water 1000.0 cc.
Constituents: 2. FeS04 0.03 g.
1. Water 1000.0 cc. 3. Potassium silicate 0.02 g.
2. Glucose (2.0%) 20.0 g. 4. Glycerol 20.0 g.
3. Asparagin (0.25%) 2.5 g. 5. Mannitol 5.0 g.
4. K2HPO4 (0.1%) 1.0 g.

6. Glucose 5.0 g.
5. MgS04 (0.05%) 0.5 g. 7. Asparagin 4.5 g.
6. Alcohol
8. (NH4)2C03 1.0 g.
Preparation 9. NaNOs 1.0 g.

10. MgSOi 0.25 g.

11. KHSO4 0.25 g.
(3) To each 10.0 cc. lot add 5, 10, 15 or 12. KH2PO4 0.5 g.
20 drops of alcohol. Preparation

Sterilization: Not specified. (1) Dissolve 2, 3, 4, 5, 6, 7, S, 9, 10, 11
Use: To study volutin production by yeast and 12 in 1.
and Oidium Inctis. (2) Adjust to pH = 6.9.
Reference: Zikes (1922 p. 34). Sterilization: Not specified.

456. Gosio's Glycerol Aspartic Acid Solution Reference: Borrel, de Coulon, Boez and
Quimaud (1922 p. 389).
Constituents
1. Water 800.0 cc.
459. Miiller, Thurgan and Osterwalder's
2. Glycerol 15.0 g.
Malate Asparagin Solution
3. NaCl 2.5 g.
4. NasCOs 2.5 g. Constituents:
5. K2HPO4 1.0 g. 1. Water 1000.0 cc.
6. Aspartic acid 1-5 g. 2. Malic acid 3.0 g.
7. Grape sugar 50.0 g. 3. Asparagin 10.0 g.
8. CaCOa 20.0 g. 4. MgS04 0.4 g.

Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7 and 5. K2HPO4 2.0 g.
8 in 1. 6. NaCl 2.0 g.
Sterilization: Not specified. 7. Levulose 40.0 g.
Use: To study fermentation by Yihrio Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
cholerae asiaticae. in 1.
Reference: Gosio (1894 p. 25). Sterilization: Not specified.

Use: Cultivation of Bacterium mannito- lizedNa2HP04l2H20 for two weeks.

poeum and bacteria from wines. There Use 0.88 per cent of Na2HP042H20.
is very little growth on this medium. Sterilization: Heat in an autoclave for 15
Organisms grow better if peptone and minutes after the pressure has reached
potassium malate be added to the 15 pounds, provided the total time of
medium. e.xposure to heat is not more than one-
Reference: Midler, Thurgan and Oster- half hour.
walder (1912-13 p. 161). Use: Differentiation of colon-aerogenes
group.
460. Maassen's Glucose Asparagin Solution References: Clark and Lubs (1915, 160),
Constituents: Levine (1921 p. 118), Committee Ameri-
1. Water 1000.0 cc. can Public Health Assoc. (1925 p. 11).
2. MgS04 (crystal-
0.4
462. Remy and Sugg's Citrate Asparagin
line) g.
Solution (Proskauer and Beck)
4. CaCl2 0.01 g. Constituents
5. Na2HP04 2.0 g. 1. Water 1000.0
6. NaCOs (crystalline). 2.5 g. 2. Glucose (0.1%)
7. Glucose 5.0 to 10.0 g. 3. Asparagin (0.5%)
8. Malic acid 7.0 g. 4. Citric acid (0.075%)...
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 5. K3PO4 (0.5%)
7 and 8 in 1. 6. MgSO4(0.25%)
Sterilization: Method not given. 7. K2SO4 (0.25%)
Use: As a general synthetic culture 8. NaCl (0.15%)
medium.
Reference; Klimmer (1923 p. 172).
461. Clark and Lub's Solution

Constituents
2. NajHPOi (anhydrous) 7.0 g.
or Na2HP04-2H20 8.8 g.
3. KH phthalate 2.0 g.
4. Aspartic acid 1.0 g.
5. Glucose 4.0 g.
Preparation
(1) Dissolve 2, 3, 4 and 5 in 800.0 cc. of
warm distilled water.
(2) Cool the solution and make up to a
liter.
(3) The hydrogen ion concentration of

the medium is fixed by the composi-
tion. should be very close to pH
It
7.0, slightlyred with phenol red. All
materials should be recrystallized or
if used from stock furnished by manu-
facturers, should be carefully exam-

ined. The di-sodium hydrogen phos-
phate may be used either as the
anhydrous salt obtained by dessica-
tion in vacuo in 100C. or else as the
salt containing two molecules of
water of crystallization. This is
obtained by exposing the recrystal-
ammonium lactate, 3.4 g. asparagin, 4. MgS04 0.2 g

40.0 g. glycerol, 5.0 g. NaCl, 0.2 g. 5. Ammonium lactate. 0.8 g
MgS04, but did not specify the 6. NaCl 1.0 g
amount of K2HPO4. 7. KNO3 0.3 g
(c) Vaughan specified the use of 1.0 g. 8. Glycerol 10.0 g.
NaCl, 0.4 g. K2HPO4 and 6.0 g. Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7 and
ammonium lactate. 8 in 1.
(d) Hadley specified the use of 6.5 g. Sterilization: Not specified.

ammonium lactate, 0.32 g. asparagin, Use: General culture medium. The author
0.3 g. MgSOi, 2.5 g. K2HPO4 and 6.0 g. reported that about 40 variaties developed
NaCl. on this medium including chromogenic
(e) Tanner used 3.4 g. asparagin, 10.0 g. bacteria and pathogenic forms.
ammonium lactate, 5.0 g. NaCl, Variants
0.2 g. MgS04-7H20, 1.0 g. K2HPO4 (a) Hadley substituted 25.0 g. glycerol
and 40.0 cc. glycerol. for 10.0 g.
(f) Bezangon and Dopter and Sacquepee (b) Magnusson used 5.0 g. NaCl, 0.5 g.
used 3.0 to 4.0 g. asparagin. ammonium lactate, 1.0 g. of sodium
(g) Harvey used 6.0 g. NaCl, 0.1 g. CaCl, asparaginate and 0.2 g. NaNOs. He
0.3 g. MgS04, 2.5 g. K2HPO4, 8.0 g. used this medium to study slime and
ammonium lactate, 3.5 sodium as- capsule production by Bacterium
paraginate, and 35.0 g. glycerol. lactis viscosum, and streptococci.
(h) Harvey used 3.4 g. asparagin, 10.0 g. Slime and capsules were produced.
ammonium lactate, 5.0 g. NaCl, 0.2 g. Reference: Sullivan (1905-06 p. 115), Had-
MgS04, 0.1 g. CaCls, 1.0 g. K2HPO4, ley (1907 p. 101), Magnusson (1918 p. 467).
and 40.0 g. glycerol,
465. Hadley's Glycerol Asparagin Solution
(i) Harvey used 5.0 g. NaCl, 0.1 g. NaCl,
No. V
0.2 g. MgS04, 2.0 g. K2HPO4, 6.0 g.
ammonium lactate, 3.0 g. potassium Constituents:
or sodium asparaginate and 30.0 g. 1. Distilled water 1000.0 cc.
glycerol, 2. Ammonium lactate 5.0 g.
(j) Roux and Rochaix used 3.0 to 4.0 g. 3. Ammonium phosphate 10.0 g.
asparagin instead of sodium asparag- 4. Asparagin 10.0 g.
inate. 5. reP04 0.2 g.
(k) Besson used 30.0 g. glycerol, 5.0 g. 6. Glycerin 40.0 g.
NaCl, 0.2 g. MgS04, 2.0 g. K2HPO4, 7. MgS04 0.3 g.
6.0 g. ammonium lactate and 3.0 g. 8. K2HPO4 3.0 g.
asparagin. 9. NaCl 1.0 g.
References: Uschinsky (1893 p. 316), Cra- Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7,
mer (1894 p. 171), Vaughan (1896 p. 312), 8 and 9 in 1.
Smith (1905 p. 197), Hadley (1907 p. 101), Sterilization: Not specified.

Roux and Rochaix (1911 p. 246), Kalb and Use: Growth and toxin production of
Wasserman (1912 p. 394), Lohnis (1913 Bacillus diphtheriae. Author reported
p. 44), Tanner (1919 p. 69), Bezangon slight growth.
(1920 p. 116), Besson (1920 p. 37), Dopter Reference: Hadley (1907 p. 101).
and Sacquepee (1921 p. 121),Harvey
466. Lowenstein's Glycerol Asparagin
(1921-22 pp. 102, 103), Klimmer (1923
Solution
p. 394).
Constituents:
464. Sullivan's Glycerol Nitrate Asparagin 1. Asparagin 6.0 g.
Solution
2. Ammonium lactate 0.0 or 6.0 g.
Constituents: 3. Sodium phosphate
1. Water 1000.0 cc. (neutral) 3.0 g.
2. Asparagin 15.0 g. 4. NaCl 6.0 g.
3. K2HPO4 1.0 g. 5. Glycerin 40.0 g.
Preparation: (1) Original article does not 3. C&Clz 0.4 g

specify the use of water as a solvent. 4. Glycerin 170.0 g
The ammonium lactate may be omitted 5. Glycocoll 5.0 g
if desired. 6. MgS04 1.6
g
Sterilization: Not specified. 7. K2HPO4 12.5 g
Use: Cultivation of tubercle bacilli. 8. NaCl 30.0 g
Reference: Lowenstein (1913 p. 591). 9. Urea 25.0 g
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7,
467. Lockemann's Citrate Glycerol Aspara-
8 and 9 in sufficient water to make
gin Solution (Lowenstein)
1000.0 cc.
1. Water 1000.0 cc. Use: Growth of Bacillus diphtheriae and
2. KH2PO4 5.0 g. study of to.xin production.
3. MgS04 0.6 g. Reference: Hadley (1907 p. 101).
4. Magnesium citrate 2.5 g.
470. Beijerinck's Urea Asparagin Solution
5. Asparagin 5.0 g.
6. Glycerol 20.0 g. Constituents:
7. Soda about 2.5 g. 1. Water 1000.0 cc.
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 2. KH2PO4 0.25 g.
in 1. 3. Asparagin 2.5 g.
Sterilization: Not specified. 4. Urea 50.0 g.
Use: Cultivation of tubercle bacilli. Preparation: (1) Dissolve 2, 3 and 4 in 1.
Variants : Sterilization: Not specified.

(a) Lockemann added 3.0 g. NaH2P04 Use: Cultivation of Planosarcina ureae;
and omitted the soda. Urobacillus p isteurii did not develop in
(b) Harvey used 2.5 g. NaOH instead this medium.
of the soda. Reference: Beijerinck (1901 p. 53).
(c) Mueller used 2.6 g. magnesium citrate
471. Beijerinck's Urea Asparagin Solution
and adjusted the reaction to pH =
7.0 using NaOH instead of soda. Constituents:
References: Lowenstein (1913 p. 592), 1. Water 1000.0 cc.
Lockemann (1919 p. 421), Harvey (1921- 2. KH2PO4 0.9 g.
22 p. 103), Mueller (1926 p. 3). 3. (NH4)2C03 0.2 g.
468. Frouin's Glycerol Asparagin Solution
5. Urea 1.0 g.
(Bezangon)

1. Water 1000.0 cc Use: Cultivation of urea organisms from
2. MgS04 1.0 g the soil, Urococcus ureae and Urobacillus
3. Potassium phosphate. 1.0 g pasteurii. A soil culture will change all
4. Asparagin 5.0 g the urea after incubation of 10 days.

5. Lactose 6.0 g Reference: Beijerinck (1901 p. 53).
6. Sodium citrate 3.0 g
472. Sears' Uric Acid Asparagin Solution
7. Glycerol 40.0 g
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 Constituents:
in 1. 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Asparagin. 10.0 g.
Use: Cultivation of tubercle bacilli. 3. Na^CO, .. 2.5 g.
Reference: Bezanôn (1920 p. 548). 4. Na2HP04. 2.0 g.
5. MgS04.... traces
469. Hadley's Urea Glycocoll Solution
6. CaCl2 traces
Constituents 7. Uric acid. 0.902 g.
1. Distilled water 1000.0 cc. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
2. Ammonium lactate 32.5 g. in 1.

CULTURE MEDIA FOR CULTIVATION OF MICRO ORGANISMS 133
sterilization: Not specified. (2) Add one of the combinations given

Use: To determine production of urea and under added nutrients to (1) and heat
study of nitrogen metabolism. Sears in flowing steam for 15 minutes.
detected urea by urease method of Van (3) Add enough N/10 NaOH to give final
Slyke. Ammonia determined by Folin's reaction pH = 8.0 to 8 2.
aeration method, amino acid by Van (4) Steam again for 15 minutes and check
Slyke's method, total nitrogen by Kjel- reaction colorimetrically (using
dahl-Gunning-Arnold method. phenolsulphonphthalein and stand-
Reference: Sears (1916 p. 132). ardized HaBOs-KCl-NaOH solutions.
(5) Distribute as desired.
473. Kappen's Cyanamide Asparagin
Sterilization: Sterilize for 20 minutes at
Solution
115C.
Constituents: Use: Cultivation of Bad. diphtheriae for
1. Water 1000.0 cc. production of to.xin. Authors report
2. Calcium cyanamide (crude). 15.0 g. better toxin production if 10.0% bouillon
3. K2HPO4 (b.5) 5.0 g. be added. All amino acids prepared
4. Asparagin (0.1%) 1.0 g. specially, in pure form. Cultures must
5. Glucose (0.1%) 10.0 g. become accustomed to medium by adding
Preparation small portions of it to bouillon.
(1) Grind 15.0 g. of crude calcium cyana- Added nutrients and variants: The authors
mide to a powder and dry sterilize for added the following compounds to the
3 hours at 160C. basic solution:
(2) Dissolve (1) in sterile water. (a) tyrosine 1.0
g
(3) Filter thru a sterile folded filter into leucine
g 3-0
a sterile flask. glutaminic acid hydrochloride. 1.6
g
(4) Mix with a sterile solution containing glycocoll 0.4 g
0.5% K.HPO4, 0.1% asparagin and creatin 0.1 g
0.1% dextrose. The final volume creatinin 0.1 g
should be 1000.0 cc. sodium asparaginate 1.4 g
(5) All the flasks, filters, funnel, etc., NaCl 40g
should be sterile and the entire K2HPO4 3.0g
process of preparation carried out (b) Used 4.0 g. tryptophane in the basal
under aseptic conditions. solution and added the following:
Sterilization: Method of sterilization of tyrosine 2.5
solutions and apparatus not specified.
Use: To study cyanamide decomposition glutaminic acid hydrochloride. 1.9 g
by bacteria. glycocoll 0.85 g
Variants: The author suggested the follow- creatin o.I g
ing solution: creatinin o.I g
1. Water 1000.0 cc. sodium asparaginate 1.2 g,
2. Asparagin (0.1%) 1.0 g. histidine dichloride 0.3 g,
3. K.2HPO4 (0.5%) 5.0 g. NaCl 40 o-
4. Glucose (1.0%) 10.0 g. K2HPO4 3^0 g

5. Cyanide (0.3%) 3.0 g. MgS04 0.5 g
Reference: Kappen (1909 pp. 385, 391). KNO3 0.2 g
(c) Used 0.6 g. tryptophane in the basal
474. Davis and Ferry's Basal Cystine
solution and added:
Tryptophane Solution
tyrosine 0.8 g.
Constituents leucine 3.0 g.
1. Water 1000.0 cc. glutaminic acid hydrochloride 2.5 g.
2. Tryptophane 0.3 g. creatin 0.2 g.
3. Cystine 0.4 g. glycocoll 0.8 g.
Preparation histidine dichloride 0.5 g.
(1) Add 2 and 3 to 1. glucosamine hydrochloride. . 1.5 g.
sodium asparaginate 0.5 g. 2. K2HPO4 2.0g.

K2HPO4 3.0 g. 3. MgS04 1.0 g.
MgS04 0.4 g. 4. Glycocoll 1.5 g.
KNO3 0.2 g. 5. Sodium asparaginate 1-0 g.
(d) Used 1.25 g. tryptophane in the basal 6. Glutamic hydrochloride. . . 1.0 g.
solution and added: 7. Alanine 1-0 g.
histidine dichloride 1.25 g. 8. Histidine chlorohydrate . . . 1.0 g.
glucosamine hydrochloride. . . 2.75 g. 9. Tyrosine 0.5 g.
NaCl 2.50 g. 10. Tryptophane 0.5 g.
K2HPO4 3.0 g. 11. Leucine 0.6 g.
MgS04 0.4 g. 12. Phenylalanine 0.6 g.
(e) Used 0.6 g. tryptophane in the basal 13. Guanidine hydrochloride. . 0.4 g.
solution and added: 14. Creatine 0.4 g.
tyrosine 1.25 g 15. Glucosamine hydrochloride 0.4 g,
leucin 3.00 g 16. Cystine 0.2 g.
glutaminic acid hydrochloride 2.50 g Preparation
glycocoll 0.85 g (1) Dissolve 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
sodium asparaginate 1-00 g 12, 13, 14, 15 and 16 in 1.
histidin dichloride 0.5 g (2) Adjustment of reaction not specified.
creatin 0.2 g Sterilization: Sterilize with ether (method
creatinin 0.08 g not given).
xanthin 0.05 g Use: Toxin production hy Proteus vulgaris.
hypoxanthin 0.05 g, This medium will not permit successive
NaCl 4.0 g, growth of the organism.
K2HPO4 3.0 g Reference: Berthelot (1914 p. 917).
MgS04 0.50 g
476. Armand-Delille, et al, Glycocoll Argi-
glucose 1.5 g
KNO3 0.20 g
nine Solution (Synthetic Medium 164)
(f) Used tryptophane in the basal

1.6 g. Constituents:
solution and added: 1. Water 1000.0 cc.
glutaminic acid hydrochloride. 2.5 g. 2. NaCl 5.0 g.
glucosamine hydrochloride. 1.6 . . g. 3. KH2PO4 5.0 g.
NaCl 2.5 g. 4. Magnesium citrate 2.4 g.
K2HPO4 3.0 g. 5. Glucose 4.0 g.
MgS04 0.4 g. 6. Glycerol 40.0 g.
(g) Used 1.0 g. tryptophane in the basal 7. Glycocoll 4.0 g.
solution and added: 8. Arginine 2.0 g.
NaCl 2.50 g. Preparation
K2HPO4 3.00 g. (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
MgS04 0.4 g. (2) Add 4.0 cc. of N/100 NaOH to (1)
(h) Used 1.6 g. tryptophane in the basal after sterilization.
solution and added: Sterilization: Method not given.
glutaminic acid hydrochloride 2.5 g Use: Cultivation of Koch's bacilli.
glucosamine hydrochloride. 1.0 . . g Medium gives good growth.
histidine dichloride 1-20 g References: Armand-Delille, Mayer,
NaCl 2.5 g Schaeffer and Terroine (1913 p. 274),
K2HPO4 3.0 g Harvey (1921-22 p. 104).
MgS04 0.4 g,
477. Armand-Delille, et al, Synthetic
Reference: Davis and Ferry (1919 pp. 226,
Solution 104
227, 228, 229, 230).
Constituents:
475. Berthelot's Amino Acid Solution
1. Water 20.0 cc.
1. Water 1000.0 cc. 3. Magnesium citrate 0.6 g.
4. NaH.P04 1.25 g Preparation:

5. Glycocoll 0.50 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1 (amount
6. Aspartic acid 0.5 g. of asparagin not specified).
7. Carnosine nitrate 0.1 g, (2) Adjustment of reaction not specified.
8. Creatinin .. 0.1 g (3) Tube in 10.0 cc. lots.
9. Sarcosine 0.1 g Sterilization : Method not given.
10. Glucose 0.5 g Use: Study of indol production.
11. Inosite .. 0.1 g Variants

12. Glycerol 10.0 g (a) Zipfel used 0.3 or 0.5% tryptophane
In similar
(Water given as 20.0 cc. instead of 0.1%.
media 389 and 391 water given as (b) Barthel used 5.0 g. asparagin and
250.0 cc.) 0.3 g.Tryptophane.
Preparation (c) Barthel used 5.0 g. asparagin, 0.3 g.
11 tryptophane and omitted the 5.0 g.

(1) Dissolve 2, 3, 4, 5, 6, 7, 8, 9, 10,
and 12 in 1. ammonium lactate.
(2) Add 1.0 cc. of N/100 NaOH to (1) References: Zipfel (1912 p. 74), Barthel
after sterilization. (1921 p. 85).
Sterilization: Method not given. SUBGROUP I-C. SECTION 6

Use: Cultivation of Koch's bacilli.
Armand-Delille, Mayer, Liquid media, or basal solutions with
Reference:
constituents of known chemical composi-
Schaeffer and Terroine (1913 p. 273).
tion containing organic nitrogen other than
amino acids; amino acids not present.
478. Armand-Delille, et al, Glycocoll Al.'* Organic nitrogen supplied as cyanogen
Aspartic Acid Solution (Synthetic or its compounds.
Medium 118) Bi. Nitrogen supplied as cyanamide.
Kappen's Glucose Cyanamide Solu-
Constituents: 480
tion
1. Water 1000.0 cc.
Vierling's Glucose Cyanamide Solu-
2. NaCl 5.0 g.
tion 481
3. Magnesium citrate 2.4 g.
Bs. Nitrogen supplied as dicyanamide.
4. NaHiPO^ 5.0 g.
Perotti's Dicyandiamide Solution 482 . . ,
5. Glycocoll 2.0 g.
Dicyandiamide
Perotti's Glucose
6. Aspartic acid 2.0 g.
483
Solution
7. Glucose 2.0 g.
Dicyandiamide
Kappen's Glucose
8. Scombrine sulphate 2.0 g.
484
Solution
9. Glycerol 40.0 g.
Bs. Nitrogen supplied as ferrocyanamide.
Preparation
Doryland's Basal Ferrocyanide Salt
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1.
Solution 485
(2) Add 4.0 cc. of N/100 NaOH to (1)
Bokorny's Sucrose Ferrocyanide
after sterilization.
Solution 486
A2.'*Organic nitrogen supplied as urea.
Use: Cultivation of Koch's bacilli.
Bi. Incomplete or basal solutions, em-
References Armand-Delille,
: Mayer,
ployed with additional nutrients.
Schaeffer and Terroine (1913 p. 273).
Stutzer and Hartleb's Basal Urea
Solution 487
47Q. Zipfel's Tryptophane Asparagin Urea Salt Solution. 488
Beijerinck's Basal
Solution Waksman's Basal Urea Salt Solu-
tion 489
Constituents: Christensen's Basal Urea Salt Solu-
1. Distilled water 1000.0 cc. 490
tion
2. Asparagin Bj. Complete media.
Ci. Containing no additional organic com-
4. K,HP04 2.0 g.
pounds.
5. MgS04 0.2 g.
6. Tryptophane (0.1%) 1.0 g. *See also A3, A4, As and As on page 136.
Fremlin's Urea Solution 491 Variants:

Wherry's Urea Solution 492 (a) The author used 1.0 or 2.0% cyana-
Evans' Urea Solution 493 mide instead of 0.5%.
Percival's Urea Solution 494 (b) The author used 0.1, 0.5 or 1.0%
Ca. Containing additional organic com- glucose with 0.5% cyanamide.
pounds. Reference: Kappen (1910 p. 635).
Di. Carbohydrates added.
481. Vierling's Glucose Cyanamide Solution
Murray's Glucose Urea Solution .... 495
Vierling's Glucose Urea Solution. 496 . . . Constituents:
Thomas' Sucrose Urea Solution 497 1 Water 1000.0 cc.
Hugounenq and Doyon's Sucrose 2. K2HPO4 1.0 g.
Urea Solution 498 3. CaCl2 0.1 g.
D2. Alcohols added. 4. MgS04 0.1 g.
Hadley's Glycerol Urea Solution 499 5. FeCls trace
Harvey's Glycerol Urea Solution. 500 . . . 6. NaCl trace
A3. Organic nitrogen supplied as uric acid. 7. Calcium cyanamide 0.2 g.
Koser's Glycerol Uric Acid Solution. 501 . 8. Glucose 5.0 g.
Lohnis' Uric Acid Solution 502 Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
A4. Organic nitrogen supplied as amines in 1.
and amides. Sterilization: Not specified.

Bokorny's Amine Solution 503 Use: Cultivation of m3^cobacteria.
Omeliansky's Amine Solution 504 Reference: Vierling (1920 p. 202).
Beijerinck and Minkman's Glucose
482. Perotti's Dicyandiamide Solution
Amine Solution 505
As. Organic nitrogen supplied as hippuric Constituents:
acid. 1. Water 1000.0 cc.
Went's Basal Hippuric Acid Solution. 506 2. K2HPO4 0.5 g.
Stapp's Hippurate Solution 507 3. NaCl 0.5 g.
Lohnis' Hippurate Solution 508 4. CaS04 0.5 g.
Ae. Organic nitrogen supplied other than 5. MgS04 0.5 g.
Ai to As. 6. Iron chloride drops
Stoklasa's Glucose Lecithin Solution. 509 7. C2N4H4 (dicyandiamide)... 0.5 g.
Stoklasa's Nucleic Acid Solution. 510 . . . Preparation
Went's Basal Creatin Solution 511 (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Stutzer's Glucose Creatin Solution. 512 . (2) Distribute in flasks.

Meyers' Taurine Solution 513 Sterilization: Sterilize on 3 successive days
Wherry's Theobromin Solution 514 at 120^C. for 10 minutes, each day.
Bokorny's Picric Acid Solution 515 Use: To study influence of dicyandiamide
on field plants. Author reported that the
480. Kappen's Glucose Cyanamide Solution nitrogen content was increased. As a
Constituents control KNO3 was used instead of
1. Water 1000.0 cc. dicyandiamide.
2. Glucose (1.0%) 10.0 g. Reference: Perotti (1908 p. 225).
3. KH2PO4 (0.5%) 5.0 g.
483. Perotti's Glucose Dicyandiamide
4. Cyanamide (0.5%,) 50 g.
Solution

Use: To study decomposition of cyanamide 1. Water 1000.0 cc.
by Penicillium brevicaule, Stysanus 2. dicyandiamide 2.0 g.
stemonitis and Cladosporium. Cyana- 3. Dextrose 0.1 g.
mide in 2.0% strength was toxic for some 4. K2HPO4 0.5 g.
of the types studied. Using 0.5% cyana- Preparation
mide and varying the dextrose content, (1) Dissolve 2, 3 and 4 in 1. (Use tap
the most NH3 was produced from the water or distilled water, with traces
cyanamide with 0.5 to 1.0% dextrose. of NaCl, Na2S03 and CaClj.)
(2) Distribute 200.0 cc. of 1 in a one liter distilled water, using phenolphthalein
flask. as indicator.
(3) Inoculate with 0.2 g. of garden earth. (2) Add to HCl the following salts:
Sterilization: Not specified. MgS04 0.5 g.
Use Cultivation of dicyandiamide bacteria
: Fe2(S04)3 0.01 g.
from the soil. CaO 0.01 g.
Variants: The author gives the following MnS04 0.01 g.
solution: K6Fe2(CN)i2, amount not given.
1. Water 1000.0 cc. (3) Standardize (2) against silicate solu-
2. Potassium phosphate 1.0 g. tion so that 1.0 cc. is equivalent to
3. CaCl. 0.1 g. 1.0 cc. using methyl orange as indi-

4. MgS04 0.3 g. cator.
5. NaCl 0.1 (4) Standardize a solution of H2SO4 in
6. Iron chloride 0.01 same way as HCl omitting salts.
7.Glucose (0.5%) 5.0 g. (5) Standardize H3PO4 in similar manner
8. Dicyandiamide 0.1, 1.0, 5.0 or 10.0%. as HCl omitting the salts and using
Reference: Perotti (1908, vol. 21, p. 207), phenolphthalein as indicator.
(1908, vol. 20, p. 516). (6) ]\Iix the acids in the following ratio:
HCl 153.5 cc.
484. Kappen's Glucose Dicyanamide H2SO4 77.0 cc.
Solution H3PO4 116.0 cc.
Constituents:
1. Water 1000.0 cc.
KOH and N/0.2578 NaOH.
(8) 1.0 cc. of (7) should neutralize 1.0 cc.
2. K2HPO4 (0.05%) 5.0 g.
of (6), phenolphthalein as indicator.
3. MgS04 (0.5%) 5.0 g.
4. Dicyanamide (1.0%) 10.0 g.
(9) Draw acid and base solution into
separate burettes and allow to stand
5. Glucose (10.0%) 100.0 g.
several hours to sterilize.

Sterilization: Method specified in prepa-
ration.
Use: To study decomposition of cyana-
Use: Synthetic culture medium.
mide by Penicillium bremcaule, Stysanus
stemonitis and Cladosporium.
Added nutrients: Acetic acid or glucose
were used as additional carbon source.
Reference: Kappen (1910 p. 637).
When using acetic acid mix with H3PO4
485. Doryland's Basal Ferrocyanide Salt
in step (5). When using glucose add a
sufficient quantity of a sterile aqueous
Solution
glucose solution to a mixture of equal
Constituents: parts of (6) and (7) so that there will be
1. Distilled water 500.0 cc. 10.0 g. of glucose per liter of solution.
2. HCl dilute Reference: Doryland (1916 p. 148).
3. MgS04 0.5 g.
4. CaO 0.01 g. 486. Bokomy's Sucrose Ferrocyanide
5. Fe2(S04)., 0.01 g. Solution
6. MgS04 0.01 g. Constituents:
7. KGFe2(CN)i2, amount not given. 1. Water 1000.0 cc.
8. H2SO4 2. Sucrose 30.0 g.
9. H3PO4 3. Potassium ferrocvanide
N/0.2578 NaOH
^
10. (K4Fe(CN),) 60 g.
11. N/0.6205 KOH 4. K2HPO4 1.0 g.
(1) Dilute the HCl so that 1.0 cc. is not 6. CaCl2 0.08 g.
quite neutralized by 1.0 cc. of silicate Preparation: (1) Dissolve 2, 3, 4, 5 and 6
solution made by dissolving 24.0 g. in 1.
KSiOi and 8.4 g. NajSiOa in 500.0 cc. Sterilization: Not specified.

Use: Cultivation of Schizomycetes. Au- (e) Sohngen used 0.05% K2HPO4 and
thor reported that mold spores did not 3.0% urea in the basic solution and
develop in this medium. Schizomycetes added 1.0% of calcium malate or cal-
developed producing lactic acid. The cium citrate to study urea decomposi-
acid produced may decompose the tion by bacteria from the soil.
K4Fe(CN)o. Potassium ferrocyanide was (f) Sohngen used a 0.5% urea and 0.05%
a suitable nitrogen source for schizomy- K2HPO4 solution without additions
cetes but not for molds. to cultivate Bacillus erythrogenes.
Variants Bokorny also used the following
: (g) Percival used 5.0% urea and 0.25%
solution: K2HPO4 in the basic solution and
1. Water 1000.0 cc. added 1.0% (NH4)2S04 to enrich
2. Sucrose 20.0 g urea splitting organisms found in the
3. Potassium ferrocyanide . . 2.0 g soil and in urine.
4. K2HPO4 2.0 g (h) Giltner used 5.0% urea and 0.05%
5. MgS04 0.052 g K2HPO4 in the basic solution and
6.CaCl2 0.012 g added 0.5 to 1.0% of one of the
Reference: Bokorny (1917 pp. 343, 344). following:
ammonium malate
487. Stutzer and Hartleb's Basal Urea calcium malate
Solution calcium citrate
Constituents: ammonium citrate
1. Water . 1000.0 cc. He used the media to show urea
2. Urea . 20.0 g. decomposition.
3. Glycerin . 10.0 g. References: Stutzer and Hartleb (1897
4. Potassium phosphate. 1.0 g. p. 404), Sohngen (1909 pp. 91, 93, 94, 95),

Percival (1920 p. 225), Giltner (1921 p.
Sterilization: Not specified. 314).
Use: Cultivation of the organisms from

488. Beijerinck's Basal Urea Salt Solution
cases of foot and mouth disease.
Added nutrients and variants: Constituents:
(a) The authors used the basic solution 1. Water 1000.0 cc.
as a medium. 2. Urea 5.0 g.
(b) Sohngen used 5.0% urea and 0.025 3. KH2PO4 0.025 g.
K2HPO4 as the basic solution and Preparation
added 1.0% of one of the following: (1) Dissolve 2 and 3 in 1.
ammonium oxalate (2) Add 1.0% of one of the added nu-

sodium acetate trients.
Rochelle salt Sterilization: Not specified.
ammonium citrate Use: Cultivation of urea organisms from
ammonium malate the soil, Urobacillus pasteurii, Urococcus
He used the media to study urea ureae. Author reported that about 2.0%
decomposition by bacteria from the of the urea was changed by a soil culture
soil. using o.xalate as carbon source, 2.0%
(c) Sohngen used 0.05% K2HPO4 and using sodium acetate, 2.0% using Seig-
2.0% urea in the basic solution and nette salt, 3.0% using ammonium citrate
added 5.0 to 61.5 mg. of nitrogen in and 4.% using ammonium malate.
the form of asparagin or ammonium Added nutrients and variants:
malate to study urea decomposition (a) 1.0% of one of the following carbon
by Urobacillus Jakschii. sources were added:
(d) Sohngen used 0.05%, K2HPO4 and ammonium oxalate 1-0 g.
5.0% urea in the basic solution and sodium acetate

added 5.0 to 61.5 mg. of nitrogen in Seignette salt
the form of asparagin or ammonium (potassium sodium tartrate) 1.0 g.
malate to study urea decomposition Ammonium citrate 1-0 g.
by Bacillus erythrogenes. Ammonium malate 1-0 g.

(b) Lohnis used 50.0 g. urea and 0.5 g. Sterilization: Not specified.
K2HPO4 in the basic solution, and Use: To study urea decomposition by soil
added 0.5 to 1.0% ammonium or cal- forms. Ammonia formation was appar-
cium malate, or calcium citrate or ent after 14 days when humus was added
tartrate. and after 5 days with glucose. If humua
(c) Percival used 2.5% KH2PO4 and 5.0% or glucose were not added, there was
urea and added 1.0% sodium acetate. apparently no ammonia formation.
References: Beijerinck (1901 p. 37), Lohnis Added nutrients: One of the following ma-
(1913 p. 108), Percival (1920 p. 225). terials were added:
(a) Humus acid small amount. Add
489. Waksman's Basal Urea Salt Solution
NaOH
sufficient dilute to keep the
Constituents: humus acid in solution.
1. Water 1000.0 cc. (b) Potassium salt of humus acid 2.0%.
2. Glycerol 30.0 g. (c) Glucose 1.0%.
3. K2HPO4 1.0 g. (d) "Buchenrohhumas" 0.1%.
4. KCl 0.5 g. (e) Xylose 1.0%.
5. MgS04 0.5 g. (f) Sodium formate 0.2%.
6. FeS04 0.01 g. (g) Meat extract 2.0%.
7. Urea 5.0 g. (h) Potassium humate 0.1% (see (8)
Preparation below),
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (i) Humin 0.2% (see (10) below).
(2) Add 2.0 g. of one of the added nu- Materials in (h) and (i) prepared as
trients. follows:
(3) Tube in 10 to 12 cc. quantities. (1) Dissolve 300.0 g. saccharose in
Sterilization: Sterilize at 15 pounds for 420.0 cc. water.
15 minutes. (2) Add 15.0 g. concentrated H2SO4
Use: To study metabolism of Actinomij- to (1).
cetes. (3) Place on a boiling water bath for
Added nutrients: Waksman added 2.0 g. 7 or 8 hours, adding the water lost
of one of the following nitrogen sources: by evaporation.
NaN03 (NH4).S04 (4) After completing the heating a
NaN02 (NH4)2C03 voluminous sediment forms at the
Reference: Waksman (1920 p. 3). bottom.
(5) Filter and carefully wash.
490. Christensen's Basal Urea Salt with an
(6) Treat the precipitate
Solution
excess of 5.0% KOH solution. A
Constituents: portion of the humus acid is dis-
1. Distilled water 1000.0 cc. solved. The humin does not

2. Urea 20.0 g. dissolve.
3. K2HPO4 (1.0% soln.) 20.0 cc. (7) Filter.
4. CaCOa (8) Utilize the filtrate to prepare a
5. MgS04 small amount neutral potassium humate (method
6. FeS04 small amount not given).
Preparation (9) Wash the precipitate to remove all
(1) Prepare a 1.0% solution of K2HPO4. traces of humus acid.

(2) Add 2.0 g. of urea (Kahlbaum's I), (10) Dry (9) and pulverize humin.
2.0 cc. of (1), some CaCOa, and very Reference: Christensen (1910 pp. 340, 348).
small quantities of MgS04 and FeS04
(amount not specified) to 1000.0 cc.
491. Fremlin's Urea Solution
distilled water.
(3) Add one of the added nutrients. Constituents
(4) Distribute into 450.0 cc. Erlenmeyer 1. Water 1000.0 cc.
flasks. 2. (NH4)2S04 1.0 g.
(5) Inoculate with a little soil infusion. 3. Potassium phosphate . 1.0 g.

4. MgC03 10.0 g. 2. (NH4)2S04 2.0 g.

5. Urea 0.1 to 1.0 g. 3. K2HPO4 1.0 g.
Preparation 4. NaCl 2.0 g.
(1) Dissolve 2 and 3 in part of 1. 5. MgS04 0.5 g.
(2) Dissolve 4 in remainder of 1. 6. FeS04 0.4 g.
(3) After sterilizing (1) and (2) mix 7. MgCOs 10.0 g.
under aseptic conditions. 8. Urea (0.01%) 0.1 g.
(4) Add various quantities (0.1 to 1.0 g.) Preparation
sterile urea. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Sterilization: Method not specified. (2) Distribute in 50.0 cc. into conical
Use: To study nitrogen oxidation by flasks.
Nitrosohacter. (3) Add 0.5 g. of basic MgCOj to each
Reference: Fremlin (1903 p. 369). flask.
(4) To each flask add 0.01% urea.
492. Wherry's Urea Solution Sterilization: Not specified.
Constituents: Use: To study nitrification by soil forms.
1. Redistilled water 1000.0 cc. Variants: Percival added 0.03, 0.3, 1.0 or
2. NaoCOs 1.0 g. 3.0% urea to each flask.
3. KCl 1.0 g. Reference: Percival (1920 p. 144).
4. MgS04 1.0 g.
495. Murray's Glucose Urea Solution
5. CaCl2 1.0 g.
6. Urea 2.0 g. Constituents:
7. KH2PO4 2.0 g. 1. Water 1000.0 cc.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. 3. K2HPO4 50.0 g.
(2) Adjustment of reaction not given. 4. MgS04 0.5 g.
Sterilization: Sterilize in autoclave, time 5. Urea 10.0 g.
not given. Preparation
Use To study acid proofness of B. tubercu-
: (1) Dissolve 2, 3, 4 and 5 in 1.
losis. Author reported that after the (2) Tube.

sixth day all rods were non-acid-fast. Sterilization: Not specified.
Reference: Wherry (1913 p. 151). Use: To study ammonification under either
anaerobic or aerobic conditions.
493. Evans' Urea Solution Reference: Murray (1916 p. 602).
Constituents:
496. Vierling's Glucose Urea Solution
1. Water 1000.0 cc.
3. K2HPO4 2.0 g. 1. Water 1000.0 cc.
4. Urea 2. K2HPO4 lOg.
Preparation 3. CaCl2 0.1 g.
(1) Dissolve 2 and 3 in 1. 4. MgS04 0.1 g.
(2) No adjustment of reaction given. 5. FeCla trace

(3) Tube. 6. NaCl trace
(4) After sterilization add a small piece 7. Glucose 10.0 g.
of urea to each tube. 8. Urea 10.0 g.
Sterilization: Method of sterilization of the Preparation: (1) Dissolve 2, 3, 4, 5, 6,
solution not specified. Sterilize a piece 7 and 8 in 1.
of solid ureaby heating for 5 hour in a Sterilization: Not specified.

toluene oven at 105C. Use: Cultivation of mycobacteria.
Use: To determine decomposition of urea. Reference: Vierling (1920 p. 206).
Reference: Evans (1916 p. 445).
497. Thomas' Sucrose Urea Solution
494. Percival's Urea Solution Constituents
1. Water. 1000.0 cc. 2. Sucrose 200.0 g.

3. KH2PO4 2.25 g. 500. Harvey's Glycerol Urea Solution

4. MgS04 0.45 g.

2, 3
2. NaCl 0.02 g.
(2) Prepare a 20.0% urea solution in 3. MgSOi 0.02 g.

water. 4. K2HPO4 0.5 g.
(3) Add 20.0 cc. of sterile (2) to cool 5. FeCla trace

sterile (1) under aseptic conditions. 6. Glycerol 10.0 g.
Sterilization: Sterilize (1) at 120C. Steril- 7. Urea 1.0 g.
ize (2) by filtration. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7

Use: Cultivation of yeast. in 1.
Reference: Thomas (1919 p. 800). Sterilization: Not given.

As other
498. Hugounenq and Doyen's Sucrose Urea sources of carbon, saccharose, glucose,
Solution mannite, calcium lactate, etc., were used.
Constituents: As other sources of nitrogen, sodium
1. Water 1000.0 cc. hippurate, uric acid, asparagin, ammo-
2. Glycerol 45.0 g.
nium sulphate, ammonium acetate, am-
3. NaCl 7.0 g. monium butyrate, and ammonium lac-
4. Ammonium lactate 10.0 g. tate were used.
5. CaClo 0.1 g. Reference: Harvey (1921-22 p. 104).
6. MgS04 0.2 g.
7. Potassium biphosphate 2.0 g. 501. Koser's Glycerol Uric Acid Solution
8. Uric acid 0.02 g.
10. Sucrose 5.0 g. 1. Distilled water (NH3 free). 1000.0 cc.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8, 2. NaCl 5.0 g.
9 and 10 in 1. 3. MgS04 0.2 g.
Sterilization: Not specified. 4. CaCU 0.1 g.
Use: General culture medium. Reported 5. K2HPO4 1.0 g.
as not being a suitable medium for 6. Glycerol 30.1 g.
diphtheria bacilli. 7. Uric acid 0.5 g.
Reference: Hugounenq and Doyon (1896 Preparation
p. 401). (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.

499. Hadley's Glycerol Urea Solution
(3) Tube.
Constituents: Sterilization: Tube and sterilize at 13 to
1. Distilled water 100.0 cc. 15 pounds extra pressure for 15 minutes.
2. Ammonium lactate 3.2 g. A slight turbidity appears after auto-
3. CaCl, 0.05 g. claving but upon cooling the solution
4. Glycerol 15.0 g. becomes clear.
5. MgS04 0.15 g. Use: B. aerogenes soon produces a dense
6. K0HPO4 1.25 g. clouding. B. coli fails to develop. Care
7. NaCl 3.0 g. must be taken to keep the medium
8. Urea 1.6 g. ammonia free.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, Variants Ecker and Morris used 0.2 g. uric
:
7 and 8 in sufficient water to make ac'd instead of 0.5 g.

100.0 cc. References: Koser (1918 p. 377), Levine
Sterilization: Not specified. (1920 p. 118), Harvey (1921-22 p. 104),
Use: Growth of Bacillus diphlheriae and Ecker and Morris (1924 p. 480), Com.
production of toxin. American Public Health Asso. (1925
Reference: Hadley (1907 p. 101). p. 112).
502. Lohnis' Uric Acid Solution 2. Potassium phosphate 10 g.
Constituents :
3. MgSOi 0.5 g.
4. NaCl 2.0 g.
5. Methyl amine 0.5 g.
2. Uric acid 3.0 g.
Preparation :
3. K2HPO4 0.5 g.
Preparation :
(3) Add the usual amount of sterile
(2) Distribute in small Erlenmeyer flasks.
MgCOs to the sterile fluid.
Sterilization: Sterilize by heating at 115C.
Use: Cultivation of B. ^Morescens and uric
for 30 minutes.
acid bacteria.
Use: To
study nitrification by nitrite
formers. The methyl amine or dimethyl
503. Bokorny's Amine Solution amine was not oxidized.
Variants: The author used 0.5 g. dimethyl-
Constituents :
amine instead of methyl amine.

1. Water 1000.0 cc.
2. K.HPO4 2.5 g.
3. MgP04 0.2 g.
505. Beijerinck and Minkman's Glucose
4. CaCh 0.05 g.
Amine Solution
5. Methyl amine hydro-
chloride (0.5%) 5.0 g.
Constituents:
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Glucose amine (0.5%) 5.0 g.
Use: To study the constituents essential 3. KNO3 10.0 g.
for growth of molds. Molds grew readily Preparation: (1) Dissolve 2 and 3 in 1.
on this medium. Sterilization: Not specified.
Variants: The author used the following Use : To study denitrification by soil forms,
amines and amides instead of methyl Bacillus nitroxus.
amine hydrochloride: Reference: Beijerinck and Minkman (1910
(a) 1.0% propyl amine hydrochloride. p. 37).
Certain molds developed on this

506. Went's Basal Hippuric Acid Solution
medium.
(b) 0.5% oxamide. Yeast did not grow Constituents:
in this medium. 1. Water 1000.0 cc.
(c) The salt concentration was changed 2. Hippuric acid (0.66%) 6.6 g.
and materials added. The medium Preparation :
used had the following composition: (1) Prepare a 0.66% solution of hippuric
1. Water 1000.0 cc. acid.
2. Trimethyl amine (2) Prepare a 5.0% solution of one of the
acetic acid 5.0 g. added nutrients.
3. Sucrose 50.0 g. (3) Mix equal volumes of (1) and (2).
5. MgSOi 0.2 g. Use: Cultivation of Monilia sitophila
6. CaCl2 0.2 g. (Mont.) Sacc.Maltose, glucose, lactose,
H3PO4
7. 0.0 or 1.0% sucrose and glycerol was the order of
Trimethyl amine served as a nitrogen vigor of growth for the carbon sources
source for bacteria and without the studied.
H3PO4 Schizomycetes developed then Added nutrients: One of the following
the molds. In the presence of H3PO1 carbon sources was employed:
molds developed at once. maltose glucose
Reference: Bokorny (1917 pp. 337, 338). lactose glycerol
sucrose
504. Omeliansky's Amine Solution
Variants: No additional carbon source was
Constituents : added. No growth occurred.
1. Water 1000.0 cc. Reference: Went (1901 p. 593).
507. Stapp's Hippurate Solution Use: To study the cycle of the phosphate
ion in the soil. Author used Bacillus
Constituents:
mycoides, Bacillus subtilis, Bacillus pro-
1. Water 1000.0 cc.
teus vulgaris. Amount of phosphoric
2. KH2PO4 1.25 g.
anhydride formed by each organism was
3. MgSOi 0.625 g.
about the same after 60 days. If the
4. Sodium hippurate 3.1 g.
Preparation
NH4NOJ be omitted the nitrogen con-
tent is too low to give good growth.
Variants: Author omitted the NH4NOS
and used only 0.8 g. NaCI instead of 1.0 g.
Erlenmeyer flasks.
Reference: Stoklasa (1911 pp. 423, 424).
Use: Isolation of uric acid splitting bac-
510. Stoklasa's Nucleic Acid Solution
teria from feces and soil. Bac. cobayae,
Bac. capri, Bad., guano, Bac. musculi, Constituents:
Bac. hollandicus. 1. Distilled water 8000.0 cc.
Reference: Stapp (1920 p. 4). 2. K2SO4 8.0 g.
3. MgCli 4.0 g.
508. Lohnis' Hippurate Solution
4. NaCl 1.0 g.
Constituents: 5. Al2(S04)3 + FeS04 0.12 g.
1. Water (tap) 1000.0 cc. 6. Nuclein acid 8.0 g.
2. Sodium hippurate 3.0 g. 7. Glucose 80.0 g.
3. K2HPO4 0.5 g. Preparation:
Preparation (1) Prepare nucleic acid from jêast by
(1) Dissolve 2 and 3 in 1. Herlant's method (method not given)
(2) Distribute in small Erlenmeyer flasks. (2) Dissolve 2, 3, 4 and 5 in 1.
Sterilization: Not specified. (3) Distribute in 250.0 cc. lots in large
Use: Cultivation of hippuric acid bacteria. Erlenmeyer flasks.
Author reported that fermentation took (4) Add 0.25 g. nucleic acid and 2.5 g.
place rather slowly when the medium was d-glucose to each flask.
inoculated with manure. (5) Heat at 45 to 50C. for 20 hours.
Reference: Lohnis (1913 p. 96). Sterilization: Method not given.
509. Stoklasa's Glucose Lecithin ion in the soil. Author used Bacillus
Solution mycoides, Bacillus subtilis, Bacillus
Constituents: proteus vulgaris. Bacillus mycoides util-
1. Distilled water 8000.0 cc ized the largest amount of phosphorous.
2. NH4NO3 20.0 Reference: Stoklasa (1911 p. 428).
g
3. K2SO4 8.0 g
511. Went's Basal Creatin Solution
4. MgClj 4.0 g
5. NaCl 1.0 g Constituents
6. Al2(S04)3 + FeS04 0.12 g 1. Water 1000.0 cc.
7. d-glucose 80.0 2. Creatin (0.66%) 6.6 g.
8. lecithin 8.0 Preparation
Preparation (1) Prepare a 0.66% creatin solution.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (2) Prepare a 5.0% solution of one of the
(2) Distribute into large 2 liter Erlen- added carbohydrates.
meyer flasks in 250.0 cc. lots. (3) Mix equal volumes of (1) and (2).
(3) Adjustment of reaction not specified. Sterilization: Not specified.
(4) After sterilization add 0.25 g. leci- Use: Cultivation of Monilia sitophila
thin to each flask. Heat at 45 to (Mont.) Sacc. Maltose, glucose, lactose,
50 for 20 hours. sucrose and glycerol was the order of
Sterilization: Sterilize thoroughly (method vigor of growth for the carbon sources
not given). studied.
Added nutrients: One of the following car- Sterilization: Sterilize in autoclave, time
bon sources was employed: not given.
maltose sucrose Use: To study acid proofness of B. tuber-
glucose glycerol culosis. On 19th day rods and diplococ-
lactose coids were non acid proof. No growth
Variants: The author used the basic solu- using caffein instead of theobromin.
tion withoutany added materials. Reference: Wherry (1913 p. 151).
515. Bokorny's Picric Acid Solution
512. Stutzer's Glucose Creatin Solution
Constituents:
Constituents: 1. Water 1000.0 cc.
1. Water 1000.0 cc. 2. Ca(N03) 2 (0.02%) 0.2 g.
2. Creatin 2.5 g. 3. KH2PO4 (0.02%) 0.2 g.
3. K2HPO4 1.0 g. 4. MgS04 (0.02%) 0.2 g.
4. KNO3 3.0 g. 5. Picric acid (0.5%) 5.0 g.
Preparation Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
(1) Dissolve 2, 3 and 4 in 1. Sterilization: Not specified.

(2) Reaction to be slightly alkaline. Use: Cultivation of fungi, algae, amobae
(3) Distribute in 10.0 cc. lots. and infusoria. No growth with 0.5%
Sterilization: Method not given. or 0.1% picric acid.
Use: To study denitrification by B. agilis, Variants: Bokorny used 0.1, 0.05, or 0.01%
B. nitrovorus, B. Stutzeri, B. Hartlebi. picric acid instead of 0.5%.
Nitrate is not reduced. Reference: Bokorny (1917 p. 346).
Variants : The author added 10.0 g. glucose.
Liquid media or basal solutions, contain-

513. Myers' Taurine Solution
ing organic carbon only as commercial
Constituents digests; additional material, if any, in-
1. Distilled water 1000.0 cc. organic.
2. Taurine 5.0 g. Ai. Not containing peptone.
Preparation Robinson and Rettger's Opsine Solu-
(1) Prepare taurine according to Hawk tion 516
(1918). Linde's Nahrstoff-Heyden Solution. . 517
(2) Dissolve 2 in 1. Lichtenstein's Cenovis Solution 518
(3) No adjustment of reaction specified. A2. Containing peptone.
(4) Suspend a sterile piece of filter paper, Bi. Containing no added salts. (Exclusive
that has been soaked in 10.0% solu- of indicators or dyes.)
tion of lead acetate, in each flask. Dunham's Peptone Solution (Comm.
Sterilization : Filter thru a Berkefeld filter. A. P. H. A.) 519
Use: To study HoS production. Levine's Crystal Violet Peptone Solu-
Variants: Myers added 10.0 g. glucose. tion 520
Reference: Myers (1920 p. 249). Signorelli's Indicator Peptone Solu-
tion 521
514. Wherry's Theobromin Solution
Steam's Gentian Violet Peptone
Constituents: Solution 522
1. Redistilled water 1000.0 cc. Rivas' Trypsinized Peptone Solu-
2. NaoCOs 1.0 g tion 523
3. CaCls 1.0 g B2. Containing added salts.
4. KCl 1.0 g Ci. Containing salts of monovalent cations
5. MgS04 1.0 g only.
6. Theobromin 0.05 g Di. Only one salt added
7. KH2PO4 2.0 g El. Sodium chloride only salt added.
Preparation Fi. No indicator added.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. Dunham's Peptone Solution 524
(2) Adjustment of reaction not given. Huss' Peptone Solution 525
Nencki's Peptone Solution 526 Will's Peptone Solution 555

Brussoff's Peptone Solution 527 Gersbach's Trypsinized Peptone
Redfield's Peptone Solution (Meyers). 528 Solution 556
Berman and Rettger's Peptone Solu- D2. Not containing calcium.
tion 529 El. Containing iron salts.
Migula's Peptone Solution 530 Kappen's Peptone Solution 557
Heap and Cadness' Basal Peptone Percival's Ammonium Sulphate Pep-
Solution 531 tone Solution 558
F2. Indicator added. E2. Not containing iron salts.
Browning, Gilmour and Mackie's Fremlin's Ammonium Sulphate Pep-
Brilliant Green Peptone Solution. 532 . tone Solution 559
Harvey's Telluric Acid Peptone Solu- Mortensen's Peptone Solution 560
tion 533 Kligler's Nitrate Peptone Solution. . 561
Smith's Rosalie Acid Peptone Solu- Percival's Phosphate Peptone Solu-
tion 534 tion 562
Bronfenbrenner's China Blue Pep- Capaldi and Proskauer's Peptone
tone Solution 535 Solution 563
E2. Potassium (or sodium) nitrate onlj' Ritter's Peptone Solution 564
salt added. Speakman's Basal Peptone Solution
Diedudonne's Nitrate Peptone Solu- (McCoy et al.) 565
tion 536
Harvey's Nitrate Peptone Solution. 537 .
516. Robinson and Rettger's Opsine
Solution
E3. A salt other than Ei or E2 added.
Cohen and Clark's Phosphate Pep- Constituents:
tone Solution 538 1. Water 1000.0 cc.
Enlow's Basal Agar Peptone Solu- 2. Opsine (1.0%) 10.0 g.
tion 539 Preparation
Beijerinck's Ammonium Sulphate (1) Dissolve 1.0% opsine in water over a
Peptone Solution 540 free flame.
Redfield's Peptone Solution (Tan- (2) Adjust the reaction to -|-0.6 to phenol-
ner) 541 phthalein.
Nicolle, Raphael and Debains' Pep- (3) Boil two or three minutes.
tone Solution 542 (4) Filter and distribute in 20.0 cc. lots
Levine's Boric Acid Peptone Solu- Erlenmeyer flasks.
in 150.0 cc.
tion 543 Sterilization: Sterilize at 12 to 14 pounds
D2. More than one salt added. pressure for 15 minutes.
El. Containing phosphates. Use: Cultivation of B. diphtheriae and
DeBord's Phosphate Peptone Solu- study of to.xin production. Using 2%
tion 544 opsine M.L.D. = 0.7 cc.
Stutzer's Nitrate Peptone Solution.. 545 Variants
Kendall, Day and Walker's Fat Free (a) Used 2.0% opsine.
Peptone Solution 546 (b) Used 1.0% or 2.0% opsine with 1.0%
Kligler's Peptone Solution 547 glucose, and 0.5% NaCl.
Klecki's Basal Ammonium Phosphate (c) Used 1.0% or 2.0% opsine with 0.5%
Peptone Solution 548 NaCl.
E2. Not containing phosphates. Reference: Robinson and Rettger (1917
Pergola's Nitrate Peptone Solution. . 549 p. 363).
Klimmer's Nitrate Peptone Solution. 550
Bleisch's Nitrate Peptone Solution. 551 .
517. Linde's Nahrstofif Heyden Solution
Logie's Nitrite Peptone Solution. . 552

. . Constituents:
C2. Containing salts of divalent cations. 1. Water 1000.0 cc.
Di. Containing calcium. 2. Niihrstoff Heyden 5.0 g.
Gottheil's Peptone Solution 553 Preparation: (1) Dissolve 2 in 1.
Guyenot's Peptone Solution 554 Sterilization: Not given.

Use: Cultivation of Cladothrix. Author Merck peptone solution to study

reported good growth for 2 or 3 days. decomposition of peptone by bac-
Then growth stopped. teria from the soil,
Reference: Linde (1913 p. 386). (d) Linde used a 0.1 or 0.5% peptone
solution to cultivate Cladothrix.
518. Lichtenstein's Cenovis Solution Good growth was obtained for 2 or
Constituents 3 days.
1. Water References: Committee A. P. H. A. (1905
2. Cenovis p. 110), Heinemann (1905 p. 25), Buhlert
Preparation and Fickendey (1906 p. 400), Molisch

(1) Add cenovis (a fine yellow yeast (1910 p. 36), Remy and Rosing (1911 p. 39),
preparation) to water (amount not Linde (1913 p. 386), Tanner (1919 p. 47).
given). Mix well.
Peptone
520. Levine's Crystal Violet
(2) Allow to stand at room temperature
Solution
for 30 minutes and then steam for
2 hours. Constituents:
(3) Neutralize with soda solution and 1. Water 1000.0 cc.
sterilize pin like steamer for another 2. Peptone (1.0%) 10.0 g.
hour. 3. Crystal violet

(4) Filter. Filtrate is clear golden yel- Preparation
low fluid. (1) Dissolve 2 in 1.
Sterilization: Not specified. (2) Add varying amounts of crystal
Use: General culture medium. Substitute violet.
for meat peptone. Sterilization: Not specified.
Reference: Lichtenstein (1923 p. 390). Use: Differentiation of Bad. coli and Bad.
aerogenes. The author reported that
519. Dunham's Peptone Solution (Committee 1-100,000 crystal violet prevented growth
A. P. H. A.) of Bod. coli, while Bad. aerogenes grew
Constituents: heavily. Decreasing the concentration
1. Water (tap) 1000.0 cc. of peptone to 0.5% increased markedly
2. Peptone 10.0 g. the inhibitory action of the dye.
Preparation Reference: Levine (1911 p. 22).
(1) Weigh a sauce pan and measure Peptone Solution

521. Signorelli's Indicator
1000.0 cc. of tap water into it.
(2) Dissolve 10.0 g. of peptone in (1) Constituents:

when hot. 1. Peptone water 1000.0 cc.
(3) Make up the loss due to evaporation 2. Dahlia (1.0% soln.) 5.0 cc.
by adding water. Preparation

(4) Filter twice thru the same filter. (1) Exact composition of peptone water
(5) Distribute into tubes. not given.
Sterilization: Autoclave for 10 minutes (2) Adjust (1) to neutral.
at 120C. (3) Tube in 10.0 cc. lots.
Use: General culture medium. Indol test. (4) Add 0.05 cc. of 1.0% dahlia solution
Variants to each tube.
(a) Buhlert and Fickendey used 1.5% Sterilization: Not specified.
peptone solution to show peptone Use: To show adsorption of dye by cholera

decomposition by bacteria from the vibrio. The author reported that the
soil. vibrios developed rapidly and fell to the
(b) Molisch used a 0.05% manganese bottom of the tube. They were highly
peptone solution for the cultivation colored with dahlia and the medium was
of iron bacteria Leptothrix ochraceae, decolorized. With the other dyes the
etc. vibrio developed rapidly but did not
(c) Remy and Rosing used a 0.8% solu- color so strongly. The media were not
tion of Witte's peptone or a 1.0% decolorized.
Variants: Signorelli prepared 1.0% solu- 524. Dunham's Peptone Solution

tions of erythrosin or sapronia and
Constituents:
added 1.0 cc. of one of the solutions to
1. Water 1000.0 cc.
each tube of culture medium.
2. NaCl (0.5%) 5.0 g.
Reference: Signorelli (1912 p. 472).
3. Peptone (1.0%) 10.0 g.
522. Steam's Gentian Violet Peptone Preparation
Solution (1) Autoclave 1, 2 and 3 to two atmos-
pheres.
Constituents:
(2) Filter thru paper.
1. Water 1000.0 cc Sterilization: Not specified.
2. Peptone 20.0 g. Use: Detection of cholera vibrio. Author
3. Gentian violet 1:200,000 reported that when concentrated H2SO4
Preparation
was poured down the side of a tube con-
taining a culture of the cholera bacillus,
(2) Add 1 to 200,000 parts gentian violet.
a red ring developed where the solutions
(3) pH from 6.8 to 6.5.
came together. A 5.0 or 10.0% peptone
solution gave better and quicker results.
Use: To study behavior of the colon aero-
Variants
genes group with dyes. Author reported (a) Dunham used 5.0 or 10.0% peptone.
that the more basic the medium the more
(b) Smith used the medium to determine
of the basic dye was absorbed by bac-
indol and phenol production.
terial protein.
(c) Sears used 2.0% peptone.
Variants:The author added 2.0 g. lactose. (d) Myers used 3.0% Witte, Difco or
Reference: Stearn and Stearn (1923 p. 568).
Fairchild peptone. Used to study
production of HjS.
523. Rivas' Trypsinized Peptone Solution
(e) Bezangon specified the use of Chapo-
Constituents: teaut peptone.
1. Water 1000.0 cc. (f) Percival used 2.0% peptone. Used to
2. Peptone (Witte) 10.0 g. test for indol production.
3. Trypsin 5.0 g. (g) Norton and Sawyer specified Armours
Preparation peptone.
(1) Dissolve 10.0 g. Witte's peptone with (h) Harvey used from 1.0 to 2.0% pep-
gentle heating in 200-300 cc. of water. tone. At a pH = 8.0 to 9.0 the
(2) Dissolve 5.0 g. trypsin in 10-20 cc. medium was used for the isolation of
water by shaking and heating not to V. cholerae.
exceed 40C. (i)Lohnis and Stitt specified the use of
(3) Add (2) to (1). Witte's peptone.
(4) Incubate (3) at 38-40C. for 2 to 3 (j) Wherry dialyzed the peptone for 2
hours, stirring gently every 15 to 20 days before using in the solution.
minutes. (k) Bristol used a 25% Difco peptone
(5) Neutralize the reaction after the 2 or 3 solution with 0.5% NaCl to study the
hours if necessary. metabolism of B. botulinus.
(6) Add water to make up to 1000.0 cc. (1) Almy and James used a 3.0% peptone
(7) Boil and filter. and 0.5% NaCl solution to study the
(8) Distribute in tubes. volatile sulphur compounds pro-
Sterilization: Sterilize in the steamer or duced by Salmonella aertrycke and
autoclave. Proteus vulgaris. They reported
Use: Study of indol production. thatwhen cysteine was added to this
Variants: Norton and Sawyer adjusted the medium it was completely decom-
reaction to +1.0 to phenolphthalein.
References: Rivas (1912 p. 549), Roddy References: Dunham (1887 p. 338), Smith
(1917 p. 42), Norton and Sawyer (1921 (1902 p. 94), Frost (1903 p. 64), Smith
p. 473). (1905 p. 195), Wherry (1905 p. 439), Abel
(1912 p. 23), Lohnis (1913, p. 44), Sears 527. Brussoff's Peptone Solution
(1916 p. 110), Roddy (1917 p. 42), Besson Constituents:
(1920 p. 29), Myers (1920 p. 242), Bezan- 1. Distilled water 1000.0 cc.
Qon (1920 p. Ill), Percival (1920 p. 113), 2. Peptone (Witte) 10.0 g.
Buchan Norton and Sawyer
(1910 p. 108), 3. Iron peptone (Merck) 1.0 g.
(1921 p. 473), Harvey (1921-22 p. 101), 4. NaCl 10.0 g.
Giltner (1921 p. 42), Abbott (1921 p. 140), Preparation
Dopter and Sacquepee (1921 p. 119), (1) Dissolve 2, 3 and 4 in 1 by heating in
Bitfield (1922 p. 118), Stitt (1923 p. 35), streaming steam for 30 minutes.
Park, and Krumwiede (1924
Williams Filter.
(2)
p. 120), Cunningham (1920 p. 18), Bristol Adjustment of reaction not specified.
(3)
(1925 p. 467), Almy and James (1926 Sterilization: Sterilizein the autoclave
p. 323). under 1.5 atmospheres pressure for 10
minutes.
525. Huss' Peptone Solution
Use: Cultivation of iron bacteria (sludge
Constituents forms), Ferribacterium duplex.
1. Water 1000.0 cc. Reference: Brussoff (1918 p. 196).
2. Peptone 10.0 g.
3. NaCl 5.0 g. 528. Redfield's Peptone Solution (Myers)
Constituents
1. Water 1000.0 cc.
Use: To study indol production by aroma 2. Peptone (Witte or Difco) . . 300.0 g.
producing bacteria. Bacillus esterifi-
3. NaCl 75.0 g.
cans, Maassen, and Pseudomonas Tri- Preparation
folii. Reaction for indol was negative Boil 2 in 700.0 cc. tap water and
(1)
wlien using H2SO4 and several drops of 75.0 g. NaCl until as much of peptone
1.0% KNO2 as a test for indol. as possible has gone into solution.
Variants: Brussoff used this solution to (RedfieldusedKCl.)
cultivate iron bacteria from sludge,
Cool rapidly and make up to 1 liter.
(2)
Ferribacterium duplex. A yellow mem- Heat to boiling in a flask, plug and
(3)
brane formed after 3 days. cool rapidly.
Reference: Huss (1907 p. 61), Brussoff
(4) Add 0.5% NaCl (to clear).
(1918 p. 195). cold thru paper.
(5) Filter
(6) Distribute in 150.0 cc. Erlenmeyer
526. Nencki's Peptone Solution flasks in 10.0 cc. lots.
Constituents Sterilization: Sterilize at 15 pounds for
1. Water 900.0 cc. 15 minutes.
2. Peptone 100.0 g. Use: Detection of H2S by bacteria from
3. NaCl 20.0 g. animal feces. Used lead acetate paper
Preparation to detect HoS production.
(1) Dissolve 100.0 g. peptone in 900.0 cc. Reference: Myers (1920 p. 232-235).
water.
529. Berman and Rettger's Peptone
(2) Add 20.0 g. NaCl to (1).
Solution
(3) Filter.
(4) Distribute into test tubes. Constituents:
Sterilization: Sterilize in the autoclave. 1. Water 1000.0 cc.
Use: To study cause of cattle plague. 2. Peptone (Witte) 5.0 g.

When the medium was inoculated with 3. NaCl 5.0 g.
material containing the causative organ- Preparation:

ism of cattle plague, round cells 1 to 3^ (1) Dissolve 2 and 3 in 1.
developed after 2 days. (2) Tube in 10.0 cc. lots.

Reference: Nencki (1898 p. 530). Sterilization: Not specified.
Use: To study bacterial nutrition and me- (f) 0.025% glucose.

tabolism. (g) 0.025% glucose + 0.42% NaoHPO^.
Variants (h) 0.01, 0.25, 0.05 or 2.0% maltose.
(a)Authors used 1.0% of VVitte's pep- (i) 0.05, 0.1 or 2.0% xylose,
tone. Other peptones were also (j)2.0% sucrose.
used. Digestive Ferments Co., Park Variants: The authors used the basic me-
Davis, Armour, Eimer and Amend dium without any additions.
and Arlington Chemical Co.'s "ami- Reference: Heap and Cadness (1924-25
noids" were used. p. 80-90).
(b) Authors used 0.5% or 1.0% of one of
the above mentioned peptones and 532. Browning, Gilmore and Machle's
added 0.25 g. Liebig's beef extract. Brilliant Green Peptone Solution
Reference: Berman and Rettger (1918
Constituents:
pp. 371-377, 392).
530. Migula's Peptone Solution 2. Peptone (Witte or Rostock). 20.0 g.
3. NaCl 5.0 g.
Constituents:
4. Brilliant green
(Bayer's brilliant green extra cryst.)
2. NaCl 0.5 g.
Preparation
3. Peptone 5.0 to 15.0 g.
(1) Add 2 and 3 to 1.
Preparation:
(2) Steam in a Koch sterilizer for J of
(1) Add 0.5 g. of NaCl to 600.0 cc. of dis-
an hour.
tilledwater and boil for 30 minutes.
(2) Add from 5.0 to 15.0 g. of peptone
to hot (1).
(5) Prepare a 1.0% stock solution of
(3) Filter.
brilliant green (Bayer's brilliant
(4) Tube.
green extra cryst), in distilled water.
Sterilization: Steam for 15 minutes.
(6) Just before use of medium a 1:10,000
Use: Chiefly for the detection of typhoid
dilution is prepared by adding 0.1 cc.
and cholera bacilli.
of (5) to 9.9 distilled water.
Reference: Migula (1901 p. 19).
Sterilization: Sterilize at 120C. for 15
531. Heap and Cadness' Basal Peptone minutes (medium reacts faintly alkaline
Solution to litmus).
Use: Enrichment of Bacillus typhosus.
Constituents:
Variants: Harvey used 10.0 to 20.0 g. pep-
1. Water 1000.0 cc.
tone and added 25.0 cc. of a 1-10,000
2. Peptone (Witte) (3.0%). . . . 30.0 g.
brilliant green solution.
3. NaCl (0.25%) 2.5 g.
References: Browning, GilmourandMackie
Preparation
(1913-14 p. 338), Harvey (1921-22 p. 91).
(1) Dissolve 2 and 3 in 1 by steaming.
(2) Filter.
533. Harvey's Telluric Acid Peptone
(3) Adjust to pH = 7.6.
Solution
Sterilization: Autoclave at 115C. for 20
minutes. Constituents:
Use: To study hydrogen sulphide produc- 1. Water 1000.0 cc.
tion by B. aertryke. 2. NaCl 5.0 g.
Added nutrients: The authors added one of 3. Peptone 10.0 to 20.0 g.
the following combinations: 4. Brilliant green (1-10,000 soln.)
(a) 2.0% glucose (sterilized by steaming 5. Telluric acid (1-1000 soln.)
as a 50% solution). Preparation
(b) 0.42% Na2HP04. (1) Dissolve 2 and 3 in 1.
(c) 0.42% Na2HP04 + 2.0% glucose. (2) Reaction of pH = 7.2.
(d) 0.05% glucose. (3) Tube in 10.0 cc. quantities.
(e) 0.05% glucose + 0.42% NajHPOi. (4) Add 0.1, 0.2, 0.35, 0.5 or 0.7 cc. of a
150 CULTURE MEDIA' FOR CULTIVATION OF MICROORGANISMS
freshly prepared 1:10,000 brilliant Sterilization: Not specified.
green solution to each tube. Use: Enrichment of colon typhoid group.

(5) Add 0.2 cc. of a 1:1000 telluric acid Using a mixture of dyes (china blue and
solution to each tube. rosalic acid). The author reported that
Sterilization: Not specified. the rosolic acid was the dye that inhibited
Use: Enrichment medium. The inositol the gram positive rods.
fermenters were suppressed. Variants: The author prepared a stock
Reference: Harvey (1921-22 p. 91). china blue and rosolic acid solution by
dissolving 10.0 g. Grubler's china blue
534. Smith's Rosolic Acid Peptone Solution and 2.0 g. Grubler's rosolic acid in
Constituents 100.0 cc. of 50.0% alcohol. This mixture
1. Water 1000.0 cc. of dyes (0.25%) was added to the medium
2. NaCl 5.0 g. instead of just china blue solution alone.
3. Peptone 10.0 g. Reference: Bronfenbrenner (1920 p. 184).
4. Rosolic acid (0.5% soln. in
80.0% alcohol) 2.0 cc. 536. Diedudonne's Nitrate Peptone
Preparation Solution
Constituents:
(2) Filter.
Water 1000.0 cc.
(3) Prepare a 0.5% rosolic acid solution 1.
in80.0% alcohol. 2. KNO3 (0.01%) 0.1 g.
Add2.0cc. of (3)to(2). 3. Peptone (1.0%) 10.0 g.

(4)
Tube. Preparation
(5)
fractional (1) Prepare a 1.0%, slightly
alkaline pep-
Sterilization: Sterilize the
method. tone solution.
Use: To detect acid and alkali production. (2) Add 0.01% KNO3 to (1).
Colorless with Distribute in 10.0 cc. lots.

Medium is pale pink. (3)
acid and red with alkaline reaction.
Enrichment of colon typhoid group. Use: Study of nitrate reduction by colon
Variants typhoid group.
Harvey used 10.0 to 20.0 g. peptone. Variants:
(a)
(a) Author used 0.001% or 0.0001%
(b) Bronfenbrenner added 0.05 to 62.5
cc. of a 2.0% solution of Grubler
KNO3.
rosolic acid in 50.0% alcohol to the (b) Grimbert specified the use of Colas
peptone solution. He recommended peptone and used 1.0 g. KNO3.
the use of 0.25% stock rosolic acid (c) Maassen used 5.0% peptone and 0.5%
solution for enrichment of the in- KNO3.
testinal bacteria. (d) Maassen used 5.0% peptone, 0.5%
References: Smith (1902 p. 94), Ball (1919 KNO3 and added 1.0% glycerol.
(e) Heinemann used 1.0 g. peptone and
p. 83), Bronfenbrenner (1920 p. 84),
Harvey (1921-22 p. 88).
added 5.0 cc. of a 2.0% KNO3 solution
instead of 0.1 g. KNO3.
535. Bronfenbrenner's China Blue Peptone (f) Committee A. P. H. A. and Harvey
Solution used peptone and 2.0 g. KNO3.
1.0 g.
(g) Killer used 10.0 g. KNO3.

Constituents
1000.0 cc. (h) Rogers, Clark and Davis, Tanner,
1. Water
Ball, Giltner, and Park, Williams
2. NaCl (c.p.) 5.0 g.
Peptone (Difco) 10.0 g. and Krumwiede used 1.0 g. peptone
3.
China blue
andO.2g.KNO3.
4.
Preparation (i) Committee S. A. B. used 2.0 to 5.0 g.

solution by dissolv- peptone and 2.0 to 5.0 g. KNO3.
(1) Prepare a stock
ing 1.0 g. of Grubler's china blue in (j) Tanner used 1.0 g. Witte's peptone
100.0 cc. of 50.0%, alcohol.
and2.0g. KNO3.
(k) Conn and Breed used 0.2 or 2.0 g.
nitrate.
(3) Add 0.25% of (1) to (2).
(1) Conn and Breed used 0.2 or 2.0 g. 539. Enlow's Basal Agar Peptone
and added 10.0 g. glucose.
nitrate Solution
(m) Conn and Breed used 0.2 g. KNO3 Constituents
with 2.0 or 5.0 g. peptone, 1. Water (tap or distilled) 2000.0 cc.
(n) Conn and Breed used 0.2 g. KNO3, 2. Peptone 10.0 g.
2.0 or 5.0 g. peptone and added K2HPO4
3. 17.0 g.
10.0 g. glucose. 4. Agar 2.0 g.
References: Diedudonne (1894-95 p. 510), 5. Indicator (Brom thymol blue)
.
Grimbert (1898 p. 385), Maassen (1902 Preparation
p. 28), Heinemann (1905 p. 131), Com- (1) Dissolve 2, 3 and 4 in 1800.0 cc.
mittee A. P. H. A. (1905 p. 109), Killer water in the Arnold sterilizer by
(1913 p. 521), Rogers, Clark and Davis heating for 40 minutes or autoclave
(1914 p. 415), Committee S. A. B. (1918 for 10 minutes or autoclave for 10
p. 116), Tanner (1919 p. 90), Conn and minutes at 15 pounds pressure.
Breed (1919 pp. 273-275), Tanner (1919 (2) Filter through paper while hot.
p. 45), Ball (1919 p. 76), Giltner (1921 (3) Dilute to 2000.0 cc. by the addition
p. 42), Harvey (1921-22 pp. 102, 108), of hot distilled water.
Park, Williams and Krumwiede (1924 (4) Adjust the hydrogen ion concentra-
p. 124). tion as desired.
(5) Add 0.5% of one of the added nu-
537. Harvey's Nitrate Peptone Solution trients.
(6) Add any desired indicator. (The
Constituents: author added 3 drops of Brom thymol
1. Distilled water 1000.0 cc. blue to each 500.0 cc. of medium.
2. NaNO, 1.0 g. The indicator solution was prepared
3. Peptone 10.0 g. by grinding 0.1 gram Brom thymol
Preparation: (1) Dissolve 2 and 3 in 1. Blue and 3.2 cc. of N/20 NaOH in an
Sterilization: Not specified. agate mortar. When solution of the
Use: Study of nitrate reduction. dye was complete, add 3.0 cc. of dis-
Reference: Harvey (1921-22 p. 107). tilled water.
(7) Tube.
538. Cohen and Clark's Phosphate Peptone Sterilization: Sterilize for 30 minutes in
Solution the Arnold sterilizer on each of 3 succes-
sive days.
Constituents:
Use: Used as a basic sugar free medium.
1. Water 1000.0 cc.
Variants The author used 14.5 g. Na2HP04
:
2. Peptone (Dif co) 10.0 g.

and 1.4 g. KH2PO4 instead of 17.0 g.
3. K2HPO4 5.0 g.
K2HPO4. The combination of Na2HP04
Preparation
and KH2PO4 gave a medium with a pH
of between 7.2 and 7.4.
(2) Adjust to any desired pH value by Added nutrients: The author added 0.5%
the addition of HCl or NaOH. of any desired sugar, alcohol, glucose, etc.
Reference: Enlow (1923, p. 2130).
Use: To study effect of reaction (pH) on
growth of the colon-aerogenes group, and 540. Beijerinck's Ammonium Sulphate
to study ammonification. Peptone Solution
Variants: Cunningham studied ammoni- Constituents:
fication using the following soil and 1. Water (ditch) 1000.0 cc.
manure forms, Bac. mycoides, Bad. 2. Peptone 5.0 g.
fluorescens, Bad. vulgar e, Bad. coli, 3. (NH4)2S04 1.0 g.
Sarcina lutea, Bac. putrificus. He speci- Preparation
fied the use of tap water and used 0.05% Dissolve 2 and 3 in ditch water
(1)
K2HPO4 instead of 0.5%. ("Grabenwasser ) '
'
Reference: Cohen and Clark (1919 p. 410). (2) Make alkaline with NsaCO,.
Sterilization: Not specified. 2. Peptone (1.0%) 10.0 g.
of sulphates by 3. Boric acid (H3BO3 0.63%) 6.3 g.

Use: To study reduction
. .
Spirillum tenue, generally the more Preparation: (1) Dissolve 2 and 3 in 1.
organic material that is present the Sterilization: Not specified.
greater the reduction. Use: To determine the efltect of boric acid

Variants Cathelineau studied pigment and
:
on the growth of Bad. coli and Bad.
aerogenes. The author reported that
fluorescent production by Bacillus varidis
(Lesage) and used Beijerinck's solution
Bad. aerogenes died off in this medium
with one of the following:
but that Bad. coli multiplied slowly.
glucose 5.0 g. Reference: Levine (1911 p. 22).
sodium phosphate 5.0 g.

544. DeBord's Phosphate Peptone Solution
sodium succinate 5.0 g.
Cathe- Constituents:
Reference: Beijerinck (1895 p. 107),
lineau (1896 p. 235).
2. Peptone (Dif co) 32.0 g.
541. Redfield's Peptone Solution (Tanner) 3. KH2PO4 (M/2 solution) .... 16.0 cc.
Constituents: 4. NajHPOi (M/2 solution) .

. . 144.0 cc.
Distilled water 1000.0 cc. Preparation

1.
(1) Dissolve peptone in 900.0 cc. cold
2. Peptone 300.0 g.
distilled water.
3. KCl 74.0 g.
(2) Add 16.0 cc. half molar KH2PO4 and
Preparation
144.0 cc. of half molar Na2HP04
(1) Add 2 and 3 to hot 700.0 cc. distilled
solutions.
water.
solution is complete. (3) Dilute to 1500.0 cc.
(2) Stir until
(4) Heat for 15 minutes at 15.0 pounds
(3) Cool.
box over pressure.
(4) Allow to stand in the ice
(5) Filter and make up loss in weight.
night.
(6) Add 100.0 cc. distilled water.
(5) Filter.
Sterilization: Sterilize to 15 pounds pres-
(6) Make up to one liter.
(7) Tube.
Sterilization: Not specified. Use: To study nitrogen metabolism of B.
coli, Ps. pyocyanea, C. sporogenes
and
Reference: Tanner (1919 p. 59). P. subtihs.
Variants: The author added 100.0 cc. of a
542. NicoUe, Raphael and Debaens Peptone sterile glucose solution prepared by
Solution dissolving 16.0 g. glucose in distilled
water instead of the 100.0 cc. distilled
Constituents:
1. Distilled water 1000.0 cc. water.
2. Peptone (Defresne) 25.0 g. Reference: DeBord (1923 pp. 16, 17).
Soda (normal) 20.0 cc.

3.
546. Kendall, Day and Walker's Fat Free
Preparation Peptone Solution
Tube in 4.0 cc. lots. Constituents

(2)
2. Peptone (Fairchilds) 15.0 g.
minutes.
Use: Cultivation of Eberth's bacillus and 3. Na2HP04 3.0 g.
NaCl 5.0 g.
paratyphoid bacilli. Indol was not pro- 4.
Preparation
duced in this medium.
(1) Extract 15.0 g. of 2 for two weeks with
Reference: NicoUe, Raphael and Debains
ether, two weeks with alcohol,
for
(1917 p. 380).
two weeks with acetone and 10 days
543. Levine's Boric Acid Peptone Solution with petroleum ether, respectively,
in a Soxhlet extractor, the successive
Constituents
1000.0 cc. extractions occurring at intervals of
1. Water
about 12 minutes. These extractions Added nutrients : The author added 100.0 g.
to be continued for 6 hours a day and of lactose or 100.0 g. of calcium lactate.
6 days per week. Reference: Klecki (1896 p. 255).
(2) Dissolve residue from the extraction
549. Pergola's Nitrate Peptone Solution
in 1.
(3) Dissolve 3 and 4 in (2). Constituents:

(4) Distribute in 100.0 cc. lots. 1. Distilled water 1000.0 cc.
(5) Adjustment of reaction not given. 2. Peptone 10.0 g.
Sterilization: Autoclave (time not given). 3. NaCl 10.0 g.
Use: To study metabolism of tubercle 4. NaaCOs 0.2 g.
bacilli. Authors used alizarin, neutral 5. KNO3 0.1 g.
red, and phenolphthalein to study changes Preparation
in reaction. Ziehl-Neelsen stain for (1) Dissolve 2, 3, 4 and 5 in 1.
staining. (2) Filter.

Variants: Harvey used 30.0 g. of peptone (3) Distribute in 15 to 20.0 cc. lots.
(did not specify Fairchilds) and added Sterilization: Method not given.
0.1 cc. of 1.0% neutral lead acetate to each Use: Enrichment of cholera vibrio.
10.0 cc. quantity of medium. Variants
References: Kendall, Day andjWalker (1914 (a) Wolfel used 2.0% peptone, 0.5% NaCl,
p. 424), Harvey (1921-22 p. 107). 0.0075% KNO3 and 0.2 g. NasCOs in

100.0 cc. distilled water to demon-
547. Kligler's Peptone Solution strate the cholera red reaction.
Constituents: (b) Ball used 10.0% peptone, 0.1% KNO3
1. Distilled water 1000.0 cc. and 0.1% NajCO,.
2. Peptone (Witte's) 10.0 g. References: Pergola (1911 p. 85), Wolfel
3. K2HPO4 0.29 g. (1912 p. 416), Ball (1919 p. 83).
4. NaCl 5.0 g.
556. Klimmer's Nitrate Peptone Solution
Preparation
(1) Dissolve 2, 3 and 4 in 1. Constituents:
(2) Adjustment of reaction not given. 1. Distilled water 1000.0 cc.
(3) Filter thru filter paper. 2. Peptone (Witte) 100.0 g.
(4) Tube in 10.0 cc. lots. 3. NaCl 100.0 g.
Autoclave (time not given).
Sterilization: 4. KNO3 1.0 g.
Use: To study indol production. 5. NaoCOa (crystalline) 2.0 g.

Reference: Kligler (1914 p. 82). Preparation
(1) Dissolve 2, 3, 4 and 5 in 1000.0 cc.
548. Klecki's Basal Ammonium Phosphate distilled water.
Peptone Solution Tube or flask.
(2)
Constituents: Sterilization: Method not given.

1. Water 5000.0 cc. Use: Enrichment of cholera vibrio. Add
2. Peptone 10.0 g. 9 parts of water under investigation to
3. Potassium phosphate 0.29 g. 1 part of medium.
4. Ammonium phosphate. . 0.5275 g. Reference: Klimmer (1923 p. 221).
5. MgS04 .'
0.275 g.
551. Bleisch's Nitrate Peptone Solution
6. (NH4)2S04 0.115 g.
(1) Dissolve 2, 3, 4, 5, 6 and 100.0 g. of 1. Distilled water 1000.0 cc.
one of the added nutrients in 1. 2. Peptone (Witte) 20.0 g.
(2) Distribute in Pasteur fermentation 3. NaCl (c.p.) 5.0 g.

tubes. 4. KNO3 (c.p.) 0.8 g.
Sterilization: Method not given. Preparation
Use: Cultivation of B<tcillus sncchnro- (1) Dissolve 2, 3 and 4 in 1.
butyricus. Organism did not grow using (2) Boil.
calcium lactate as a carbon source. (3) Filter.

Sterilization: Method not given. 3. CaHP04 0.32 g.
of indol production by cholera 4. MgCl, 0.56 g.

Use: Study
bacteria.
5. KH2PO4 4.16 g.
6. Peptone (Chapoteaut) .... 20.0 g.
Variants
Merck's Preparation
(a) Frost and Johnson used 1.0 g.
peptone, 0.2 g. KNO3 and 0.5 g. NaCI. (1) Add 2, 3, 4 and 5 to 1.
pep- Add sufficient acetic acid to keep the
(b) Wherry used 10.0 g. of Witte's
(2)
materials in solution.
tone and 0.01% KNO3.
Dissolve 6 in (2).
(c) Wherry used 10.0 g. of Witte's pep- (3)
tone that was dialysed for two days
Use: Cultivation of Drosophila ampe-
and 0.01% KNO3.
(d) Logie used 10.0 g. peptone and lophila.
KNO3. Reference: Guyenot (1914 p. 548).

0.00283 g.
Cunningham used 10.0 g. peptone,
(e)
nitrite free 555. Will's Peptone Solution
5.0 g. NaCI and 0.5 g.
KNO3. Constituents:
(1893 112), Frost
Reference: Bleisch p.
Wherry (1905 p. 439), Logie
(1903 p. 66), 2. CaHP04 0.5 g.
Johnson (1912 p. 219),
(1919 p. 147), 3. KH2PO4 4.55 g.
Cunningham (1924 p. 18). 4. MgS04 2.1 g.
Peptone Solution 5. Witte peptone 20.0 g.

552. Logie's Nitrite

1000.0 cc. forming
1. Water Use: Cultivation of non-spore
2. Peptone 10.0 g.
yeast.
NaCI 5.0 g.
3. Variants
NaNO. 0.0023g. K2HPO4 instead of
4. (a) Will used 4.55 g.
Preparation: (1) Dissolve 2, 3 and 4 m 1.
KH2PO4 mycoderma.
to cultivate
(b) Nakozawa added 5.0% sucrose.
Use: To study the formation and
destruc-
Reference: Will (1908 p. 387), (1901 p. 3),
tion of nitrite by the dysentery
bacilli.
Nakazawa(1909p. 530).
Reference: Logie (1910 p. 147).
Peptone Solution 556. Gersbach's Trypsinized Peptone
553. Gottheil's
Solution
Constituents:
1000.0 cc. Constituents:
1. Distilled water cc.
Water 1000.0
2 Potassium phosphate
10 g. 1.
Peptone (Witte) 10-0 g.

O.lg. 2.
3.CaCU
0.3 g. 3. NaCI 0.5 g.
4. MgS04
O.lg. 4 Calcium phosphate 0.2 g.
5. NaCI
t^^ 5. MgS04 0.02 g.
6. Iron
10-0 g- Preparation:
7. Peptone
6 and 7 (1) Prepare a 1.0% solution of Witte's
Preparation: (1) Dissolve 2, 3, 4, 5,
peptone and add 0.5% NaCI to the
inl.
liter.
Boil one hour and cool to 40C.
for organ-
Use: General culture medium
(2)
Neutralize and make slightly alkaline
roots and
isms found in the soil, on
(3)
with soda.
rhizomes. fitting glass
(4) Place in flask with a well
stopper.
554. Guyenot's Peptone Solution Add 0.2 g. of Griibler's trypsin, 0.2 g.
(5)
calcium phosphate 0.02 f. MgS04,
Constituents:
10.0 cc. chloroform and 10.0 cc.
of
1000.0 cc.
1 Redistilled water
toluol.
2. NaaS04 0.16g.
(6) Shake thoroly, removing the stopper 559. Fremlin's Ammonium Sulphate
occasionally. Peptone Solution
(7) Place a paper around the flask and Constituents:
place in the incubator at 37C. for
1. Water 1000.0 cc.
24 hours.
2. (NH4)2S04 1.0 g.
(8) Shake thoroly. 3. Potassium phosphate. 1.0 g.
(9) Filter thru a sterile moistened filter
4. MgCOa 10.0 g.
paper. Peptone
5. 0.1 to 0.5 g.
Sterilization: Sterilize for 45 minutes
Preparation
(method not given).
Use: Study of indol production. Culture
(2) Dissolve 4 in the remainder of 1.
medium used in water analysis.
and (2) under aseptic
(3) Mi.x sterile (1)
Reference: Gersbach (1922 p. 146).
conditions and add from 0.1 to 0.5 g.
557. Kappen's Peptone Solution peptone.
Constituents
Use: To study nitrogen o.xidation by
1. Water 1000.0 cc.
nitroso bacteria.
2. Peptone (0.5%) 5.0 g.
Reference: Fremlin (1903 p. 368).
3. K2HPO4I
4. NaCl I 560. Mortensen's Peptone Solution
,- Tv,r CI/-. } usual amounts
5. MgS04 Constituents:
6. FeS04 J 1. Water 1000.0 cc.
Preparation: Prepare a 0.5% solution
(1) 2. Peptone (Witte (1.0%)) .... 10.0 g.
of peptone in water containing the usual 3. Cobalt salts
amounts of K2HPO4, NaCl, MgSOi, and Preparation
FeS04 (amounts not given). (1) Dissolve 2 in 1.
Sterilization: Method not given. (2) Add cobalt salts in varying amounts
Use: To study decomposition of peptone from 4 to 1/32%.
by cyanamide decomposing bacteria. Sterilization: Not specified.
Reference: Kappen (1909 p. 395). Use: To study the effect of cobalt salts on
Aspergillus niger. The author reported
558. Percival's Ammonium Sulphate
that the toxic properties of cobalt were
Peptone Solution
different in different media. A 0.5%
Constituents: cobalt chloride solution in a liquid
1. Water 1000.0 cc. medium was as toxic as 1.0% cobalt
2. (NH4)2S04 2.0 g. chloride in a gelatin medium.
3. K2HPO4 1.0 g. Reference: Mortensen (1909 p. 523).
4. NaCl 2.0 g.
5. MgS04 0.5 g. 561. Kligler's Nitrate Peptone Solution
6. FeS04 0.4 g. Constituents:
7. MgC03 10.0 g. 1. Distilled water 1000.0 cc.
8. Peptone (0.01%) 0.1 g. 2. Peptone 10.0 g.
Preparation 3. K2HPO4.. 0.2 g.
(2) Distribute in 50.0 cc. into conical 5. NaCl 0.2 g.
flasks. 6. KNO3 0.2 g.
(3) Add 0.5 g. of basic MgCOj to each Preparation: (1) Dissolve 2, 3, 4, 5 and 6
flask. in 1.
(4) Add 0.01% peptone to each flask. Sterilization: Not specified.

Sterilization: Not specified. Use: To study denitrification by cocci.
Use: To study nitrification. Author reported that cocci reduced
Variants: The author used 0.03, 0.3, 1.0 or Ammonia was
nitrates to nitrites only.
3.0% peptone instead of 0.01%. formed but as a decomposition product
Reference: Percival (1920 p. 144). of the peptone.
Variants: The KNO3 was omitted. 3. MgS04 2.5 g.

Reference: Kligler (1913 p. 790). 4. NaCl 2.5 g.
Peptone (0.1%)
5. 1.0 g.
562. Percival's Phosphate Peptone Solution References Capaldi and Proskauer (1896
:
Constituents p. 460), Percival (1920 p. 112).

564. Ritter's Peptone Solution
2. KH2PO4 2.0 g.
3. Magnesium phosphate 0.1 g. Constituents:
4. Peptone 10.0 g. 1. Distilled water 1000.0 cc.
Preparation 2. Peptone (Witte) 10.0 g.
(1) Dissolve 2 and 3 in 1. 3. MgS04 1.0 g.
(2) Add 4 to (1). 4. K2HPO4 1.0 g.
(3) Heat to boiling. Preparation: (1) Dissolve 2, 3 and 4 in 1.
(4) Filter. Sterilization: Not specified.

Adjustment of reaction not specified. Use: Cultivation of Bacillus prodigiosus
(5)
(6) Tube in 10 to 15.0 cc. quantities. yeast and iron bacteria.
(7) After sterilization inoculate with con- Variants
tents of a nodule of a pea, runner (a) The author added 10.0 to 20.0% of
bean, red clover, and broad bean by glucose and specified that other
means of a sterile knife. sugars might be added instead of
Sterilization: Sterilize on 3 successive days glucose if desired.
(time not specified). (b) Zikes cultivated Cladothrix dicho-
Use: Cultivation of bacteria from the toma and Cladothrix natans in the
nodules of legumes. following solution.
Reference: Percival (1920 p. 204). 1. Water 1000.0 cc.
2. MgS04 1.0 g.
563. Capaldi and Proskauer's Peptone 3. K2HPO4 0.25 g.
Solution Peptone 1.25 g.
4.
Constituents: He substituted a nearly ash and iron

1. Water 1000.0 cc. free peptone or a "Manganpeptone"
2. NaCl (0.02%) 0.2 g. for ordinary peptone and reported
3. MgS04 (0.01%) 0.1 g. slightly poorer growth using the ash
4. K2HFO4 (0.2%) 2.0 g. and iron free peptone.

5. Peptone (Witte 0.5%) 5.0 g. (c) Bokorny cultivated yeast in the
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. following solutions:
Sterilization: Not specified. 1. Water 1000.0 cc.
Use: Cultivation of colon-typhoid group. 2. Peptone 50.0 g.
Variants 3. KH2PO4 1.0 g.
(a) The author used 2.0% peptone for 4. MgS04 1.0 g.
0.5% and used either 0.5 or 2.0% of or

one of the following instead of 1. Water 1000.0 cc.
Witte's peptone. 2. KH2PO4 4.0 g.
Peptone (e Carne Konig) 3. MgS04 1.0 g.
Peptone (Aschman) Peptone

4. 25.0 g.
Peptone (Chapoteaut) References: Ritter (1900 p. 207), Zikes

Peptone, driisen (Kiihne) (1915 p. 542), Bokorny (1917 p. 370),
Hemialbumose (Griibler) (1920 p. 23).

Somatose
565. Speakman's Basal Peptone Solution
Peptone (Glutin (Paal))
(McCoy et al.)
(b) Percival used the following solution
to determine ammonia production Constituents:
by B. mycoides: 1. Water 1000.0 cc.
1. Water 1000.0 cc. 2. K2HPO4 0.5 g.
2. K2HPO4 5.0 g. 3. KH2PO4 0.5 g.

4. MgS04 0.2 g. Clark and Lubs' Basal Phosphate

5. NaCl 0.01 g. Peptone Solution 577
6. MnS04 0.01 g. C2. Salts of divalent cations employed.
7. Peptone 5.0 g. Grimbert's Basal Peptone Solution. 578 .
Preparation B2. More than one salt added.

(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. Ci. Salts of only monovalent cations added.
(2) Dissolve 3% of one of the added Kendall, Walker and Day's Basal
nutrients. Peptone Salt Solution 579
(3) Adjust the reaction to pH = 7.0. Berman and Rettger's Basal Peptone
Sterilization: Not specified. Salt Solution 580
Use: Cultivation of acetone butyl alcohol Co.* Salts of only mono and divalent
organisms. cations added.
Added nutrients: The authors added 3% of Di. Containing calcium salts.
any desired carbohydrate. Palladin's Basal Peptone Salt Solu-
Reference: McCoy, Fred, Peterson and tion 581
Hastings (1926 p. 460). Boekhout and Ott de Vries' Basal
Peptone Salt Solution 582
SUBGROUP I-C. SECTION 8 Will's Basal Peptone Salt Solution. 583 .
D2. Not containing calcium salts.

Incomplete or basal solutions containing
Maassen's Basal Peptone Salt Solu-
peptone as the sole organic nitrogen and
tion 584
carbon source; employed with the addition
Calmette, Massol and Breton's Basal
of a variety of materials.
Peptone Salt Solution 585
Ai. No added.
salts
Hansen's Basal Peptone Salt Solu-
Bi. Indicators employed.
tion 586
Morishima's Basal Phenol Red China
C3. Salts of mono, di and trivalent cations
Blue Peptone Solution 566
added.
Robinson's Basal Azolitrain Peptone
Stoklasa and Vitek's Basal Peptone
Solution 567
Salt Solution 587
B2. Indicator not employed.
Vierling's Basal Peptone Salt Solu-
Capaldi and Proskauer's Basal Pep-
tion ., 588
tone Solution 568
A2. Salts added. 566. Morishima's Basal Phenol Red China
Bi. Only one salt added. Blue Peptone Solution
Ci. Salts of only monovalent cations
Constituents:
employed.
1. Peptone solution 1000.0 cc.
Di. Sodium salt added.
2. Phenol red (0.02% soln.) 50.0
. . . cc.
El. Indicator employed.
3. China blue decolorized
Barsiekow's Basal Salt Peptone Solu-
(1.0% soln.) 12.0 cc.
tion 569
Preparation
Cunningham's Basal Fuchsin Pep-
(1) Method of preparation of 1 not given.
tone Solution 570
Any fluid medium used to determine
Harvey's Basal Neutral Red Peptone
fermentation may be used.
Solution 571
(2) Prepare a 1.0% solution of China blue
Abba's Basal Phenolphthalein Pep-
in distilled water, and keeping the
tone Solution 572
water hot on a water bath, normal
E2. Indicators not employed.
NaOH solution is slowly added until
Mendel's Basal Peptone Solution... 573
the blue color disappears and the
Nicolle, Raphael and Debains' Basal
solution becomes brown. (About
Peptone Solution 574
3.5 cc. N/1 NaOH is required per
Robinson and Rettger's Basal Opsine
100.0 cc. of China blue.) Keep in
Solution 575
rubber stoppered flask.
D2. Potassium salt added.
(3) Prepare a 0.02% solution of phenol red.
Elser and Huntoon's Basal Nahr-
stofT-Heyden Solution 576 * See C3.
(4) Adjust (1) to desired pH. References: Robinson (1915 p. 407), Harvey
(5) Add 5.0 cc. of (3) and 1.2 cc. of (2) (1921-22 pp. 101, 109).
to (4) for every 100.0 cc. of (4).
568. Capaldi and Proskauer's Basal Peptone
Solution
sure for 10 minutes. Constituents:
Use: To determine fermentation of sugars. 1. Water 1000.0 cc.
Pink color indicates alkali formation. 2. Peptone (Witte) (0.5
Acid indicated by first a bright green and or 2.0%) 5.0 or 20.0 g.
then changing to deep blue. Preparation: Dissolve 2 and 0.1% of one
(1)
Added nutrients: Author suggested the use of the added nutrients in 1.
of any carbohydrate or fermentable ma- Sterilization: Not specified.
terial. The carbohydrate solution may Use: To determine fermentation of sugars
be sterilized separately and added aseptic- by the colon-typhoid group. Different
ally to sterile (5), or the sugar can be investigators added various materials to
added to sterilized (5) and the mixture the basic solution, or a modification of it,
be heated a second time in the autoclave and used the media for a variety of
at 10 pounds for 10 minutes. purposes.
Reference: Morishima (1920 p. 43). Added nutrients and variants:
(a) The authors added 0.1% of one of
567. Robinson's Basal Azolitmin Peptone
the following materials:
Solution
glucose raffinose
Constituents: levulose dextrin
1. Water 1000.0 cc. mannose inulin
2. Peptone (Witte) 10.0 g. galactose mannitol
3. Azolitmin 0.5 g. sorbose adonitol
Preparation lactose sorbitol
(1) Prepare 500.0 cc. of a 2.0% solution of maltose isodulcitol
Witte' s peptone. melibiose erythritol
(2) Adjust to a reaction of -f-0.6 to trehalose dulcitol
phenolphthalein. They reported growth poor with a
(3) Prepare 500.0 cc. of a solution con- M.L.D. = 1.0-f- cc.
taining 2.0% of an added nutrient (b) Matzuschita used 10.0 g. Koch's
(sugar). meat peptone per liter water as a
(4) Mix sterile (2) and (3) in equal basic solution and added one of the
amounts, and add 0.05% azolitmin. following materials or combinations:
(5) Tube in sterile tubes. NaCl 0.5, 0.7 to 10.5%.
Sterilization: Sterilize (2) in the autoclave. NaCl and glucose 0.5 to 50.0%.
5.0 g.
Filter (3) thru Berkefeld candles into NaCl 5.0 g., glucose 2.0% and from 0.2
sterile flasks. Incubate 24 hours to to 15.0% Na.COs. Add the NajCOs
insure sterility. to the neutral medium.
Use To determine fermentation by typhoid
: NaCl 5.0 g., glucose 2.0%, glycerol
and paratyphoid bacilli. 6.0%.
Added nutrients and variants NaCl 5.0 g., glucose, 2.0% and from 0.1
(a) Robinson used 10.0 g. of any desired to 0.4% HCl. Add the HCl to the
sugar. neutral medium.
(b) Harvey used 2.0 to 10.0 g. peptone, No additions to the basic solution.
50.0 cc. of litmus solution and 10.0 g. These media were used to cultivate
of any desired carbon source or fer- Clostridium butyricum, Bacillus
mentable material. oedematis maligni, Bacillus anthracis
(c) Harvey used 20.0 g. peptone, 50.0 cc. sym-ptomaiici, Bacillus sporogenes,
of litmus solution and 10.0 g. of any Bacillus botulinus.
carbon source or fermentable ma- (c) Revis did not specify the exact com-
terials per 850.0 cc. of distilled water. position of the basic solution (pep-
t:)ne water) and added one of the added to lactic acid until a pH = 7.0
following materials: is reached. In case of phos-
the
mucic acid (sodium salt) 1-0% phates, mono and di-sodium phos-
saccharic acid (potassium salt). . 1.0% phates are mixed in the proper
gluconic acid (potassium salt)... 0.5% proportions to give a pH of approxi-
The media were used to study the mately 7.0. NaCl is used also in
fermentation of acids by the colon 0.4M and MgCU in O.IM concentra-
groip. The author reported that tions. The media were used to show
with 1.0% of the acid sodium salt of the effect of salts on the growth of
mucic acid no gas was produced with B. coli. The authors reported that
B. coll or B. lactis aerogenes, but generally the order of growth accele-
slightly more acid was produced. ration of B. coli by these salts was
With the normal sodium salt and in approximately the same as their
the presence of Mg(0H)2 acid and order in the lyotropic series,
gas were produced by B. coli and B. (f) Hotchkiss used 10.0 g. peptone per
lactis aerogenes. liter in the basic solution and added
Same thing was true with saccharic a number of salts to study the effect
acid. (Acid and gas produced when of various cations on a communis
medium was alkaline.) Gluconic type of B. coli. He used the follow-
acid was attacked when present as ing salts and reported that the
the potassium salt orwhen alkalinity concentrations here indicated stimu-
was increased to 1.0 cc. N/1 NaOH lated growth. NaCl, KCl, CaCU and
per 100.0 cc, or when the acid itself MgCl2 may be added to the peptone
was employed. before sterilization. Other salt solu-
(d) Davis and Ferry cultivated Bad. tions were made up and stored until
diphtheriae and studied toxin pro- sterile and added under aseptic
duction using 20.0 g. of peptone in the conditions to the sterile peptone
basic solution and adding one of the solution. pH for all solutions except
following materials: NH4CI were from 6.6 to 7.0. NH4CI
glycocoll 0.75 g solution pH = 6.0 to 6.4.
leucine 30.0 g NaCI 0.25 molar concentration in the
histidine dichloride 0.5 g media.
glutaminic acid hydrochloride. 2.5 g KCl 0.25 molar concentration in the
tyrosine 20.0 g media.
cystine 0.5 g NH4CI 0.25 molar concentration in the
tryptophane 0.6 g media.
sodium asparaginate 1.5 g CaCU 0.05 molar concentration in the
creatine 0.2g + creatinine 0.15g. media.
xanthin 0.05 g.+ hypoxanthine 0.05 g MgClo 0.05 molar concentration in the
glucose amine hydrochloride . . 2.0 g media.
(e) Holm and Sherman used 10.0 g. of SrCl2 0.025 molar concentration in the
peptone per liter in their basic solu- media.
tion and added one of the following BaCl2 0.05 molar concentration in the
salts in sufficient quantities to make media.
the medium 0.2 molar for each salt. TiCl2 molar concentration in
0.0005
NaCl Na citrate the media.
NaXOs Na fluoride NiCl2 0.0001 to 0.00005 molar concen-
NaHP04 KCl tration in the media.
Nal CaCl2 PbCla 0.0005 to 0.00005 molar concen-
Na2S04 MgClo tration in the media.
Na lactate FeClj SnCl2 0.00005 to 0.000005 molar con-
Na o.xalate NH4CI centration in the media.
Na acetate ZnCl2 0.00005 to 0.00001 molar con-
In case of sodium lactate, NaOH is centration in the media.
CeCl2 0.00001 molar concentration in (16) Filter into test tubes.

the media. (17) Sterilize for 2 days at lOctc. and
HgCl2 0.000001 molar concentration in allow the temperature p reach
the media. 120C. on the third day (Ind then
References: Capaldi and Proskauer (1894 extinguish the flame.
p. 467), Matzuschita (1902 p. 286), Revis References; Barsiekow (1901 pp. 823-825),
(1912 p. 427), Davis and Ferry (1919 Harvey (1921-22 p. 108).
p. 235), Holm and Sherman (1921 p. 515),
Hotchkiss (1923 pp. 142-161).
570. Cunningham's Basal Fuchsin Peptone
Solution
569. Barsiekow's Basal Salt Peptone
Constituents:
Solution
1. Water 1000.0 cc.
Constituents 2. Peptone 10.0 g.
1. Distilled water 1000.0 cc. 3. NaCl 5.0 g.
2. Lackmus tincture 100.0 g. 4. Andrades indicator (1.0%). 10.0 cc.
3. Peptone (1.0%) 10.0 g. Preparation:
4. NaCl (0.5%) 5.0 g. (1) Dissolve 2 and 3 in 1 by autoclaving
Preparation: (1) Dissolve 2, 3, 4 and 1.0% to 2 atmospheres pressure.
of one of the added nutrients in 1. (2) Filter thru paper.
Sterilization: Not specified. (3) Dissolve 0.5% of one of the added
Use: Differentiationof colon typhoid nutrients and 1.0% of Andrades
group. Colon forms turned the medium indicator in (2).
red, typhoid and alkaligenes types blue. (4) Tube with Dunham's fermentation
Added nutrients and variants: tubes.
(a) The author added 1.0% glucose or (5) Stand the tubes in a water bath and
1.0% lactose. heat to boiling.
(b) Harvey used a similar basic solution (6) Plug.
and prepared it as follows Sterilization: Sterilize in the autoclave at
(1) Dissolve 10.0 g. peptone, 5.0 g. 22.5 pounds pressure. (Do not exceed
NaCl in a liter of water by 22.5 pounds pressure.)
steaming. Use: To observe fermentation.
(2) Make faintly alkaline to litmus by Added nutrients: The author added 0.5%
the addition of sodium carbonate. of any desired sugar.
(3) Filter. Reference: Cunningham (1924 p. 18).
(4) Sterilize.
571. Harvey's Basal Neutral Red Peptone
(5) Place solid commercial litmus in a
Solution
flask and pour 96% alcohol over it.
(6) Close with a rubber stopper.

(5) Constituents:
(7) Keep in an incubator at 30C. for 1. Water 1000.0 cc.
2 days, shaking occasionally. 2. NaCl 5.0 g.
(8) Pour off the alcohol and add 3. Peptone 10.0 g.
fresh. 4. 1.0% neutral red 2.5 cc.
(9) Continue the process until the Preparation
alcohol extracts no more color. (1) Dissolve 2 and 3 in 1 by steaming.
(10) Filter off the alcohol. (2) Filter.
(11) Dry the residue. (3) Add freshly prepared 1.0% neutral
(12) Dissolve (11) in distilled water to red 2.5 cc.
saturation. (4) Prepare a 10.0% solution of one of
(13) Add sufficient 1-1000 sulphuric the added nutrients.
acid to produce the slightest color. (5) Add sufficient amount of sterile (4)
(14) Dissolve 10.0 g. of any desired to sterile (3) so that the concentra-
carbon source of fermentable ma- tion of theadded nutrient is 1.0%.
terial in 50.0 cc. of (13). (6) Distribute in test tubes containing
(15) Add (14) to (4). fermentation tubes.
Sterilization: Sterilize (3) in the autoclave. (2) Add 1.0% of one of the added nu-
Sterilize (4) at 105C. for 10 minutes. trients (see sterilization).
Sterilize the tubes at 105C. for 10 Sterilization: Sterilize for 30 minutes on
minutes or at 100 on each of 2 successive each of 2 successive days. On the third
days for 20 minutes. day add 1.0% of one of the added nutri-
Use: To observe fermentation by bacteria. ents, and sterilize for 15 minutes.
Added nutrients: The author did not Use: To study decomposition of sugars.
specify the sugars used. One per cent of Other investigators used variants of the
any desired carbon source may be em- medium to cultivate sludge forms and to
ployed. study proteolysis.
Reference: Harvey (1921-22 p. 108). Added nutrients and variants:
(a) Mendel added 1.0%, of one of the
572. Abba's Basal Phenolphthalein Peptone following:
Solution glucose lactose
Constituents sucrose maltose
1. Water 1000.0 cc. (b) Rettger, Berman and Sturges studied
2. Peptone 100.0 g. proteolysis used 5.0 g. NaCl and 2.5,
3. NaCl 50.0 g. 5.0 or 20.0 g. of Witte's peptone in
4. Phenolphthalein (1.0% alco- the basic solution and adding the
holic soln.) 0.5 cc. following materials individually or in
Preparation combination:
(1) Dissolve 2, 3 and 200.0 g. of one of the glucose 10.0 g.
added nutrients in 1. beef extract 5.0 g.
(2) Boil in the steamer for 30 minutes at (NH4)2S04 2.5 g.
100C. (c) Brussoff used 10.0 g. NaCl in the
(3) Filter. basic solution. He used the media
(4) Distribute into 100.0 cc. lots into to cultivate Ferribacterium duplex,
sterile containers. and other iron bacteria from sludge.
(5) After sterilization add 0.5 cc. of He added one of the following ma-
phenolphthalein solution and Na2C03 terials to the basic solution.
solution until the solution is perma- Iron ammonium citrate 0.5 g.
nently red (see use). K,C03 1.0 g.
Sterilization: Not specified. K2SO4 1.0 g.
Use: Isolation of Bacillus coli communis Potassium acetate 1.0 g.
from water. About 1 liter of the water Potassium citrate 1.0 g.
under investigation is added to each flask References: Mendel (1911 p. 297), Brussoff
of medium. Bacillus coli communis (1918 pp. 195, 208) Rettger,, Burman and
decolorized the solution after from 12 to Sturges (1916 p. 20-22).
24 hours (using glucose). Using lactose
574. Nicolle, Raphael and Debains' Basal
B. coli caused decolorization after about
Peptone Solution
8 to 24 hours. Typhoid bacilli require a
greater length of time. Constituents:
Added nutrients The author added 200.0
: g. 1. Distilled water 1000.0 cc.
of lactose or glucose. 2. Peptone (Defresne) 1.0 g.
Reference: Abba (1896 p. 13), (1896 p. 224), 3. Soda (normal) 20.0 cc.
(1895 ^176). Preparation
(1) Dissolve 2, 3 and one of the added
573. Mendel's Basal Peptone Solution
nutrients in 1.
Constituents: (2) Distribute in test tubes containing
1. Distilled water 1000.0 cc. fermentation tubes.
2. Peptone (Witte) 10.0 g. Sterilization: Sterilize at 110 for 15
3. NaCl 5.0 g. minutes.
Preparation Use: To study the fermentation of sugars
(1) Dissolve 2 and 3 in 1. by typhoid and paratyphoid bacilli.
Authors report that glucose may or may Use: To study fermentation by meningo-
not be fermented, lactose never. cocci.
Added nutrients: Authors added 1.0% of Added nutrients: The author added 10.0 g.
glucose or lactose. of one of the following:
Reference: Nicolle, Raphael and Debains glucose sucrose
(1917 p. 379). galactose mannitol
levulose dulcitol
575. Robinson and Rettger's Basal Opsine
lactose inulin
Solution
maltose dextrin
Constituents: Reference: Elser and Huntoon (1909
1. Water 1000.0 cc. p. 404).
2. Opsine 10.0 g.
577. Clark and Lubs' Basal Phosphate
3. NaCl 5.0 g.
Peptone Solution
4. Glycerol 50.0 g.
Preparation : Constituents:
(1) Dissolve 2, 3 and one of the added 1. Distilled water 1000.0 cc.
nutrients in 1 by heating. 2. Peptone (Witte) 5.0 g.
(2) Adjust the reaction may be used 3. K2HPO4 (c.p.) 5.0 g.
either slightly acid or basic to litmus. Preparation
(3) Boil over a flame a few minutes. (1) Dissolve 2, 3 and one of the added
(4) Filter and tube. nutrients in 800.0 cc. distilled water
Sterilization: Sterilize at 12 pounds extra by heating for 20 minutes over a
pressure for 15 minutes. steam bath. Stir occasionally.
Use: General culture medium. Opsine is (2) Filter thru a Schleicher and SchuU
a protein free trypsin, erepsin and pepsin No. 588 folded filter.
digest of certain proteins. The medium (3) Cool to 20C. and make up e.xactly
supported the growth of some pathogenic to 1000.0 cc.
forms. (4) Tube in 10.0 cc. quantities in clean
Added nutrients: The authors added 5.0% sterile test tubes.
glycerol or 1.0% glucose. by the intermittent
Sterilization: Sterilize
Reference: Robinson and Rettger (1918 method.
p. 212). Use: Used to study fermentation of sugars.
Added nutrients and variants:
576. Elser and Huntoon's Basal Nahrstoflf-
(a) Clark and Lubs added 5.0 g. c.p.
Heyden Solution
glucose.
Constituents: (b) Levine added 5.0 g. of one of the
1. Distilled water 1000.0 cc. following materials:
2. Nahrstoff Heyden. . . . 10.0 g. fructose raffinose
3. NaCl 5.0 g. galactose mannitol
4. Litmus (Merck's maltose glycerol
soln) 5.0 to 7.5 cc. lactose salicin
Preparation : sucrose dextrin
(1) Dissolve 2 and 3 in 1. The media were sterilized in the
(2) Add 5.0 to 7.5 cc. of a watery solution autoclave.
ofMerck's highly sensitized litmus. (c) Rogers, Clark and Evans used 10.0 g.
(3) Prepare a 10.0% solution of one of Witte's peptone instead of 5.0 g. and
the added nutrients in distilled water. added 10.0 g. of one of the following:
(4) Mix sterile (2) and sterile (3). glucose inulin
(5) Tube in sterile tubes. lactose mannitol
(6) Incubate for 3 days to detect acci- sucrose dulcitol
dental contamination. raffinose adonitol
Sterilization: Sterilize (2) in the usual melibiose glycerol
manner, method not given. Sterilize arabinose
(3) by heating at 100C. for 10 minutes. The media was tubed in 10.0 cc.
quantities and sterilized by the inter- Rettger (1917 p. 165), Kligler (1918 p. 467),
mittent method in flowing steam. Tanner (1919 p. 47), Winslow, Rothberg
(d) Levine used Digestive Ferments and Parsons (1920 p. 151), Levine (1921
Co. peptone instead of Witte's, and p. 117), AyeTs, Rupp and Mudge (1921
added 5.0 g. of glucose. The medium p. 258), Giltner (1921 p. 383), Cunning-
was sterilized in the autoclave. ham (1924 p. 88), Wilson and Blair (1925
(e) Burton and Rettger added 5.0 g. p. 112), Committee A. P. H. A. (1925
glucose to the basic solution. The p. 111).
reaction was adjusted to neutral to
578. Grimbert's Basal Peptone Solution
litmus (pH = 7.0 or +1.0). Method of
sterilization not specified. Constituents:
(f) Kligler used 10.0 g. of peptone in- 1. Water 1000.0 cc.
stead of 5.0 g. Witte's peptone, added 2. Peptone (dry) 20.0 g.
5.0 g. NaCl, 1.0 g. of glucose and 3. CaCOs
added any organic test material in Preparation
any desired quantity. The ma- (1) Dissolve 2 and one of the added nu-
terials used were added to test their trients in 1.
antiseptic ability. Sterilize the basic (2) Add a sufficient quantity of CaCOa.
solution with the glucose in the auto- Sterilization: Not specified.
clave. Then add one of the ma- Use: Determine fermentation of Fried-
terialsunder aseptic conditions. lander's pneumobacillus.
(g) Winslow, Rothberg and Parsons Added nutrients and variants:
added 27.0 cc. of a 0.04% alcoholic (a) The author added 30.0 g. of any
solution of brom cresol purple and fermentable sugar.
5.0 g. of any carbon source or fer- (b) Pottevin used 10.0 g. peptone, 12.0 g.
mentable material. The basic solu- of CaCOa in the basic solution to
tion was adjusted to pH = 7.4, brom study the lactic fermentation by
cresol purple added and autoclaved lactic acid forming organisms. He
at 15 pounds pressure for 15 minutes. added one of the following:
The sterile added nutrient was added lactose 8.86 g
under aseptic conditions. sucrose 10.1 g
(h) Ayers, Rupp and Mudge used 40.0 g. maltose 11.4 g
of Bacto peptone instead of 5.0 g. glucose 10.0 g
Witte's peptone and added 2.0 g. Invert sugar 11.2 g
glucose. Method of sterilization not galactose 9.2 g
specified, mannose 8.96 g
(i) Cunningham autoclaved the K2HPO4, mannitol g 10.0
peptone and water to 30 pounds dulcitol g 10.0
pressure, filtered, made up to glycerol 10.0 g
1000.0 cc, distributed in 5.0 cc. malic acid 10.0 g
quantities and autoclaved at 22.5 Reference: Grimbert (1895 p. 843), Pot-
pounds pressure, tevin (1898 p. 54).
(j) Wilson and Blair made up the basic
579. Kendall, Walker and Day's Basal
solution with 5.0 g. glucose in double
Peptone Salt Solution
strength. They used the medium to
enrich streptococci in water analyses. Constituents:
Method of sterilization not given. 1. Redistilled water 1000.0 cc.
An equal volume of water under 2. Peptone (Fairchild) 5.0 g.
investigation was added to the 3. Na2HP04 2.0 g.
medium. 4. NaCl 5.0 g.
References: Clark and Lubs (1915 p. 169), Preparation
Levine (1916 pp. 160, 161), Committee (1) Extract 5.0 g. of Fairchild's peptone
A. P. H. A. (1917 p. 107), Rogers, Clark for 2 weeks with ether, 2 weeks with
and Lubs (1918 p. 234), Burton and alcohol, 2 weeks with acetone and 10
days with petroleum ether, respec- bacteria. In presence of carbohydrates,

tively, in a Soxhlet extractor, the buffers aided nitrogen metabolism.
successive extractions occurring at Added nutrients One : of the following com-
intervals of about 12 minutes. These binations were added:
extractions were continued for 6 (a) glucose, 2.0 to 10.0 g.
hours a day for 6 days per week. (b) lactose, 2.0 to 10.0 g.
(2) Dissolve the residue from (1), 3, 4 (c) glucose, 2.0 to 10.0 g. + beef extract
and one of the added nutrients in 1. 2.5 g.
(3) Distribute in 100.0 cc. lots. (d) lactose, 2.0 to 10.0 g. + beef e.xtract
Sterilization: Not specified. 2.5 g.
Use: To study lipolytic and proteolytic Reference: Berman and Rettger (1918
activity of tubercle bacilli. To deter- p. 392).
mine lipolytic activity, the authors placed

1.0 cc. of the bacteria free filtrate of the 581. Palladin's Basal Peptone Salt Solution
culture in freshly boiled dist. H2O, then
Constituents:
added 0.25 ethylbutyrate and 0.5 cc.
cc.
1. Water 1000.0 cc.
of toluene and incubated
at 37C. The
2. Peptone 10.0 g.
acid increase expressed in terms of X/50
NaOH was a measure of the lipolytic 4. MgS04 1.0 g.
activity.
5. CaCl, 1.0 g.
6. FeCl. trace
(a) The authors added 10.0 g. glucose or
Preparation
20.0 g. glycerol.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(b) Kendall, Day and Walker used 15.0 g.
(2) Add one of the added nutrients so
of Fairchild's peptone (extracted)
that the carbohydrate is present in
and 3.0 g. NaoHPO^ in the basic solu-
fourth normal concentration.
tion. They added 10.0 g. glucose or
(3) Distribute in thin layers in Erlen-
30.0 g. glycerol. The solution was
meyer flasks.
used to study the metabolism of
tubercle bacilli. Used
a alizarin,
Use: To study fermentation by Cloro-
neutral red and phenolphthalein to
thccium saccharophilum.
study changes in reaction, and Ziehl-
Added nutrients : One of the following were
Neelsen stain for staining.
added in fourth normal concentration:
(c) The authors used the same basic solu-
sucrose glucose
tion without extracting the peptone,
mannitol raffinose
adding 10.0 g. glucose or 30.0 g.
Reference: Palladin (1903-04 p. 146).
glycerol.
References: Kendall, Walker and Day
582. Boekhout and Ott de Vries' Basal
(1914 p. 463), Kendall, Day and Walker
(1914 p. 424),
Constituents:
580. Berman and Rettger's Basal Peptone
1. Water 1000.0 cc.
Salt Solution 2. Peptone (2.0%) 20.0 g.
Constituents 3. Bicalcium phosphate
1. Water 1000.0 cc. (0.2%) 2.0 g.
2. Peptone (Witte) 2.5 or 5.0 g. 4. CaCli (concentrated soln.). 5 drops

3. NaCl 5.0 g. 5. NaCl (0.5%) 5.0 g.
4. K2HPO4 5.0 to 8.0 g. Preparation: (1) Dissolve 2, 3, 4, 5 and one

Preparation of the added nutrients in 1

(2) Add one of the combinations listed Use: Cultivation of organisms found in
under added nutrients to (1). cheese.
Sterilization: Not specified. Added nutrients: The authors added 0.5%
Use: To study nitrogen metabolism of of glucose or galactose.
Reference: Boekhout and Ott de Vries acids by organisms. The author used 51
(1918 p. 134). organisms and reported that 41 organisms
developed in the presence of malic acid
583. Will's Basal Peptone Salt Solution and 38 in the presence of citric and
Constituents: fumaric acids. These acids allowed the
1. Distilled water 1000.0 cc. development of the greatest number of
2. CaHP04 0.5 g. organisms.
3. KH2PO4 4.55 g. Added nutrients: One tenth equivalent
4. Peptone (Witte) 20.0 g. weight of one of the following acids was
Preparation added:
(1) Dissolve 2, 3 and 4 in 1. formic acid malonic acid
(2) Add one of the added nutrients. acetic acid tartaric acid
Sterilization: Not specified. propionic acid fumaric acid
Use: Cultivation of non-spore forming glycolic acid malic acid
yeast. lactic acid "trikarballysaure"
Added nutrients and variants: butyric acid citric acid
(a) The author added one of the glyceric acid aconitic acid
following: "Oralsaure" mucic acid
sucrose 5.0% malic acid quinic acid
organic acid, carbohydrate or alcohol succinic acid mandelic acid
(b) Grosbiisch added one of the fol- Reference: Maassen (1895-6 p. 341).
lowing:
585. Calmette, Massol and Breton's Basal
alcohol 5.0%
formic acid 0.1%
acetic acid 0.2% Constituents:
tartaric acid . .-
0.3% 1. Water lOOO.O cc.
lactic acid 4.0% 2. NaaCOa 1.0 g.
citric acid 8.0% 3. FeS04 0.04 g.
malic acid 10.0% 4. MgS04 0.05 g.
succinic acid 11.0% 5. K0HPO4 1.05 g.
The amounts of acids given was the 6. NaCl 8.5 g.
largest amount at which Torida 7. Peptone 10.0 g.
rubefaciens developed. Preparation
Reference: Will (1908 p. 387), (1912 p. 3), (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Grosbiisch (1920 p. 311). (2) Add one of the combinations given

under added nutrients to (1).
584. Maassen's Basal Peptone Salt Solution
Constituents: Use: Cultivation of tubercle bacilli. The
1. Water 1000.0 cc. authors reported the best growth in the
2. Peptone 10.0 g. presence of both glucose and glycerol.
3. KH2PO4 1-5 g. Added nutrients: One of the following
4. NaCl 1.0 g. was added:
5. MgS04 0.3 g. (a) glycerol 40.0 g.
Preparation (b) glucose 10.0 g.
(1) Dissolve 2, 3, 4 and 5 in 800.0 cc. of 1. (c) glycerol 40.0 g. + glucose 10.0 g.
(2) The reaction of (1) is acid. Reference: Calmette, Massol and Breton
(3) Dissolve one tenth the equivalent (1909 p. 580).
weight of one of the added nutrients
586. Hansen's Basal Peptone Salt Solution
in 200.0 cc. of 1.
(4) Add (3) to (1). Constituents:

(5) Neutralize and make slightly alkaline 1. Water 1000.0 cc.
to litmus. 2. Peptone 1.0 g.
Sterilization: Not specified. 3. KH2PO4 3.0 g.
Use: To study the utilization of organic 4. MgS04 2.0 or 5.0 g.
Preparation :(1) Dissolve 2, 3, 4 and one of 3. K2SO4 0.2 g

the added nutrients in 1. (Use 5.0 g. 4. NaaCOa 0.25 g
MgS04 using maltose and 2.0 g. with 5. FeP04 0.2 g

glucose.) 6. MgCl2 0.1 g
Sterilization: Not specified. 7. CaCl2 0.05 g
Peptone 0.1 g
Use: Cultivation of Saccharomyces cere- 8.
Preparation
visiae, Saccharomyces Pastorianus and a
wine yeast. (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
Added nutrients and variants: (2) Distribute in 100.0 9,0. lots.

(3) Add one of the added nutrients
of maltose to
(a) The author added 5.0 g.
each flask (Neutralize and make the
.
or glucose.
cultivated the causative acids slightly alkaline by the addi-
(b) Stormer
organism of water rust in flax (Plec- tion of NazCOs).
tridium pectinovorum) using 5.0 g. (4) Add 2.0 g.CaCOs to each flask.
KH2PO4, 2.5 g. MgS04 and 5.0 g. of Sterilization: Method not given.
peptone in the basic solution. One Use: To study nitrogen assimilation by
Bacillus megatherium (Alinit bacteria).
of the following materials was added
starch 5.0 to 10.0 g Authors reported that organisms showed
glucose 5.0 to 10.0 g greater development in medium contain-
arabinose 5.0 to 10.0 g ing organic acids as a source of carbon
dextrin 5.0 to 10.0 g than d-glucose. Greatest development

galactose 5.0 to 10.0 g took place with saccharose, 1-arabinose
pectin substances 2.5 to 5.0 g and l-.xylose. No growth took place using
d-laevulose or d-galactose. Lactic acid
All the solutions were sterilized in
is the best suited acid for B. megatherium
the autoclave except the one con-
taining pectin. Sterilize this solu-
development.
Added nutrients: The authors added 10.0%
tion at 100C. Incubate under anae-
robic conditions, using pyrogallic
d-glucose maltose
acid and KOH.
of d-laevulose tartaric acid
(c) Buromsky studied the effect
d-galactose citric acid
organic acids upon the growth of
d-xylose malic acid
yeast, Saccharomyces ellipsoid, and
lactic acid
Saccharomyces pastorian. He used 1-arabinose
KH2PO and saccharose butyric acid
0.5 g. MgS04, 1.0 g.
10.0 g. peptone in the basic solution
of the following: p. 268).

and added one
citric acid 10% 588. Vierling's Basal Peptone Salt Solution
tartaric acid 10%
malic acid 10% Constituents:
Water 1000.0 cc.
quinic acid 0.5% 1.
succinic acid 10% 2. K2HPO4 1-0 g-
1-0% 3. CaCU 01 g-
glycerol
mannitol 1-0% 4. MgS04 0.1 g.
5. FeCl3 trace
He reported no growth using tartaric
NaCl trace
acid. Citric acid also inhibited most 6.
7. Peptone 10-0 g.
yeast.
Stormer Preparation
References: Hansen (1899 p. 6),
(1) Dissolve 2, 3, 4, 5, 6
and 7 in 1.
(1904 p. 177), Buromsky (1914 p. 532).
(2) Add one of the
combinations given
587. Stoklasa and Vitek's Basal Peptone under added nutrients.
Salt Solution Distribute in 5.0 cc. quantities in
(3)
tubes.
Constituents:
1000.0 cc. Sterilization: Not specified.
1. Water
Na2HP04 0.25 g. Use: To study nitrate reduction by myco-
2.
bacteria. To test for nitrite production Henneberg's Glucose Peptone Salt

add a 2.0% acidified Kt solution to the Solution 594
tube. A blue color will develop in the Ej. Not containing phosphates.
presence of nitrites. The presence of Fi. Salts of monovalent cations only added.
nitrite may be detected also by dissolving Harvey's Glucose Peptone Solution. 595 .
0.1 g. napthylamine in 150.0 g. dilute Eijkman's Glucose Peptone Solution. 596

acetic acid, and 0.5 g. of sulfanilic acid Matzuschita's Glucose Peptone Solu-
in 150.0 g. dilute acetic acid. Mix 1.0 tion 597
CO. of each solution and heat to boiling. F2. Salts of divalent cations with or without
Add 1.0 CO. of the mixture to the culture. other cations added.
If nitrite be present a red color will be Gosio's Glucose Peptone Solution... 598
found. Sears' Glucose Peptone Solution 599
Added nutrients and variants: Ringer's Solution Broth (Park, Wil-
(a) Starch 10.0 g. + KNO3 5.0 g. liams and Krumwiede) 600
(b) Peptone 20.0 g. in the basic solution Ci.* Disaccharides added.
and added 10.0 g. of starch. This Di. Lactose employed.
medium was used to study amylase El. Inorganic salts not added.
production by mycobacteria. No re- Grimbert's Lactose Peptone Solu-
ducing sugars were found to be present tion (Robin) 601
using Fehling's solution for the test. Levine's Lactose Peptone Solution.. 602
(c) Glucose 10.0 g. This solution was Wagner and Monfort's Lactose Pep-
used to study catalase production by tone Solution 603
mycobacteria. Catalase was pro- Baginsky's Lactose Peptone Solution. 604
duced. A 14 day culture quickly E2. Inorganic salts added.
decomposed 1.0 cc. of a 15.0 H2O2 % Fi. Indicators added.
solution, of o.xygen with the libera- Robin's Lactose Peptone Solution. . . 605
of oxygen. Klein's Lactose Peptone Solution. . . . 606
Reference: Vierling (1920 pp. 201, 204, Harvey's Lactose Peptone Solution. . 607
207). Olszewski and Kohler's Lactose Pep-
tone Solution 608
Bacto Neutral Red Medium (Dehy-
Complete or basal liquid media contain- drated) 609
ing peptone or similar digests as sole sources F2. No indicators added.
of nitrogen; additional organic carbon Fischer and Andersen's Lactose Pep-
supplied. tone Solution 610
Ai.* Carbohydrates present. Baginsky's Lactose Peptone Salt
Bi. Carbohydrate only added. Solution 611
Ci. Monosaccharides added. D2. Sucrose employed.
Di. Levulose added. El. Containing salts of monovalent cations
Thurgau and Osterwalder's
Miiller, only.
Levulose Peptone Solution 589 Bendick's Phenolphthalein Sucrose
D2. Glucose added. Solution 612
El. Containing phosphates. Smith's Sucrose Peptone Solution
Fi. Containing salts of monovalent cations (Owen) 613
only. Briaudat's Sucrose Peptone Solution. 614
Kligler's Glucose Peptone Solution. . 590 Bokorny's Sucrose Peptone Solution. 615
Lohnis' Glucose Peptone Solution... 591 E2. Containing salts of mono and divalent
F2. Containing salts of mono and divalent cations.
cations. Mutchler's Sucrose Peptone Solution. 616
Mueller's Glucose Peptone Salt Solu- Waksman's Sucrose Peptone Solution. 617
tion 592 Zikes' Sucrose Peptone Solution 618
Schukow's Basal Glucose Peptone Behrens' Basal Sucro.se Peptone Solu-
Salt Solution 593 tion 619
* See ne.xt page for A2 and B2. *See C3 next page.

Bokorny's Sucrose Somatose Solution. 620 Freudenreich and Jensen's Lactate

E3. Containing salts of mono, di and tri- Peptone Solution 644
. valent cations. Baginsky's Lactate Peptone Solu-
Trommsdorff's Sucrose Peptone Solu- tion 645
tion 621 Harvey's Tartrate Peptone Solution. 646
Vierling's Sucrose Peptone Solution. 622 Harrison and Vanderleck's Aesculin
C3. Polysaccharides added. Peptone Solution 647
Baginsky's Starch Peptone Solution. 623
589. Miiller, Thurgau and Osterwalder's
Dumas' Glycogen Peptone Solution. 624
Levulose Peptone Solution
Kodama and Takeda's Starch Pep-
tone Solution 625 Constituents:
Khouvine's Cellulose Peptone Solu- 1. Water 1000.0 cc.
tion 626 2. Levulose 10.0 g.
Peptone Solution. 627
Vierling's Cellulose 3. Peptone 10.0 g.
B2. Carbohydrates added together with 4. K2HPO4 0.5 g.

other organic carbon compounds. 5. MgS04 0.1 g.
Ci. Alcohols employed. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
Matzuschita's Pyrogallic Acid Bouil- Sterilization: Not specified.
lon 628 Use: Cultivation of bacteria from wine and

Bezangon's Glycerol Peptone Solu- fruit wine, Micrococcus malolacticus
tion 629 Reference: Miiller, Thurgau and Oster-

walder (1912-13 p. 155).
C2. Organic acids employed.
Behrens' Tartrate Peptone Solution. 630 590. Kligler's Glucose Peptone Solution
Dombrowski's Glucose Peptone Solu-
631 Constituents:
tion
1. Water 1000.0 cc.
Zumstein's Citric Acid Peptone Solu-
632 2. Peptone (0.5%) 5.0 g.
tion (Killer)
3. Glucose (0.5%) 5.0 g.
Seitz's Double Sugar Peptone Solu-
633 Preparation: (1) Dissolve 2 and 3 in 1.
tion (Harvey)
Thurgau and Osterwalder's Sterilization: Not specified.
Miiller,
634 Use : To study metabolism of colon-typhoid
Malic Acid Peptone Solution
group.
Ayers, Rupp and Johnson's Glucose
635 Variants: Kligler gives the following solu-
Peptone Solution
tions:
Ao. Carbohydrates not present.
(a) Glucose 0.5% + 1.0% peptone.
Bi. Additional organic carbon supplied as (b) Glucose 0.5% + 1.5% peptone.
alcohols. (c) Glucose 0.5% + 0.5% peptone +
Marmier's Glycerol Protease Solu- 0.5% NaoHPOi.
tion 636 (d) Glucose 0.5% + 1.0% peptone +
Frankland and Fox's Glycerol Pep- 0.5% NaoHPOi.
tone Solution (Owen) 637 (e) Glucose 0.5% + 1.5% peptone +
Harvey's Glycerol Peptone Solu- 0.5% Na2HP04.
tion 638 (f) Glucose 0.3% + 1.0% peptone -t-
Waksman's Basal Glycerol Peptone 0.2% NaoHP04.

Solution 639
(g) Glucose 0.3% + 1-0% peptone +
Capaldi and Proskauer's Mannitol 0.3% Na2HP04.
Peptone Solution 640 (h) Glucose 0.3% + 1.0% peptone +
B2. Additional organic carbon supplied as 0.4% Na2HP04.
acids or their salts. (i) Glucose 0.4% + 1.0% peptone +
Omeliansky's Formate Peptone Solu- 0.2% Na2HP04.
tion 641 (j) Glucose 0.4% + 1.0% peptone +
Voges and Proskauer's Basal Citrate 0.3% Na2HP04.
Peptone Solution 642 (k) Glucose 0.4% + 1.0% peptone +
Behrens' Basal Citrate Acid Peptone 0.4% Na2HP04.
Solution 643 Reference: Kligler (1916 pp. 668-669).
591. Lohnis' Glucose Peptone Solution 5. CaCla 0.2 g.
Constituents:
6. K0HPO4 2.0 g.
7. Glucose 2.0 g.
1. Water tap 1000.0 cc.
8. Phenol red (0.02% soln.) ... 2.0 g.
2. Glucose (4.0%) 40.0 g.
Preparation
3. Peptone (0.1%) 1.0 g.
(1) Dissolve 2 in 1000.0 cc. of 1.
4. K2HPO4 (0.05%) 0.5 g.
(2) Dissolve 3, 4, 5, 6, 7 and 8 in 1000.0 cc.
of 1.
(3) Mix (1) and (2).
Use: Cultivation of aerobacter and amylo-
(4) Add NaOH until the medium is red
bacter. Tiie medium was inoculated
(pH = 7.8).
with 10.0% soil. Similar media were used
Sterilization: Sterilize in autoclave at 10
to study the fermentation of glucose, for
pounds for 10 minutes.
methyl red test.
Use: To study the constituents essential
Variants
for the growth of streptococci and pneu-
(a) Rogers, Clark and Evans prepared
mococci. The author reported this as a
the medium as follows:
poor medium.
(1) Dissolve 10.0 g. glucose, Merck's
Reference: Mueller (1922 pp. 317, 325).
highest qi\ality, 10.0 g. Witte's pep-
tone and 5.0 g. K2HPO4 in 800.0
593. Schukow's Basal Glucose Peptone Salt
cc. distilled water, heating over the
Solution
steam for 20 minutes, and occa-
sionally stirring. Constituents:
(2) Filter thru a Schleicher and Schull 1. Water 1000.0 cc.
No. 588 folded filter. 2. Peptone 10.0 g.
(3) Allow the filtrate to cool to 20C. 3. Dextrose 100.0 g.
and make up to 1000.0 cc. 4. Potassium phosphate 5.0 g.
(4) Sterilize in the steamer on each of 5. MgS04 0.5 g.
3 successive days. Preparation

(b) Clark and Lubs used the same (1) Dissolve 2, 3, 4 and 5 in 1.
medium as in variant (a), but omitted (2) Add 9.0 to 10.0% of one of the added
the K2HPO4 with or without the nutrients to (1).
addition of H3PO4 until the solution (3) Adjustment of reaction not specified.
is 0.075 molar NaOH. (4) Distribute into fermentation flasks
(c) Clark and Lubs used the same (sealed with H2SO4).
medium as in variant (a) but used 5.0 Sterilization: Sterilize in a steamer.
to 8.0 g. glucose instead of 10.0 g. Use: To study the utilization of acids by

(d) Bunker, Tucker and Green gave the yeast. After 85 days 0.124 g. of tartaric
same formula as variant (a) but used acid and 0.27 g. of malic acid were used.
5.0 g. glucose and 5.0 g. peptone and Acid determined by titration using
omitted the phosphate. litmus as an indicator.
References: Lohnis (1913 p. 104), Rogers, Added nutrients: The author added 9.0 or
Clark and Evans (1914 p. 103), Clark and 10.0% of malic or tartaric acid.
Lubs (1915 pp. 162, 163), Bunker, Tucker Reference: Schukow (1896 p. 608).
and Green (1918 p. 493), Committee 594. Henneberg's Glucose Peptone Salt
American Public Health Association
Solution
(1923 p. 107), (1925 p. 111).
Constituents:
592. Mueller's Glucose Peptone Salt 1. Distilled water 1000.0 cc.
Solution 2. KH,P04 (0.8%) 8.0 g.
Constituents: 3. MgS04 (0.1%) 1.0 g.
1. Water 2000.0 cc. 4. Glucose (5.0%) 50.0 g.
2. Peptone 20.0 g. 5. Peptone (1.0 to 5.0%). 10.0 to 50.0 g.
3. NaCl 10.0 g. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.

Use: Cultivation of lactic acid bacteria. ascitic fluid might be added. Pneu-
Reference: Henneberg (1903 p. 8). mococci grew very well without the
ascitic fluid however.
595. Harvey's Glucose Peptone Solution (b) Stewart and Stitt, determined the
Constituents: Voges Proskauer reaction using 5.0 g.
1. Distilled water 960.0 cc. NaCl, 20.0 g. glucose and 10.0 g.
2. Peptone 10.0 g. Witte's peptone per liter.
3. NaCl 5.0 g. (c) Meyers studied H2S production using
4. Glucose 10.0 g. 30.0 g. of Witte's, Difco or Fairchild's
5. Litmus solution 40.0 cc. g. glucose, 5.0 g. NaCl,
peptone, 5.0
Preparation and 0.0 or 5.0 g. sucrose per liter.
(1) Dissolve 2 and 3 in 1. (d) Klimmer used 10.0 g. peptone, 5.0 g.
(2) Filter sterile (1) and adjust the NaCl and 10.0 g. of glucose per liter.
reaction if necessary. References: Eijkman (1904 p. 745), Truche
(3) Dissolve 10.0 g. glucose in 40.0 cc. of and Cotoni (1911 p. 480), Stewart (1917-18
litmus solution. p. 294), Myers (1920 p. 242), Stitt (1923
(4) Mix (2) and (3). p. 36), Klimmer (1923 p. 221).
597. Matzuschita's Glucose Peptone
(6) Steam 10 minutes.
Solution
(7) Tube sterile (6) into sterile test tubes.
Sterilization: Sterilize (1) in the autoclave. Constituents:
Sterilize (6) by thru a candle.
filtering 1. Water 1000.0 cc.
Incubate to test sterility before use. 2. Meat peptone
Use: General culture medium. (Koch) 10.0 g.
Reference: Harvey (1921-22 p. 109). 3. Gum arable 50.0 to 300.0 g.
4. NaCl 5.0 g.
596. Eijkman's Glucose Peptone Solution 5. Glucose 20.0 g.
Constituents: Preparation:
1. Water 1000.0 cc. (1) Dissolve 2 in 1.
2. Glucose (10.0%) 100.0 g. (2) Dissolve 3, 4 and 5 in (1).

3. Peptone (10.0%) 100.0 g. (3) Neutralize (indicator not specified).
4. NaCl (5.0%) 50.0 g. Sterilization: Sterilize in the steamer on
Preparation: (1) Prepare a solution con- from 2 to 5 successive days for 15 to 30
taining 10.0% glucose, 10.0% peptone minutes. Incubate for 2 days at 37C. to
and 5.0% NaCl. test sterility.
Sterilization: Not specified. Use: Cultivation of spore forming bacilli.
Use: Water analysis and detection of Clostridium butyricum, Bacillus oedem-

B. coli. The water under investigation is atis maligni, Bacillus anthracis sijmpto-
placed into fermentation flasks and \ of matici, Bacillus sporogenes, Bacillus

its volume of the medium is added. The botulinus.
presence of uniform turbidity and gas Variants: The author used 1.0 to 3.0% gum
formation after 24 hours at 46 C. indicates tragacanth instead of 50 to 300.0 g. gum
the presence of B. coli. Indol may be arable.
formed after 48 hours. Reference: Matzuschita (1902 p. 287).
Variants
598. Gosio's Glucose Peptone Solution
(a) Truche and Cotoni enriched pneumo-
cocci in the following solution: Constituents:
1. Water 1000.0 cc. 1. Distilled water 3000.0 cc.
2. Peptone (Chapoteaut) 40.0 g. 2. Peptone 30.0 g.
3. NaCl 5.0 g. 3. Dextrose 138.0 to 150.0 g.
4. Glucose 2.0 g. 4. NaoCOs 1-6 g.
Chapoteaut's peptone cannot be 5. CaC03 75.0 g.

replaced by Witte's or Defresne's. Preparation
They specified that one-third part (1) Dissolve 2 and 4 in part of 1.
171
(2) Dissolve 3 in the remainder of (1).
(3) Mix sterile (1) and (2) when cool.

The addition of CaCOs not specified.
Use: Lactic acid production by vibrio.
solution with 30.0 cc. normal soda solution
to study fermentation by Vibrio cholera
asiaticae.
1. Water 3000.0 cc.
2. Peptone (Witte) 30.0 g.
3. Glucose 150.0 g.
4. CaCO,, 75.0 g.
Reference: Gosio (1894 p. 116, 2).
599. Sears' Glucose Peptone Solution
Constituents
1. Water 1000.0 cc.
2. Peptone 20.0 g.
3. Glucose 5.0 g.
4. NaCl 50 g.
5. CaCOs
Preparation
(2) Divide in two parts.

(3) To one part of (2) add 1.0% (5.0 g.)
of pure glucose and a little CaCOs.
(4) Distribute both portions in 200.0 cc.
lots into 300.0 cc. flasks.
Sterilization: Sterilize in autoclave for
10 minutes at 15 pounds pressure.
Use: To study nitrogen metabolism. Am-
monia determined by Folin's aeration
method, amino acid by Van Slyke's micro
method and total N by Kjeldahl-Gunning-
Arnold method.
Reference: Sears (1916 p. 118).
600. Ringer's Solution Broth (Park,

Williams and Krumwiede)
Constituents
1. Water 00.0 cc
2. NaCl
3. KCl
4. CaCls
5. Sodium bicarbonate.
6. Glucose
7. Peptone (1.0 or
2.0%) 10.0 or 20.0 g.
in 1.

Reference: Park, Williams and Krumwiede
(1924 p. 122).
2. Peptone (2.0%) 20.0 g. (6) Distribute in 10.0 cc. lots in Pasteur

3. Lactose (0.2%) 2.0 g. flasks.
4. Gentian violet (0.001%). . . 0.01 g. Sterilization: Sterilize in the autoclave at
Preparation 105 for 15 minutes.
(1) Dissolve 2, 3 and 4 in 1. Use: Differentiation of colon and typhoid
(2) The reaction is between pH 7.0 and groups. Author reported that coli strains
7.6. produced a blue colored medium, typhoid
(3) Tube and plug with cotton. cultures clouded the medium but gave no
Sterilization: Pasteurize at 70 to 80C. for change in color.
15 minutes. Reference: Robin (1897 p. 49).
Use: Presumptive test for Bad. coli in
606. Klein's Lactose Peptone Solution
water analysis Author reported that the
.
medium gave as good results as when the Constituents:

usual 1.0 or 0.5% lactose was used. 0.001 1. Distilled water 1000.0
to 0.00033% of gentian violet may be used. 2. Peptone
Variants: Used 0.001 to 0.00033% gentian 3. NaoHPOi
violet instead of 0.01%. 4. NaCl
Reference: Wagner and Monfort (1921 5. Lactose
p. 207). 6. Azolitmin
604. Baginsky's Lactose Peptone Solution
Constituents
2. Lactose 300.0 g.
3. Peptone
Preparation
(1) Distribute distilled water in 25.0 cc.
quantities in small flasks that have
been sterilized at 140C.
(2) Add 5.0 g. of lactoseand a small
amount (exact amount not given) of
peptone to each flask.
Sterilization: Sterilize in flowing steam on
3 successive days for ^ to 1 hour.
Use: To studj^ fermentation by B. lactis.
Reference: Baginsky (1888 pp. 434-462).
605. Robin's Lactose Peptone Solution

Constituents:
1. Water 1000.0 cc.
2. Peptone (Collas) 20.0 g.
4. NaCl 2.0 g.
5. Soluble blue (1.0% soln.) ... 4.0 cc.
6. Lactose 80.0 g.
Preparation
(1) Dissolve 2, 3 and 4 in 1 by boiling.
(2) Add 4.0 cc. of 1.0% aqueous solution
of soluble blue ("blue soluble") to
boiling (1).
(3) Add sufficient tenth normal KOH
solution to decolorize (2).
(4) Add 80.0 g. of lactose to (3).

(5) Filter.
4. Lactose 75.0 g. Sterilization: Not specified.
5. Litmus solution 500.0 cc. Use: To study acid production by Bac-

Preparation terium coli.
(1) Dissolve 75.0 g. peptone and 50.0 g. Reference: Fischer and Andersen (1912
NaCl in 600.0 cc. water. p. 289).
(2) Filter.
(3) Dissolve 75.0 g. lactose in 500.0 cc. 611. Baginsky's Lactose Peptone Salt
made according to
litmus solution, Solution
Kubel and Tiemann with Kahlbaum's Constituents:
litmus (method or reference not
1 Distilled water 750.0 cc.
given). 2. Lactose 36.0 g.
(4) Boil (3) for 15 minutes. 3. Peptqne (siccum) 8.0 g.
(5) Filter. 4. K2HPO4 1-6 g.
(6) Add NaOH or soda solution to neu- 5. CaCl, 0.15 g.
tralize if necessary. MgS04 0.3 g.
6.
(7) Mix sterile (2) and (6) when ready 7. CaCOs 0.02 g.
for use. Preparation
(8) Tube. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Sterilization: Method of sterilization of
Erlenmeyer
(2) Distribute into sterile
(2), (6) or (8) not given. flasks.
Use: Presumptive test for B. coli in water Sterilization: Sterilize in flowing steam for
analysis. Ten under in-
parts water hour.
4 days for | to 1
vestigation were added to one part of Use: To study fermentation by B. lactis.
medium. B. coli turned the medium red. Reference: Baginsky (1888 pp. 434-462).
Reference: Olszewski and Kohler (1922
p. 305). Sucrose
612. Bendick's Phenolphthalein
609. Bacto Neutral Red Medium Solution
(Dehydrated) Constituents:
Distilled water 2. Peptone 10.0 g.

1.
2. Peptone, Bacto 30.0 g. 3. NaCl 5.0 g.
3. Lactose, Bacto 6.0 g. 4. NaaCOa 1-0 g.
KCl 8.0 g. 5. Sucrose 5.0 g.

4.
5. Neutral red 0.08 g. 6. Phenolphthalein (Sat. 50.0%

Preparation :
ale. soln.) 5.0 cc.
(1) Dissolve 44.0 g. of Bacto neutral red Preparation:

medium in 1000.0 cc. of distilled water. (1) Dissolve 2 and 3 in 1 by heating over
(2) If sterilized 20 minutes at 15 pounds a free fl^-me.
pressure pH = 6.7. (2) Add NazCOs to neutralize to phenol-
Sterilization: Sterilize in the usual manner. phthalein.
Reference: Digestive Ferments Co. (1925 (3) Add 1.0 g. of NajCOs to (2), and boil
p. 13). over a free flame.

(4) Filter thru a double layer of filter
610. Fischer and Andersen's Lactose paper.
Peptone Solution Add 5.0 g. sucrose and 5.0 cc. of a
(5)
Constituents: 50.0% saturated alcoholic solution of
1. Water (96.5%) 965.0 cc. phenolphthalein to (4).

2. Peptone (Witte) (1.0%) 10.0 g. (6) Medium should be moderately deep
3. Lactose (2.0%) 20.0 g. red.
4. NaCl (0.5%) 5.0 g. Sterilization: Sterilize in the Arnold for
Preparation 20 minutes if the medium is to be used at
(1) Dissolve 2, 3 and 4 in 1. once. If not to be used at once, sterilize

Adjustment of reaction not given. in the Arnold for 15 minutes on each of 3
(2)
(3) Distribute in 50.0 cc. flasks. successive days.
Use: Detection of the cholera vibrio. The Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
cholera vibrio decolorized the medium. Sterilization: Not specified.
References: Bendick (1911 p. 907), (1912 Use: To study the effect of constituents of
p. 536), Park, Williams and Krumwiede the medium on the growth of yeast.
(1924 p. 130), Abbott (1921 p. 570). Variants: Bokorny gives the following
variants:
613. Smith's Sucrose Peptone Solution
(a) 50.0 g. of sucrose instead of 200.0 g.
(Owen)
(b) 2.0 g. of MgS04 instead of 1.0 g. and
Constituents added 0.1 g.H2S04.
1. Water 1000.0 cc. (c) Added 0.1 g. (or more) of H3PO4 or
2. Sucrose 100.0 or 200.0 g. lactic acid.
3. KCl 5.0 g. (d) Added 0.1 g. of HF or formaldehyde
4. Na2HP04 2.0 g. (e) Added 1.0 g. of formaldehyde, NaCl
5. Peptone 1-0 g. CaCl2 or caffeine.
Preparation (f) Used 1.0 g. peptone, 1.0 or 10.0 g
(1) Dissolve 2, 3, 4 and 5 in 1. sucrose, 1.0 g. KH.2PO4 and 0.5 g
(2) Distribute in 200.0 cc. quantities in MgS04 instead of amounts indicated
300.0 cc. Erlenmeyer flasks. in original medium and added 0.5 g
(3) After final sterilization adjust to pH CaCl2 and a trace of FeCU.
values varying from pH = 6.7 to 8.5 (g) Used 1.0 g. peptone, 1.0 or 10.0 g
by the addition of normal NaOH sucrose 1.0 g. KH2PO4 instead of
and H2SO4. The medium should still amounts indicated, omitted the
be warm. MgS04 and added 0.5 g. K2SO4, a
Sterilization: Sterilize on each of 3 suc- trace of FeCl2 and 0.5 g. of KCl or
cessive days for 30 minutes. CaCl2.
Use: To study gum formation. Author (h) Used 50.0 g. sucrose, 10.0 g. peptone,
reported the optimum pH between 6.7 5.0g.KH2PO4 or NaH2P04 and 5.0 g.
and 7.0. of MgS04 instead of the amounts
Variants: Invertase may be added to invert given in the original medium and
the sugar. Levan formation was not added 0.0 or 5.0 g. Rb2S04.
aided. (i) Used 100.0 g. sucrose, 25.0 g. peptone,
Reference: Owen (1923 pp. 423, 431). 4.0 g. KH2PO4 instead of given
amounts,
614. Breaudat's Sucrose Peptone Solution
(j) Used 1.0 g. peptone, 10.0 g. sucrose,
Constituents: 1.0 g. KH2PO4, and 0.5 g. MgS04
1. Water 1000.0 cc. instead of the given amounts, and
2. Peptone (Chapoteaut) 10.0 g. added a trace of iron chloride with
3. Sucrose 30.0 g. 0.5 g. of CaCl2, 0.5 g. KCl or 0.5 g.
4. K2CO3 4.0 g. CaCl2 + 0.5g. K2SO4.
Preparation (k) Used 50.0 g. sucrose, 1.0 g. peptone,
(1) Dissolve 2,and 4 in 1.
3 0.5 g. MgS04, omitted the KH2PO4
(2) Distribute in two liter flasks. from the original medium and added
Sterilization: Method not given. a trace of iron chloride, a trace of
Use: Acetone production by J5aci7rMs viola- CaCla with 1.0 g. KH2PO4, 0.1 g.
cius acetonicus. Rb2S04 + 1.0 g. NaH2P04 or 1.0 g.
Reference: Breaudat (1906 p. 877). NaH2P04.
References: Bokorny (1902 p. 58), (July
615. Bokomy's Sucrose Peptone Solution (1903-04 p. 20),
1903), (1903-04 p. 16),
Constituents (1911 p. 182), (1912 p. 128), (1912 p. 12S).
616. Mutchler's Sucrose Peptone Solution
2. Sucrose 200.0 g.
3. Peptone (meat) 5.0 g. Constituents:
4. KH2PO4 2.0 g. 1. Water 1000.0 cc.
5. MgS04 1.0 g. 2. Peptone 20.0 g
3. KH2PO4 100.0 g. 619. Behrens' Basal Sucrose Peptone

4. MgS04 0.2 g. Solution
5. Calcium phosphate 0.2 g.
Constituents:
6. Sucrose 150.0 g.
1. Water 1000.0 cc.
Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
3. Peptone 5.0 g.
(2) Flask.
on
4. MgS04 1.0 g.
Sterilization: Sterilize in an autoclave
5. K2HPO4 2.0 g.
successive days with temperatures of
Preparation
115 to 120C. (15 to 18 pounds pressure).
Use: Cultivation of yeast {Saccharomyces
cereviseae). Author reported a more
(3) Add one of the added nutrients as
rapid growth than in beer wort.
indicated below.
Reference: Alutchler (1905-06 p. 34).
617. Waksman's Sucrose Peptone Solution Use : acids on
To study the effect of organic
growth of Penicillium glaucum, Pencil-
Constituents:
Hum luteum and Botrytis spores.
1.
2. Peptone 20.0 g.
the following to each lot:
3. K2HPO4 1.0 g.
malic acid 0.5, 1.0, 1.5, 2.0 or 5.0 g.
4. KCl 0.5 g.
phloroglucine 0.5 or 1.0 g.
5. MgS04 0.5 g.
hydrochinon 0.5 or 1.0 g.
6. FeS04 0.01 g.
tartaric acid 7.35 g.
citric acid 6.86 g.
Preparation:
Reference: Behrens (1898 pp. 644, 739).
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
(2) Distribute (1) in 100.0 cc. portions

620. Bokorny's Sucrose Somatose Solution
in 200.0 cc. Erlenmeyer flasks.
Sterilization: Sterilize at 15 pounds for Constituents:
15 minutes. 1. Water 1000.0 cc.
Use: Cultivation of soil actinomyces and 2. Somatose 2.5 g.
fungi. 3. Sucrose 50.0 g.
Variants: Author used 1.0, 3.0, 5.0 or 20.0% 4. KH2PO4 2.0 g.
sucrose. 5. MgS04 10 g-
Reference: Waksman (1918 p. 510). Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
618. Zikes' Sucrose Peptone Solution Sterilization: Not specified.

Use: Cultivation of yeast.
Constituents: Reference: Bokorny (1917 p. 364).
2. Sucrose 100.0 g.
621. Trommsdorff's Sucrose Peptone
3. K2HPO4 1.0 g.
Solution
4. MgS04 0.3 g.
5. Peptone 2.5 g. Constituents:
(1) Dissolve 2, 3, 4 and 5 in 1. 2. MgS04 0.05 g.
(2) pH = 6.0603, CH = 8.7 X 10-^ 3. CaC04 0.05 g.
Sterilization: Not specified. 4. K2HPO4 0.1 g.
Use: Volutin formation by Saccharomyces 5. NaCi 0.1 g.
Frohberg, wine yeasts, Saccharomyces 6. FeCl3 0.001 g.

anamensis, Saccharomyces ilicis Saccharo- 7. Peptone (0.1%) (1.0 g. or
myces Will (Bajanus), Pichia membranae- 0.5%) 5.0 g.
faciens, Cholera m,ycoderma, Manilla 8. Sucrose (0.5%) 5.0 g.

Candida, Oidium lactic, Endomyces Preparation: (1) Dissolve 2, 3, 4, 5, 6,
Magnusi. 7 and 8 in 1.
Reference: Zikes (1922 p. 29). Sterilization: Not specified.

Use: Cultivation of Leptomitus. Growth

very good on this medium. Better
growth using 0.5% peptone than 0.1%.
Reference: Trommsdorff (1918 p. 65).
622. Vierling's Sucrose Peptone Solution

Constituents:
1. Water 1000.0 cc.
2. K2HPO4 1.0 g.
3. CaClo 0.1 g.
4. MgS04 0.1 g.
5. FeCls trace
6. NaCl trace
7. Peptone 10.0 g.
8. Sucrose 10.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7 and
8 in 1.

Use: To determine invertase production by
mycobacteria. Test for reducing sugars
with Fehling's solution. Very small
amounts of sugar if any were reduced.
Reference: Vierling (1920 p. 206).
623. Baginsky's Starch Peptone Solution
Constituents
CULTURE MEDIA FOR CULTIVATION OF MICRO ORG AXISMS 177
(4) Add 1 square centimeter of cellulose 5. Glycerin (5.0%) 50.0 g.

in the form of Berzelius filter paper 6. "Eikonogen" (0.1%) 1.0 g.
or a little cellulose precipitated after 7. Hydrochinone (0.1%) 1.0 g.
having been dissolved in Schweitzer's 8. Pyrogallic acid (0.1%) 1.0 g.
reagent to each tube. Preparation
Sterilization: Sterilize for 15 minutes at (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
110C. (2) Neutralize. Indicator not specified.

Use: Isolation and enrichment of B. cellu- Sterilization: Sterilize in the steamer on
lose dissolvens from 2 to 5 successive days for 15 to 30
Variants: The author gives the following minutes. Incubate for 2 days at 37^0.
variants to test sterility.
(a) Addition of 2.0 g. glucose. Use: Cultivation of spore forming bacilli.
(b) Used 0.5 g. K2HPO4 instead of 1.0 g., Clostridium butyricum, Bicillus oedem-
added 0.5 g. Na2HP04, and 2.0 g. of atis maligni, Bicillus anthracis sympto-
CaCOs. matica, Bacillus sporogenes, Bacillus
(c) Same as (b) with 2.0 g. of glucose. botulinus.
(d) Used traces of NaCl instead of 1.0 g., Reference: Matzuschita (1902 p. 287).
added 0.5 g. MgS04, traces of CaCOj
629. Bezanfon's Glycerol Peptone Solution
and some gum arable (amount not
given). Constituents:
(e) Same as (d) with 2.0 g. glucose. 1. Water 1000.0 cc.
Reference: Khouvine (1923 p. 713). 2. Glucose 23.0 g.
3. Glycerol 20.0 g.
627. Vierling's Cellulose Peptone Solution
4. Peptone 10.0 g.
Constituents Preparation: (1) Dissolve 2, 3 and 4 in 1.

2. Peptone (3.0%) 30.0 g. Use: Cultivation of Hyphomycetes, Sporo-
3. CaCOa (0.1%) 1.0 g. trichum heurmanni
4. Filter paper Reference: Bezangon (1920 p. 644).
Preparation
630. Behrens' Tartrate Peptone Solution.
(2) Place strips of filter paper in flasks. Constituents:
(3) Pour (1) into (2). 1. Water 1000.0 cc.
(4) Seal the flasks with paraffin after 2. Starch 10.0 g.
inoculation. 3. Cane sugar 20.0 g.
Sterilization: Not specified. 4. Peptone 5.0 g.
Use: Decomposition of cellulose by myco- 5. Tartaric acid 5.0 g.
bacteria. Filter paper showed no signs 6. K2HPO4 2.0 g.
of being attacked. Growth occurred, 7. MgS04 1.0 g.
however. 8. Copper sulphate 0.0, 0.1, 0.02, 0.1,
Variants: The author gave the following 0.2, 0.5 or 1.0 g. (per 100.0 cc.)
variants: Preparation
(a) Used 2.0% peptone and added 1.0% (1) DLssolve 2, 3, 4, 5, 6 and 7 in 1.
glucose. (2) Distribute in 100.0 cc. lots.

(b) Used 2.0% peptone and added 0.4% (3) Prepare a 10.0% solution of copper
KNO3. vitrol.
Reference: Yierling (1920 p. 206). (4) AddO.O, 0.1, 0.2, 1.0, 2.0, 5.0 or 10.0 cc.
of (3) to each flask.
628. Matzuschita's Pyrogallic Acid Bouillon
Constituents: Use : To show influence of copper on growth
1. Water 1000.0 cc. of Oidium fructigenum. Growth is in-
2. Meat peptone (Koch) 10.0 g. hibited by a dilution of about 1.0 g. of
3. NaCl 5.0 g. copper sulphate per 109.0 cc. (10.0 cc. of a
4. Dextrose (2.0%) 20.0 g. 10.0% solution).
Variants: Author used 50.0 g. sucrose in- Use: Substitute for litmus milk.
stead of 20.0 g. and omitted the starch. Variants: Klimmer specified the use of
Reference: Behrens (1898 pp. 775-776). Witte's peptone and used 0.25 g. Kahl-
baum's azolitmin or added 1 or 2 drops of a
631, Dombrowski's Glucose Peptone saturated watery solution of China blue.
Solution He used the medium as a whey substitute.
Constituents References: Harvey (1921-22 p. 109), Klim-
1. Water 1000.0 cc. mer (1923 p. 208).
634. Miiller, Thurgau and Osterwalder's
3. Glucose 50.0 g.
Malic Acid Peptone Solution
4. K2HPO4 2.0 g.
5. MgS04 10 g. Constituents:
6. NaCl 1.0 g. 1. Water 1000.0 cc.
7. Lactic acid 2. Peptone (Witte) 10.0 g.
Preparation 3. H2KPO4 10 g.
(2) Acidify slight!}^ with lactic acid. 5. CaCl2 0.2 g.
Sterilization: Not specified. 6. Malic acid 1.0 g.
Use: Cultivation of milk yeasts. 7. Levulose 20.0 g.
Reference: Dombrowski (1910 p. 358). Preparation

(1) Boil 10.0 liters of tap water. (Given
632. Zumstein's Citric Acid Peptone and not one.)
as ten liters
Solution (Killer)
(2) Filter.
Constituents: (3) Dissolve 2, 3, 4, 5, 6 and 7 in (2).
1. Water 1000.0 cc. (4) Tube.

Peptone 5.0 g. Sterilization: Sterilize the fractional
2.
3. Glucose 5.0 g. method.

4. Citric acid 2.0 g. Use: Cultivation of Bacterium mannito-
5. MgS04-7H20 0.2 g. poeum and bacteria from wine and fruit
6. KH2PO4 0.5 g. wine. Authors reported that when po-
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 tassium malate is present a larger ratio
inl. of the acid produced is lactic (non volatile)
Sterilization: Not specified. rather than acetic.
Use: Cultivation of flagellates, organisms Variants: Authors added 1.0% potassium
from the soil, etc. malate
Variants : Dilute the medium 5 to 10 times Reference: Miiller, Thurgau and Oster-
for cultivation of protozoa. walder (1912-13 p. 161).
Reference: Killer (1913 p. 522).
635. Ayers, Rupp and Johnson's Glucose
633. Seitz's Double Sugar Peptone Solution Peptone Solution
(Harvey) Constituents:
Constituents
1. Distilled water 1000.0 cc. 2. Pepsin (Park Davis & Co.).. 5.0 g
2. Lactose 20.0 g. 3. Peptone (Park Davis & Co.) 5.0 g
3. Glucose 0.4 g. 4. K2HPO4 3.0 g
4. Na2HP04 0.5 g. 5. NaCl 5.0 g

Glucose 2.5 g
5. Ammonium sulphate 1.0 g. 6.
6. Sodium citrate 2.0 g. 7. Soap (castor oil)

8. Peptone 0.05 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
9. Azolitmin 0.05 g. (2) Adjust to pH = 7.6.
Preparation: Dissolve 2, 3, 4, 5, 6, 7, (3) Add sufficient quantity of castor oil

(1)
8 and 9 in 1. (Method not given.) soap (sodium ricinolate) to lower the
Sterilization: Not specified. surface tension 50 and 43 dynes.
Sterilization: Method not specified. 2. Glycerol 60.0 g.

Use: To study effect of surface tension on 3. Peptone 2.0 g.
growth of streptococci. Author reported 4. Calcium carbonate (pre-
that in general when the surface tension cipitated) 30.0 g.
was reduced to from 40 to 41 dynes the 5. Potassium phosphate 0.5 g.
growth of streptococci was prevented. 6. MgS04 1.0 g.
Reference: Ayers, Rupp and Johnson (1923 7. CaCls 0.5 g.
p. 204). Preparation
636. Marmier's Glycerol Protease Solution
(2) Dissolve 2, 3 and 4 in 2000.0 cc. of (1).
Constituents: (3) Divide into liter portions.
1. Water 1000.0 cc. Sterilization: Sterilize using the inter-
2. Peptone 40.0 g. mittent method.
3. NaCl 15.0 g. Use: To determine amount of alcohol
4. Sodium phosphate 0.5 g. formed in the fermentation of glycerin
5. Potassium phosphate 0.2 g. by Bacillus saccharalis. Author reported
6. Glycerol 40.0 g. that from a liter culture 50.0 cc. of a
Preparation 1.707% alcohol solution could be obtained.
(1) Dissolve the peptone in a certain Reference: Owen (1916 pp. 239-241).
amount of water.
(2) Add enough (NH4)2S04 to (1) so that 638. Harvey's Glycerol Peptone Solution
the solution is saturated at 100C.
Constituents:
(3) Boil and then filter.
1. Peptone (2.0% soln.) 870.0 cc.
(4) Dialyse in a current of water for
40 hours.
The liquid may then be reduced to
3. NaOH(normal soln.) 30.0 cc.
(5)
Preparation:
3.0% peptone and sterilized at 115
(1) Prepare a 2.0% peptone solution.
if not ready for use.
(6) Instead of dialysing the liquid free
from (NH4)2S04 as in step (4),
Use: Cultivation of V. cholerae.
barium hydrate may be added in such
an amount so that all the sulphate be
precipitated. Filter and keep the
639. Waksman's Basal Glycerol Peptone
filtrate at a temperature near boiling
Solution
and pass a current of air thru it to
free it of ammonia. When free of Constituents:
ammonia pass a current of CO2 1. Water 1000.0
thru the filtrate to remove the excess 2. Glycerol
of barium. Filter. 3. K2HPO4
(7) Make up the ammonia free filtrate (5) 4. KCl
or (6) up to 1 liter and dissolve 3, 4, 5 5. MgS04
and 6 in it. 6. FeS04
(8) Filter and distribute the filtrate in 7. Peptone (Witte's)
250.0 cc. Pasteur flasks. Preparation:
Sterilization: Sterilize at 110C. (time not (1) Dissolve 2, 3, 4, 5, 6, 7 and 2.0 g. of
specified). one of the added nutrients in 1.
Use: Anthrax toxin production. Marmier (2) Tube in 10.0 to 12.0 cc. quantities.
reported this medium as being non-toxic Sterilization: Sterilize at 15 pounds pres-
for animals. sure for 15 minutes.
Reference: Marmier (1895 p. 536). Use: To study metabolism of actinomy-
cetes.
637. Franklin and Fox's Glycerol Peptone
Solution (Owen)
of one of the following salts:
Constituents: NaNOa
1. Water 5000.0 cc. NaNOz
(NH4)2S04 References: Capaldi and Proskauer (1894

(NH4)2C03 p. 464), (1896 p. 472), Harvey (1921-22
Reference: Waksman (1920 p. 3). p. 111).
640. Capaldi and Proskauer's Mannitol 641. Omeliansky's Formate Peptone

Peptone Solution Solution
1. Water 1000.0 cc.
1. Water 1000.0 cc.
2. Mannitol (0.1%) 1.0 g.
2. Calcium formate 20.0 g.
3. Peptone (Witte) (0.5
Peptone
3. 2.0 g.
or 2.0%) 5.0 or 20.0 g.
Preparation
Flask.
(2)
Use: To study the fermentation of man- Sterilization: Method not given.
nitol by the colon-typhoid group. Use To study decomposition of formic acid
:
Variants: The author gave the following by Bacterium formicicum. About 3

variants
times more H2 was formed than CO2.
(a) Substituted Carne, Aschman, Chapo- Inoculate and seal with a rubber stopper
teaut, Driisen, Hemialbumose, soma-
fitted with a tube containing a mercury
tose or glutin peptone (Paal) for seal. About 250.0 cc. of air was sealed
Witte's peptone. into the flask with the culture.
(b) Used 2.0% Witte's peptone and added Reference: Omeliansky (1903-04 p. 188).
sufficient litmus a color.
to give
Mannitol was fermented with strong 642. Voges and Proskauer's Basal Citrate
acid reaction by the typhoid bacilli Peptone Solution
after 20 hours. The following salts
were added in 0.1% strength. Their Constituents:
presence did not inhibit development 1. Distilled water 100.0 cc.
of the organisms to any extent: 2. Na2HP04 0.37 g.

aluminum acetate 3. KH2PO4 0.14 g.
potassium antimonate 4. CaCl2 0.04 g.
sodium arsenate 5. KCl 0.3 g.
lead acetate 6. Magnesium citrate 0.01 g.
borax 7. Peptone (1.0%) 1.0 g.
cadmium chloride Preparation

chrome alum (1) Dissolve 2, 3, 4, 5, 6, 7 and 1.0% of
iron alum one of the added nutrients.
potassium ferrocyanate (2) Neutralize with soda.
copper acetate Sterilization: Not given.
manganese sulphate Use: Study fermentation of hemorrhagic
ammonium molybdate septicemia bacteria. Growth reported
nickel sulphate as beingmore luxuriant than in bouillon.
sodium nitroprusside Added nutrients: The authors added 1.0%
palladium chloride of one of the following:
sublimate glucose dextrin
silver sulphate mannose starch (potato)
strontium nitrate levulose glycerol
thallium sucrose adonitol
uranium nitrate lactose dulcitol
bismuth citrate maltose mannitol
zinc sulphate raffinose
zinc chloride Variants: The authors used the basic solu-
(c) Harvey added 47.5 litmus solution

cc. tion withoutany additions.
to a solution containing 2.0% pep- Reference: Voges and Proskauer
tone and 0.1% mannitol. pp. 22, 28).
643. Behren's Basal Citrate Acid Peptone Preparation

Solution (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
(2) Distribute in sterile flasks.

Constituents
Sterilization: Sterilize in flowing steam on
1. Water 1000.0 cc.
3 consecutive days for 5 to 1 hour.
2. K2HPO4 2.0 g.
Use: To study fermentation by B. lactis.
3. MgS04 1.0 g.
Reference: Baginsky (1888 pp. 434-462).
4. Citric acid 10 g.
5. Peptone 20.0 g. 646. Harvey's Tartrate Peptone Solu-îon
Preparation:
Constituents:
1. Water 1000.0 cc.
(2) Distribute in 200.0 cc. lots in flasks.
2. NaCl 5.0 g.
(3) Add one of the combinations given K2HPO4
3. 3.0 g.
under added nutrients to each flask.
4. Peptone 30.0 g.
5. Ferric tartrate (2.0% soln.). 10.0 cc.
Use: Cultivation of botr3^tis spores.
Preparation:
the following to 200.0 cc. of the basic
(2) Tube in 10.0 cc. quantities.
solution:
(3) Add 0.1 cc. of a 2.0% neutral ferric
sucrose 0.5 g.
tartrate solution. A yellowish pre-
salicin 4.0 g.
cipitate forms.
salicin 1 g.
+ glycerol 1 .0 g.
glycerol 1 -0 g.
Reference: Behrens (1898 p. 578).
647. Harrison and Vanderleck's Aesculin
Peptone Solution
644. V. Freudenreich and Jensen's Lactate
Peptone Solution Constituents:
Constituents: 2. Peptone (Witte) 23.0 g.
1. Water 1000.0 cc. 3. Aesculin (Merck) 0.5 g.
2. Peptone (Witte) 20.0 g. 4. Iron citrate scales (Merck). 0.5 g.
4. NaCl 5.0 g.
5. Calcium lactate 20.0 g.
(2) Adjust the reaction to +0.6.
Use: Enrichment of propionic acid formers Use: Detection of B. coli and B. typhosus.
from cheese. Medium is inoculated with B. coli gives blackened medium. Me-
a cheese emulsion. dium without bile salts blackened quicker
References: V. Freudenreich and Jensen than with the bile salt.
(1907 p. 529), Boekhout and Ott de Vries Variants: The authors give the following
(1918 p. 130). variants:
(a) Addition of 3.0 g. sodium tauro-
cholate.
645. Baginsky's Lactate Peptone Solution
(b) Use of 10.0 g. peptone instead of
Constituents 20.0 g. Witte's peptone, 2.5 g. iron
1. Water 1000.0 cc. citrate scales (Merck) instead of
2. Sodium lactate 10.0 g 0.5 g., and 1.0 g. of aesculin instead
3. Peptone 2.5 g of 0.5 g.
4. K2HPO4 0.1 g (c) Same
as (b) but add 2.5 g. bile salt
5. CaClj 0.02 g (sodium taurocholate).
6. MgS04 0.02 g Reference: Harrison and Vanderleck (1909
7. CaC03 0.02 g pp. 616, 622).
SUBGROUP I-C. SECTION 10 Hiss' Basal Asparagin Peptone Solu-

tion 666
Liquid media or basal solutions contain- Henneberg's Basal Asparagin Pep-
ing peptone or similar commercial digests tone Solution 667
and nitrogen compounds of
additional Nicole and Refik-Bey's Asparagin
known chemical composition; organic car- Peptone Solution 668
bon compounds added. Thoinnessen's Asparagin Peptone
Ai. Organic nitrogen present only as
Solution 669
peptone. Bokorny's Asparagin Peptone Solu-
Bi. Inorganic nitrogen supplied as ammo- tion 670
nium salt.
Reichenbach's Lactose Peptone
Ci. Ammonium chloride added.
Solution (Quantz) 671
Jones' Glucose Peptone Solution. . . . 648
Frankel's Asparagin Peptone Solu-
Lustig's Ammonium Chloride Pep-
tion (Tanner) 672
tone Solution 649
C2. Amino nitrogen supplied as tyrosine.
Cathelineau's Basal Ammonium Sul- Berthelot's Tyrosine Peptone Solu-
phate Peptone Solution 650
tion 673
C2. Ammonium sulphate added. C3. Amino nitrogen supplied as trypto-
Grimbert's Basal Peptone Salt Solu- phane.
tion 651
Tryptophane Broth (A. P. H. A.). 674 . . .
Beijerinck's Malate Peptone Solu- Frieber's Tryptophane Peptone Solu-

tion 652
tion 675
Klecki's Lactose Peptone Solution. 653
Harvey's Tryptophane Peptone Solu-
. .
Seitz's Lactose Peptone Solution. . 654

. .
676
tion
C3. Ammonium salts of phosphoric acid
B2. Organic nitrogen other than amino
added. acids added.
Hauman's Pectin Peptone Solution. . 655
Ci. Containing urea.
Charpentier's Sucrose Peptone Solu- Urea Peptone Solution.. 677
Hiss' Basal
tion 656
Urea Peptone Solution
Killer's 678
B2. Inorganic nitrogen supplied as nitrate. Cunningham's Urea Peptone Solu-
Ci. Additional organic carbon supplied as tion 679
carbohydrates. C2. Containing hippuric acid.
Di. Monosaccharides added. Ayers and Rupp's Hippurate Peptone
Lustig's Nitrate Peptone Solution.. 657 Solution 680
Stoklasa's Nitrate Peptone Solution. 658 Ayers and Rupp's Pepsin Peptone
Stutzer's Nitrate Glucose Solution. . 659 Solution 681
Duchacek's Nitrate Peptone Solu- C3. Containing Bile Salts.
tion 660 MacConkey's Bile Salt Peptone Solu-
D2. Polysaccharides added. tion 682
Khouvine's Cellulose Nitrate Solu- Harrison and van der Leek's Bile Salt
tion 661 Peptone Solution 683
Omeliansky's Cellulose Peptone Solu-
tion (Khouvine) 662 648. Jones' Glucose Peptone Solution
C2. Additional Organic carbon supplied as Constituents:
alcohols or organic acids. 1. Water 1000.0 cc.
Maassen's Nitrate Peptone Solution. 663 2. NH4CI 5.0 g.
Wassermann's Nitrate Peptone Solu- 3. Glucose 5.0 g.
tion 664 4. Peptone 10.0 g.
Duchacek's Nitrate Tartaric Acid 5. Calcium lactate 10.0, 20.0, 30.0, 40.0,
Solution 665 50.0, 60.0, 70.0, 80.0, 90.0, or 100.0 g.
A2. Organic nitrogen present in addition to Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
peptone. Sterilization: Method not given.

Bi. Amino acids added. Use: To study fermentation by B. proteus.
Ci. Amino nitrogen supplied as asparagin. The author reported the more lactate
salt present, the less acid and gas were Preparation

formed. (1) Dissolve 5.0 g. of a mixture of sodium
Variants: The author used sodium or am- phosphate and sodium potassium
monium lactate instead of calcium tartrate (Seignette's salt) in 1.
lactate. (2) Dissolve 5.0 (NH4)iS04, 10.0 g. of

g.
Reference: Jones (1916 p. 40). peptone and one of the added nu-
trients in (1).
649. Lustig's Ammonium Chloride Peptone
Add an excess of CaCOa.
(3)
Solution
Constituents: Use: Cultivation of Bacillus viridis
1. Water 4000.0 cc. (Lesage).
2. Potassium phosphate 1-0 g. Added nutrients The author added 2.0% of
:
3. MgS04 (crystals) 0.2 g. one of the following materials:

4. CaClj (fusum) 0.1 g. glucose succinic acid
5. Invert sugar 5.0 g. levulose tartaric acid
6. NH4CI 5.0 g. lactose lactic acid
7. Peptone 10 g. galactose citric acid
8. CaCOs 15.0 g. maltose butyric acid
Preparation sucrose glycocolic acid
(1) Prepare a salt solution by dissolving mannitol malic acid
1.0 g. potassium phosphate, 0.2 g. glycerol aspartic acid
MgS04 (crystals) and 0.1 g. CaCh Reference: Cathelineau (1896 p. 229).
(fusum), in 1000.0 cc. water.
(2) Prepare an invert sugar solution by
651. Grimbert's Basal Peptone Salt Solution
dissolving 5.0 g. cane sugar in 1000.0
cc.water and inverting. Constituents
(3) Prepare an ammonium chloride solu- 1. Water 1000.0 cc.
tion by dissolving 5.0 g. NH4CI in 2. Ammonium phosphate 0.4 g.
500.0 cc. water. 3. MgS04 0.4 g.
(4) Mix 400.0 cc. (1), 240.0 cc. of (2) and 4. Potassium phosphate 0.2 g.
200.0 cc. of (3). 5. (NH4)2S04 0.2 g.
(5) Add and dissolve in (4), 1.0 g. pep- 6. KNO3 0.2 g.
tone and 15.0 g. of CaCOs (puriss). 7. Peptone (dry) 2.5 g.
(6) Dilute (5) to 4000.0 cc. with water. Preparation
(7) Distribute into well sterilized 100.0 (1) Dissolve 2, 3, 4, 5, 6, 7 and 3.0 to 5.0%
cc. flasks in 30 to 50.0 cc. quantities. of one of the added nutrients in 1.
Sterilization: Sterilize well on each of two (2) Tube or flask.
successive days for an hour. Sterilization: Sterilize at 120C. for 15 to
Use: To study the oxidation of ammonia 45 minutes depending on the size of the
by a red pigment producing organism flask.
isolated from water, and by bacteria from Use: To study the fermentation ability of
the soil. Bacillus orthobutylicus.
References: Lustig (1890 p. 38), Frankland Added The author added 3.0 to
nutrients:
and Frankland (1889 p. 376). 5.0% any fermentable material.
of
Reference: Grimbert (1893 p. 359).
650. Cathelineau's Basal Ammonium
Sulphate Peptone Solution
652. Beijerinck's Malate Peptone Solution
Constituents:
2. Sodium phosphate 1. Water (ditch) 1000.0 cc.
3. Sodium potassium tartrate 2. Potassium malate 2.5 g.
4. (NH4)2S04 5.0 g. 3. Peptone (siccum) 2.5 g.
5. Peptone 10.0 g. 4. Mohr salt 1.0 g.
6. CaCOa (FeS04(NH4)2S04-6H20)
Preparation :
acted as typhoid. Bacillus faecalis alcali-
ditch water genes produced an alkaline reaction.
(1) Dissolve 2, 3, and 4 in
("Grabenwasser"). Reference: Seitz (1912 p. 434).
(2) Make alkaline with Na2C03.

655. Hauman's Pectin Peptone Solution
Use: To study reduction of sulphates by Constituents
Spirillum tenue. 1. Water 1000.0 cc.
Reference: Beijerinck (1895 p. 107). 2. Pectin 10.0 g.
3. Peptone 10 g.
653. Klecki's Lactose Peptone Solution
4. Ammonium phosphate 10 g.
Constituents 5. K0SO4 0-5
1. Water 8000 to 10,000.0 cc.
6. MgS04 0-2 _
2. Calcium lactate 225.0 g. Preparation: (1) Dissolve 2, 3, 4, 5 and 6
3. Ammonium phosphate. . 0.75 g. in 1.
4. Potassium phosphate 0.4 g. Sterilization: Not specified.

5. MgS04 0-4 g- Use: Cultivation of B. coli, B. fluorescens,
6. (NH4)2S04 0-2 g- Cladosporium herbarum, Aspergillus
7. Peptone 200.0 g. niger, Penicillium glaucum.
8. Lactose 500.0 g. Reference: Hauman (1902 p. 384).
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. 656. Charpentier's Sucrose Peptone
(2)Add some chalk to each flask. Solution
Sterilization: Method not given. Constituents
Use: To study fermentations in cheese. 1. Distilled water 1000.0 cc.
The medium was inoculated with a piece 2. Peptone (Malto) 10.0 g.
of "Quargel" cheese. Vibrio butyrique,
3. Ammonium phosphate
Bacillus saccharobutyricus, and other 0.25 g.
(neutral)
organisms developed. Medium was used 4. Sucrose 100.0 g.
under anaerobic as well as aerobic
5. Arsenic Varying amounts
conditions. Preparation
Reference: Klecki (1896 p. 173). and 4 in
(1) Dissolve 2, 3 1.
Peptone Solution (2) Adjustment of reaction not given.

654. Seitz's Lactose
(3) sterile (2) add varying amounts
To
Constituents: of a sterile solution of arsenic salt.
Sterilization: Sterilize (2) at 120C. Steril-
2. Lactose 20.0 g. thru
ize the arsenic solution by filtering
3. Glucose 0.4 g.
a candle.
4. NaoHP04" 1-0 g.
Use: To show the effect of metallic salts
5. Sodium citrate (tribasic) 2.0 g.
(arsenic) on the growth of brewers' yeast.
.
6. NaCl 5.0 g.
The author reported no growth when
7. Peptone (Witte sice) 0.05 g.
arsenic was present in one part in 1500.
8. Azolitmin (Kahlbaum's) 0.25 g.
Reference: Charpentier (1915 p. 452).
. .
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9
in 1. 657. Lustig's Nitrate Pptone Solution
(2) The reaction should be neutral.
Constituents
(3) The medium is bluish violet in
color.
Water 4000.0 cc.
Sterilization: Do not sterilize longer than 1.
30 minutes at 100 C. 2. Potassium phosphate 1-0 g.
MgS04 (crystals) 0.2 g.

Use : Differential medium for colon-typhoid 3.
CaCl2 (fusum) 0.1 g.
group. Author reported that the colon 4.
Invert sugar 5.0 g.
group produced acid after 5 hours. 5.
Ca(N03)2 5.0 g.
Typhoid bacilli produced a little acid 6.
Peptone 1-0 S-
by dextrose decomposition.
after 24 hours 7.
CaCOs (puriss) 15.0 g.

Dysentery and paratyphoid organisms 8.
Preparation: Ca(N03)2 nitrate reduction was less.

(1) Prepare a salt solution by dissolving When CaCOs was omitted amount of
1.0 g. potassium phosphate, 0.2 g. nitrate reduced with 1.0 g. peptone and
MgS04 (crystals) and 0.1 g. CaCla 6.0 g. Ca(N03)2 was about 10.0%.
(fusum) in 1000.0 cc. water. Variants: Stoklasa gave the following
(2) Prepare an invert sugar solution by variants:
dissolving 5.0 g. cane sugar in (a) Omitted the CaCOj.
1000.0 cc. water and inverting. (b) Used 1.0 g. of peptone, 6.0 g. of
(3) Dissolve 5.0 g. Ca(N03)2 in 500.0 cc. Ca(N03)2, with or without CaCOs.
water. Reference: Stoklasa (1898 p. 285).
(4) Mix 400.0 cc. (1), 240.0 cc. (2) and
659. Stutzer's Nitrate Glucose Solution
240.0 cc. of (3).
(5) Dissolve 1.0 g. peptone and 15.0 g. Constituents
CaCOs (puriss), in (4). 1. Water
(6) Dilute (5) to 4000.0 cc. with water. 2. Glucose
(7) Distribute into well sterilized 100.0 cc. 3. Peptone
flasks in 30-50 cc. lots. 4. KXO3
Sterilization: Sterilize well on each of two Preparation
successive days for an hour. (1) Dissolve 2, 3 and 4 in 1.
Use: To study reduction of nitrates by a (2) Reaction to be slightly alkaline.

red pigment producing organism isolated (3) Distribute in 10.0 cc. lots.
from water, and by soil forms. Sterilization: Method not given.
Reference: Lustig (1890 p. 38), Frankland Use: To study denitrification by B. agilis,
(1889 p. 376). B. nitrovorus, B. Stulzeri, B. Hartlebi.
was the only organisms that
B. Hartlebi
658. Stoklasa's Nitrate Peptone Solution
decomposed the nitrate.
Constituents Reference: Stutzer (1901 p. 84).
1. Water 2000.0 cc.
660. Duchacek's Nitrate Glucose Solution
3. K2S()4 0.2 g. Constituents
4. MgClo 0.05 g. 1. Water 1000.0 cc.
5. Glucose 2.5 g. 2. Glucose 10.0 g.
6. Peptone 0.5 g. 3. NaNOs 4.0 g.
7. CaCO., 7.5 g. 4. Peptone 10.0 g.
Preparation 5. Sodium phosphate 0.25 g.
(1) Dissolve 0.5 g. sodium phosphate, 6. CaS04 0.2 g.
0.2 g. potassium sulphate and 0.05 g. 7. CaCla 0.05 g.
magnesium chloride in 500.0 cc. 8. MgClo 0.05 g.
water. 9. FeP04 0.05 g.
(2) Dissolve 2.5 g. glucose in 500.0 cc. Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7,
water. 8 and 9 in 1.
(3) Mix 200.0 cc. (1) and 120.0 cc. of (2). Sterilization: Not specified.
(4) Dissolve 0.5 g. peptone, 7.5 g. cal- Use: Cultivation of Bacillus typhi ab-
cium carbonate and 5.72 g. calcium dominalis and Bact. coli commune.
nitrate in (3). Reference: Duchacek (1904 p. 162).
(5) Dilute to 2 liters.
661. Khouvine's Cellulose Nitrate Solution
Sterilization: Sterilize in the steamer for Constituents:
3 hours. 1. Water 1000.0 cc.
Use : To study nitrate reduction by Bacillus 2. K2HPO4 5.0 g.
megatherium, {Bacillvs Ellenbachii) or 3. KNO3 2.5 g.
"Alinit." The author reported that the 4. NaCl., 1.0 g.
nitrate was reduced about 8 to 10% after 5. Peptone (pancreatic) 1-0 g.
19 days. Using 1.0 g. of peptone + 6.0 g. 6. Cellulose
Preparation Preparation: Dissolve 2, 3, 4, 5, 6,

(1)
(1) Dissolve 2, 3, 4, 5 in 1. 7 and 8 inThe malic acid is to be

1.
(2) Reaction is neutral. neutralized by the addition of pure soda

(3) Distribute in 5.0 cc. lots in tubes. solution.
(4) Add 1 square centimeter of cellulose Sterilization: Not specified.

in the form of Berzelius filter paper Use: To study ammonia production and
or a little cellulose precipitated after denitrification.
having been dissolved in Schweitzer's Reference: Maassen (1902 p. 52).
reagent to each tube. 664. Wassermann's Nitrate Peptone Solution
Constituents:
110.
Use: Isolation and enrichment of B. cellu-
2. Na2HP04 3.7 g.
lose dissolvens.
3. KH2PO4 1.4 g.
Variants: The author added 2.0 g. glucose.
4. CaCl2 0.4 g.
Reference: Khouvine (1923 p. 713).
5. KCl 3.0 g.
662. Omeliansky's Cellulose Peptone 6. Magnesium citrate 0.1 g.

Solution (Khouvine) 7. Nitrate
8. Peptone (Witte) (1.0%) .... 10.0 g.
Constituents
Preparation:
(1) Dissolve 2, 3, 4, 5, 6 and 8 in 1.
2. K0HPO4 10 g.
(2) Add nitrate to (1). (Amount or kind
3. MgS04 0.5 g.
not specified.)
4. KNOs 3.0 g.
Use: Differentiation of hemorrhagic septi-
6. NaCl trace
cemia bacteria on basis of nitrate reduc-
7. CaCOs trace
tion and indol production.
8. Cellulose
Variants: The author used 1.0% of Asch-
Preparation
mann's, Merck's or Carne, peptone in-
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
stead of Witte's. Also used 1.0% of
(2) Reaction is neutral.
Kuhne's driisenpeptone, albumoses from
(3) Distribute in 5.0 cc. lots in tubes.
Witte's peptone or sodium caseinate
(4) Add 1 square centimeter of cellulose instead of Witte's peptone.
in the form of Berzelius filter paper
Reference: Wassermann (1898 p. 28).
or a little cellulose precipitated after
having been dissolved in Schweitzer's 665. Duchacek's Nitrate Tartaric Acid
reagent to each tube. Solution
Sterilization: Sterilize for 15 minutes at Constituents
110. 1. Water 1000.0 cc.
Use: Isolation and enrichment of B. 2. Tartaric acid 10.0 g
cellulose dissolvens. 3. NaNOs 4.0 g
Variants: Khouvine added 2.0 g. glucose. 4. Peptone 1.0 g
Reference: Khouvine (1923 p. 713). 5. Sodium phosphate 0.25 g
6. CaS04 0.20 g
663. Maassen's Nitrate Peptone Solution CaCl2 0.5
7. g
Constituents 8. MgCl. 0.5 g
1. Water 1000.0 cc. 9. FeP04 0.5 g
2. Sodium phosphate (secon- Preparation
dary) 0.5 g (1) Dissolve 2, 8 and 9 in 1.
3, 4, 5, 6, 7,
3. NaCl 0.5 g (2) Neutralize (1) by the addition of

4. Soda 0.5 g soda solution.
5. MgS04 1-0 g Sterilization: Not specified.
6. Malic acid 7.0 g Use: Cultivation of Bacz7Zus typhi abdomi-

7. Glycerin 20.0 g nalis and Bad. coli commune.
8. Sait peter 2.5 g Reference: Duchacek (1904 p. 162).
666. Hiss' Basal Asparagin Peptone

Solution
Constituents:
2. Peptone 10.0 g.
3. Asparagin 5.0 g.
4. 5.0% litmus solution 10.0 cc.
Preparation
)0.0
(2) Dissolve 10.0 g. of one of the added

nutrients in (1).
Use: To study fermentation of sugars by
the dysentery group.
Added nutrients The author added 10.0
: g.
glucose sucrose
maltose dextrin
mannitol
Reference: Hiss (1904-05 p. 29).
667. Henneberg's Basal Asparagin Peptone

Solution
Constituents:
2. KH2PO4 (0.3%) 3.0 g.
3. MgS04 (0.01%) 0.1 g.
4. Asparagin (0.3%) 3.0 g.
5. Peptone (1.0%) 10.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and
5.0% of one of the added nutrients in 1.

Use: Determine acid production by lactic
acid bacteria using various carbon
sources. Author reported no growth
using starch or dextrin.
of a large variety of carbon sources.
668. Nicole and Refik-Bey's Asparagin

Peptone Solution
Constituents:
1. Water 1000.0 cc.
2. Peptone 20.0 g.
3. NaCI 5.0 g.
4. K2HPO4 2.0 g.
5. Asparagin 2.0 g.
in 1.

Use: Cultivation of cocco bacillus causing
2. Na2HP04 2.5 g 674. Tryptophane Broth (A. P. H. A.)

3. Asparagin 4.0 g Constituents:
4. NaCl 5.0 g 1. Water (Dist.) 1000.0 cc.
5. Peptone (Witte) 2.5 g 2. Tryptophane 0.3 g.
6. Lactose 5.0 g 3. K2HPO4 5.0 g.
7. Azolitmin 0.25 g
4. Peptone 10 g.
Preparation Preparation: Dissolve 3 and 4 in 1.
(1) 2,
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
(2) The reaction is neutral.
Use: Detection of indol production. The
Committee stated that some American
Use: Water analysis. B. coli grew rapidly brands of peptone contained certain uni-
in this medium, producing the maximum form amounts of tryptophane. If such
amount of acid in 4 days. peptone be used it is unnecessary to add
Reference: Quantz (1914 p. 202).
the tryptophane, but use 5.0 g. peptone
per liter.
672. Frankel's Asparagin Peptone Solution References: Committee A. P. H. A. (1917
(Tanner) Rogers,
p. 107), (1920 p. 107), (1923 p. 107),
Constituents: Clark and Lubs (1918 p. 233), Tanner

1. Distilled water 1000.0 cc. (1919 p. 47), Giltner (1921 p. 384), Levine
2. CaH4(P04)2-H20 2.0 g. (1921 p. 109).
675. Frieber's Tryptophane Peptone
4. Asparagin 4.0 g.
Solution
5. NaCl 5.0 g.
Constituents
6. N/1 NaOH 20.0 cc.
Peptone (4.0%) 40.0
1. Water 1000.0 cc.
7. g.
Preparation
2. NaCl (0.5%) 5.0 g.
and 3. Potassium phosphate (0.2%). 2.0 g.

(1) Dissolve 2, 3, 4, 5, 6 7 in 1.
Tube.
4. MgS04 (0.02%) 0.2 g.
(2)
5. Tryptophane (0.03%) 0.3 g.
Sterilization: Sterilize in Arnold.
6. Peptone (Witte) (0.25%) ... 2.5 g.
Use: To sulphur metabolism of
study
fluorescent bacteria, colon typhoid group
in 1.
and others.
Use: Indol production by Bad. pyocy-
aneus, Staphylococcus pyogenes, Bad.
673. Berthelot's Tyrosine Peptone Solution
pneumoniae, Bac. diphtheriae, potato
Constituents bacillus, disciformans and glanders bacil-
1. Water 1000.0 cc. lus. Author reported that the organ-
2. Peptone (Witte) 20.0 g. isms listed above gave positive reactions
3. Glucose 20.0 g. for indol when using the Salkowski
4. Tyrosine 10.0 g. (H2SO4 and nitrites) test. Ehrlich's test
Preparation: (1) Dissolve 2, 3 and 4 in 1. gave negative indol reaction for these
Sterilization: Not specified. organisms.
Use: Production of aromatic hydroxy acids Reference: Frieber (1921-22 p. 264).
and the study of phenol production by
676. Harvey's Tryptophane Peptone Solution
Bacillus phenologenes.
Variants: The author used the following Constituents:
variants to study phenol production: 1. Distilled water 1000.0 cc.
(a) 2.0 g. tyrosine in 1000.0 cc. of peptone 2. Ammonium lactate 5.0 g.
water. 3. Na2S04 2.0 g.
(b) 2.0 g. tyrosine and 10.0 g. glucose in 4. MgS04 0.2 g.
1000.0 cc. peptone water. 5. Peptone 30.0 g.
Reference: Berthelot (1909 p. 87), (1918 6. Tryptophane 1:1000 soln. 0.1, 0.3 or
p. 30). 0.5 cc.

Preparation: (1) Dissolve 2, 3, 4, 5 and 6 (4) Distribute in 50.0 cc. quantities in

in 1. .300.0 cc. flasks.
Sterilization: Not specified. Sterilization: Sterilize intermittently.

Use: Detection of indol production. Use : Urea decomposition by Bac. Pasteuri,
Reference: Harvey (1921-22 p. 102). M. ureae hgue/aciens and other soil forms.
Variants: The author added 1.0% glucose.
677. Hiss' Basal Urea Peptone Solution Reference: Cunningham (1924 p. 145).
Constituents:
680. Ayers and Rupp's Hippurate Peptone
Solution
2. Urea 5.0 g.
3. Peptone 10.0 g. Constituents
4. 5.0% litmus solution 10.0 cc. 1. Distilled water 1000.0 cc.
Preparation: (1) Dissolve 2, 3, 4 and 10.0 g. 2. Peptone (Parke-Davis) 10.0 g.
of one of the added nutrients in 1. 3. K2HPO4 1.5 g.
Sterilization: Not specified. 4. Sodium hippurate 10.0 g.
Use: To study fermentation of sugars by Preparation
the dysentery group. (1) Dissolve 2, 3 and 4 in 1.
Added nutrients: Hiss added 10.0 g. of one (2) Adjust reaction to pH = 7.2.
of the following: Sterilization: Method not given.
glucose sucrose Use: To show hydrolysis of sodium hip-
maltose dextrin purate by streptococci.
mannitol Reference: Ayers and Rupp (1922. p. 391).
Reference: Hiss (1904-05 p. 30).
681. Ayers and Rupp's Pepsin Peptone
678. Killer's Urea Peptone Solution Solution
1. Water 1000.0 cc. 1. Distilled water 1000.0 cc.
2. Peptone 10.0 g. 2. Peptone (Park Davis) 10.0 g.
3. Urea 100.0 g. 3. Pepsin 5.0 g..
Preparation: (1) Dissolve 2 and 3 in 1. CaClo 0.03 g.

Sterilization: Not specified. 5. FeCla (1.0% soln.) 1 drop
Use: Study of decomposition of urea by 6. Sodium hippurate 10.0 g.
soil forms and protozoa; production of Preparation
uric acid and study of nitrogen metab- (1) Dissolve 2, 3, 4, 5 and 6 in 1.
olism. (2) Add NaOH to give pH = 7.1.

Variants Sterilization: Method not given.
(a) Killer diluted the medium 5 to 10 Use: To show hydrolysis of sodium hip-
times for the cultivation of protozoa. purate by
hemolytic streptococci.
(b) Sears used 20.0 g. peptone and Author reported that streptococci from
10.0 g. urea to study uric acid forma- human origin did not show a great in-
tion and nitrogen metabolism. crease in volatile acids with sodium hip-
References: Killer (1913 p. 523), Sears (1916 purate. Udder types showed increase.
p. 132). Variants: The authors omitted the FeClj
and added 1.0 g. NaCl. They adjusted
679. Cunningham's Urea Peptone Solution
the pH from 8.0 to 9.0 and studied the
Constituents: effect of alkaline reactionupon hydrolysis
1. Water 1000.0 cc. of sodium hippurate by hemolytic strepto-
2. Peptone (1.0%) 10.0 g. cocci. They reported that if medium
3. K.2HPO4 (0.05%) 0.5 g. was suitable growth of strepto-
for the
4. Urea (1.0%) 10.0 g. cocci the composition or reaction had no
Preparation effect on the hydrolysis of sodium hip-
(1) Dissolve 2 and 3 in 1 by steaming. purate.
(2) Filter. Reference: Ayers and Rupp (1922 pp. 390,
(3) Dissolve 1.0% urea in 1. 393).
682. MacConkey's Bile Salt Peptone (f) Levine (1921) omitted the litmus,
Solution used 1.0% peptone, used ox bile or a
Constituents: 1.0% solution of dry ox bile instead
Water 1000.0 cc. of sodium taurocholate and used
1.
2. Sodium taurocholate (0.5%) 5.0 g. 1.0% lactose instead of 0.5% glucose.

3. Glucose (0.5%) 5.0 g. (g) Giltner (1921) specified distilled
4. Peptone (2.0%,) 20.0 g. water, used 10.0 g. of lactose instead

5. Litmus of 5.0 g. glucose and added 20.0 cc.
Preparation of 2.0% azolitmin solution instead

of litmus.
(2) Add a sufficient quantity of litmus to
(h) Harvey (1921-22) used 5.0 cc. of a
give a distinct color. 1.0% neutral red solution instead of

litmus and added 50.0 cc. of a 20.0%
(3) Tube, and place a Dunham's fer-
mentation tube in each tube. solution of any sugar instead of
glucose,
Sterilization: Method not given. (i) Harvey (1921-22) specified distilled
Use: Enrichment of colon-typhoid group water, used 4.0% peptone, 1.0%

in water and sewage analysis. The sodium taurocholate and 1.0% glu-
author reported that in this medium B. cose instead of amounts given by
coll and the Gartner group gave acid and MacConkey.
gas in 24 hours. (j) Harvey (1921-22) used 1.0%, peptone,
Variants 2.5% sodium taurocholate, omitted
(a) MacConkey (1905) specified the use the litmus and used 1.0% glycerol
of distilled water, Witte's peptone, instead of 0.5% glucose,
used 5.0 g. glucose or 10.0 g. of any (k) Cunningham (1924) specified the use
other carbohydrate and used 5.0 cc. of tap water, used 1.0% lactose
of a 1.0% neutral red solution in- instead of 0.5% glucose and used
stead of litmus. 1.0% Andrades indicator instead of
(b) MacConkey (1905) specified the use litmus.
of distilled water, Witte's peptone, References: MacConkey (1901 p. 740),
used 5.0 cc. 1.0% neutral red

of a (1905 pp. 334, 337), (1908 p. 325), Abel
solution instead of litmus, added (1912 p. 226), Roddy (1917 p. 42), Tanner
5.0 g. KI and added 10.0 g. mannitol, (1919 p. 46), Percival (1920 p. 307),
5.0 g. glucose or 5.0 g. lactose. Levine (1921 p. 110), Giltner (1921 p. 382),
(c) MacConkey (1908) specified the use Harvey (1921-22 pp. 89, 90, 109), Klimmer
of distilled water, Witte's peptone, (1923 p. 214), Cunningham (1924 p. 103).
used 2.5 cc. 1.0% neutral red
of a
683. Harrison and van der Leek's Bile Salt
solution instead litmus, added
of
Peptone Solution
0.3 g. CaCl2 and added one of the
following: Constituents:
glucose 5.0 g 1. Water 1000.0 cc.
lactose 10.0 g 2. Peptone (Witte)

sucrose 10.0 g (1.0% or 2.0%) 10.0 or 20.0 g.
dulcitol 5.0 g 3. Sodium taurocholate

adonitol 5.0 g (Commercial 0.5%,) . . 5.0 g.
inulin 5.0 g 4. Aesculin (0.1%,) 1-0 g.
tap water be used in the above

If 5. Iron citrate (0.05%) . 0.5 g.
solution omit the CaCl2. Preparation

(d) Roddy (1917) used 2.5 cc. of a 1.0%, (1) Steam 2, 3, 4 and 5 in 1 for 30 minutes.
neutral red solution and added 0.5% (2) Filter.
glucose, dulcitol or adonitol or 1.0% (3) Adjustment of reaction not given.
of any other sugar. (4) Tube.

(e) Levine (1921) used 5.0 g. lactose Sterilization: Sterilize on 3 successive days
instead of 5.0 g. glucose. in the steamer.
Use: Presumptive test for colon group in A2. Bacterial Derivatives Added.
water analysis. Colon bacilli blacken Thjotta's Basal Bacterial Infusion
the medium. Peptone Solution 700
Variants A3. Fungus derivatives added.
(a) Authors used 0.05% aesculin, and Wiegert's Fungus Infusion Peptone
0.3% sodium taurocholate instead of Solution 701
amount given above. A4. Derivatives of other plants added.
(b) Authors used 0.25% iron citrate, and Bi. Worts, malt or other derivatives added.
0.25% sodium taurocholate. Will and Wanderscheck's Sulphur
(c) Giltner used 0.1 g. aesculin instead of Wort Solution 702
1.0 g. aesculin. Gottheil's Mannitol Wort Solution. 703 .
References: Harrison and van der Leek de Kruyff's Malt Extract Peptone
(1908 p. 312), (1909 pp. 549, 616, 622), Solution 704
Giltner (1921 p. 382). Zikes'Wort Peptone Solution 705
Peklo's Wort Peptone Solution 706
B2. Materials other than worts or malts
Liquid media, or basal solutions, con- added.

taining peptone (or other commercial Otabe's Wheat Infusion Peptone
digest) and in addition, one other organic Solution 707
constituent of unknown chemical composi- Ayers and Mudge's Cabbage Infusion
tion, exclusively of plant or soil origin. Peptone Solution 708
Ai. Yeast or yeast derivatives added.
Kaufmann's Jequirity Seed Infusion
Sherman's Basal Yeast Peptone Solu- Peptone Solution 709
tion 684 Zikes' Must Peptone Solution 710
Cannon's Basal Yeast Peptone Solu- Henneberg's Prune Infusion Peptone
tion 685 Solution 711
Ayers and Mudge's Basal Yeast Pep- Miquel's Wood Ash Peptone Solu-
tone Solution 686 tion (Besson) 712
Ayers' Basal Cerevisine Peptone
684. Sherman's Basal Yeast Peptone
Solution 687
Solution
Ayers and Rupp's Yeast Infusion
Peptone Solution 688 Constituents
Ayers and Rupp's Acid Yeast Infu- 1. Water 1000.0 cc.
sion Peptone Solution 689 2. Peptone 20.0 g.
Ayers and Mudge's Yeast Infusion 3. Dried yeast 10.0 g.
Peptone Solution 1 690 4. Na.HPOi 5.0 g.
Ayers and Mudge's Autolized Yeast Preparation: (1) Dissolve 2, 3, and the
4
Peptone Solutions 691 sodium salt of one of the acids given under
Spronck's Yeast Infusion Peptone added nutrients in 1.
Solution 692 Sterilization: Not specified.
Cohen and Clark's Yeast Infusion Use: To study the production of CO2 from
Peptone Solution 693 organic salts by the kefir type of strepto-
Ickert's Yeast Extract Peptone Solu- coccus. Author reported that none of the
tion 694 organic acid salts aided in CO2 pro-
Ickert's Yeast Infusion Peptone Solu- duction.
tion 695 Added nutrients and variants
Fred and Peterson's Yeast Infusion (a) The author added the sodium salt
Peptone Solution 696 of one of the following organic acids:
Ventre's Basal Yeast Ash Peptone formic malic
Solution 697 acetic valeric
Henneberg's Glucose Yeast Infusion propionic oxalic
Solution 698 butyric tartaric
Dietrich's Glycerol Yeast Infusion caproic citric or
Peptone Solution 699 lactic succinic acid
(b) The author omitted the Na2HP04 and Added nutrients: The author added 0.5 cc.
used 10.0 g. of peptone instead of of one of the following fats or oils to each
and added 10.0 g. of lactic acid
20.0 g. 50.0 cc. lot of the basic solution:
form of sodium lactate.
in the Sesame Chia seed
Reference: Sherman (1921 p. 129), (1921 Chaulmoogra Mustard seed
p. 383). Castor Okra seed
Corn Linseed
685. Cannon's Basal Yeast Peptone
Rape seed _ , fOpen kettle
Solution
Lumbang \ Steam refined
Constituents Cocoanut Butterfat
1. Tap water 1000.0 cc.
p frefined Cod liver
2. Peptone (Armours) 10.0 g. \ crude Mineral and 2%
1
3. Yeast (dried autolyzed). ... 10.0 g. Soy bean Vaseline and 2% 1
4. NaCl 5.0 g. Olive Paraffine 1 and 2%

Preparation Reference: Ayers and Mudge (1922 p. 458).
(1) Boil 2,and 4 in 1 for 15 minutes.
3
687. Ayers' Basal Cerevisine Peptone
(2) Flask and autoclave.
Solution
(3) When cold shake well and filter until
clear through paper. Constituents:
(4) Add 1% added nutrients.
of the 1. Distilled water 1000.0 cc.
Sterilization Sterilize in the Arnold steri-
: 2. Cerevisine (a dry yeast
lizer for 20 minutes on each of 3 successive preparation) 10.0 g.
days. 3. Peptone 10.0 g.
Use Cultural study of aciduric bacteria.
: Preparation: Dissolve 2, 3 and 10.0
(1) g.
Added nutrients: The author added 1.0% of one of the added nutrients in 1.
of any carbohydrate. The aciduric bac- Sterilization: Not specified.
teria do not develop on the basic medium, Use: To study hydrogen ion concentra-
but when a suitable carbohydrate is tion in culture of streptococci. Author
added the growth is luxuriant, producing reported two limiting pH zones, pH = 4.6
a pH less than 5. to 4.8 and pH = 5.5 to 6.0.
Reference: Cannon (1924 p. 229). Added nutrients: Ayers added 10.0 g. of
one of the following materials:
686. Ayers and Madge's Basal Yeast
glucose raffinose
Peptone Solution
lactose mannitol
Constituents sucrose inulin
1. Distilled water 1000.0 cc. Reference: Ayers (1916 p. 84).
2. Peptone (Difco) 10.0 g.
688. Ayers and Rupp's Yeast Infusion
3. Yeast (Autolized) 10.0 g.
Peptone Solution
Preparation
(1) Dissolve 2 and 3 in 1. Constituents
(2) Adjust to pH = 7.4. 1. Distilled water 1000.0 cc.
(3) Distribute in 50.0 cc. quantities and 2. Dry fresh yeast 10.0 g.
add 0.5 cc. of one of the added nu- 3. Peptone 10.0 g.
trients to each flask. Preparation
Sterilization: Sterilize at 15 pounds for (1) Prepare an infusion by mi.xing 1.0%
30 minutes. of dry fresh yeast with cold distilled
Use: To study growth promoting sub- water. Allow to stand 10 minutes.
stances found in oils and fats for strepto- Steam in Arnold for 3 minutes and
cocci. Authors reported that sesame oil filter. (If not clear add
filtrate
and chaulmoogra oil inhibited growth, kieselguhr or Merck's dialysed iron
while even solid paraffin stimulated and filter again).
growth. Growth stimulation probably (2) Add 1.0% peptone to yeast extract.
not due to vitamin A, for mineral oils do (3) Adjust to pH = 7.5, heat and filter.
not contain this vitamin. (4) Tube.
Sterilization: Method not given. (5) Add 1.0% Difco peptone.

Use: General culture medium. Authors (6) Adjust to pH = 7.2.
reported good growth of streptococci. (7) Steam for 15 minutes.
Variants (8) Filter and flask.
(a) Fresh yeast may be autolysed for Sterilization: Method not given.
24 hours at 45-50 C. and then dried Use : To show influence of water-soluble B
at low temperatures. Then add pep- vitamin on growth of streptococci. This
tone and adjust to pH = 7.4. medium should not contain water-soluble
(b) Dry yeast may also be autolysed and B from yeast. It gave fairly good growth,
then used. about same as regular yeast extract
Reference: AyersandRupp (1920 p. 90-91). medium.
Reference: Ayers and Mudge (1922 p. 453).
689. Ayers and Rupp's Acid Yeast Infusion
Peptone Solution 691. Ayers and Mudge's Autolyzed Yeast
Peptone Solutions
Constituents
1. Distilled water 1000.0 cc. Constituents
2. Yeast (dry fresh) 10.0 g. 1. Water 1000.0 cc.
3. Peptone 10.0 g. 2. Yeast (autolized) 10.0 g.
Preparation 3. Peptone (Difco) 10.0 g.
(1) Add 10.0 g. dried fresh yeast to Preparation
400.0 cc. distilled HoO and 50 N/1 (1) Add 10.0 g. autolized yeast to
HCl. 500.0 cc. boiling H2O containing
(2) Heat in autoclave at 14 pounds for 0.01% acetic acid.
5 hour. (2) Stir for some time.
(3) After heating add 50.0 cc. N/1 NaOH. (3) Filter and concentrate filtrate to
(4) Cool and filter until clear. 300.0 cc. at about 80C.
(5) Adjust to pH = 7.5. (4) To this filtrate add 95.0% alcohol
(6) Make up extract to 1000.0 cc. with to get concentration of about 52.0%.
distilled water to make a 1.0% (5) Allow to stand over night at about
solution. 5C.
(7) Dissolve the peptone n (6). (6) Filter.
(8) Adjust to pH = 7.5. (7) Dissolve the precipitate in 1000.0 cc.

(9) Heat and filter. distilled water.
(10) Tube. (8) Dissolve 1.0% peptone in (6).
Sterilization: Method not given. (9) Adjust to pH = 7.2.
Use: General culture medium. (10) Filter.
Reference: Ayers and Rupp (1920 p. 95). (11)Distribute as desired.
Method not given.
Sterilization:
690. Ayers and Mudge's Yeast Infusion
Use: To study the effect of vitamins on the
Peptone Solution I
growth of streptococci. The medium
Constituents given above should contain some soluble
1. Water 1000.0 cc. B vitamin. Fair growth was obtained.
2. Yeast 10.0 g. Variants The author prepared the follow-
:
3. Peptone (Difco) 10.0 g. ing yeast infusions and used them in-
Preparation stead of the solution obtained from step
(1) Add 10.0 g. yeast to 200.0 cc. distilled (7) as above in preparing media,
water, allow to stand about 1 hour, (a) This medium should contain a con-
shaking before filtering. siderable quantity of water soluble B
(2) Add 50.0 g. Lloyd's Reagent (Fuller's vitamin.
earth) and HCl to make solution (1) Take the filtrate from step (6) above
0.01normal HCl. and concentrate to 200.0 cc.
(3) Shake every ^ hour for four hours and (2) Add sufficient 95.0% alcohol to give
filter. a 79.0% alcohol solution.
(4) Make up filtrate to 1000.0 cc. (3) Allow to stand over night at 5C.
(4) Filter. 692. Spronck's Yeast Infusion Peptone

(5) Dissolve the precipitate in 1000.0 cc. Solution
distilled water. Constituents:
(6) Use this solution for preparing the 1. Water 5000.0 cc.
medium instead of
(6) above. 2. Yeast 1000.0 g.
(b) This medium should contain little, if 3. NaCl 25.0 g.
any, water soluble B vitamin. 4. Peptone (Witte) 100.0 g.
(1) Take the filtrate from variant (a) Preparation
step (4) and concentrate to 100.0 cc. (1) Boil 1 kilogram of commercial yeast
(2) Add 95.0% alcohol to get a con- in 5 liters of water for 20 minutes,
centration of 90.0% alcohol. stirring constantly.
(3) Allow to stand over night at 5C. (2) Pour into glass cylinders and allow
(4) Filter. to stand for 24 hours.
(5) Evaporate the filtrate to dryness. (3) Decant the clear fluid.
(6) Dissolve in 1000.0 cc. distilled (4) Carefully acidulate the fluid and add
water. 5.0 g. NaCl and 20.0 g. Witte's peptone
(7) Use this solution for preparing the per liter of fluid.
medium instead of (6) in the origi- (5) Neutralize by the addition of soda
nal medium. and then add 7.0 cc. of a normal soda
(c) This medium should contain water solution for each liter of medium.
soluble B vitamin. (6) Heat, and filter on a paper.
(1) Extract 10.0 g. autolized yeast in a (7) Distribute in flasks.
Soxhlet apparatus for 8 hours with Sterilization: Sterilize at 120C. (time not
95.0% alcohol. Fresh alcohol then specified).
added and continued another 8 Use: Production of diphtheria toxin.
hours. Repeat this action for References: Spronck (1898 p. 702), Tanner
40 hours, breaking up the yeast (1919 p. 57), Besson (1920 p. 35), Harvey
clumps in thimble several times. (1921-22 p. 120).
(2) Evaporate alcohol after extraction
at 60-80 C. 693. Cohen and Clark's Yeast Infusion
Add residue to 500.0 cc. distilled Peptone Solution

(3)
water. Constituents
(4) Add (3) to 500.0 cc. of a 2.0% Difco 1. Water 1000.0 cc.
peptone solution. 2. Brewer's yeast 10.0 g.
(5) Adjust, heat, filter and sterilize as 3. Peptone (Difco) 10.0 g.

for the original medium. 4. K2HPO4 5.0 g.
(d) This medium should contain no water 5. Succinic acid 2.0 g.
soluble B vitamin. 6. Glucose 10.0 g.
(1) Dissolve the residue from variant Preparation

(c) step (1) in 500.0 cc. distilled (1) Extract 10.0 g. Brewer's yeast in
water. 1000.0 cc. of water containing Difco
(2) Add (1) to 500.0 cc. of 2.0% Difco peptone 1.0%, K2HPO4 0.5%, suc-
peptone solution and prepare the cinicacid 0.2% and glucose 1.0%
medium as in variant (c) step (4) (method not given).
and (5). (2) Filter.
(e) This medium should contain water (3) Adjust to desired pH with NaOH
soluble B vitamin. or HCl.
(1) Add 10.0 g. autolized yeast to Sterilization: Method not specified.
500.0 cc. of distilled water. Use: To study H-ion influence on growth
(2) Add (2) to 500.0 cc. of a 2.0% Difco of B. bulgaricus and B. coli.
peptone solution prepare the me- Reference: Cohen and Clark (1919 p. 421).
dium as in variant C step (4)
Yeast Extract Peptone Solution
694. Ickert's
and (5).
Reference: Ayers and Mudge (1922 pp. 450, Constituents:
451). 1. Water 1000.0 cc.
2. Yeast extract 10.0 g. Preparation

3. Peptone 10.0 g. (1) Preparation or composition of fresh
Preparation: yeast water extract not given.
(1) Stir 10.0 g. of yeast extract (commer- (2) Dissolve 2, 3 and 4 in 1.
cial product from E. stock) and boil (3) Adjustment of reaction not given.
for one hour in a steamer. (4) Flask in 250.0 cc. lots.
(2) Clarify by repeated filtering thru (5) When inoculating add a few drops of
paper, by addition of serum, or egg brom cresol purple.
white, or by the addition of 5.0 cc. Sterilization: Sterilize at 15pounds pres-
liquid Ferric oxychlorate, and then sure for 30 minutes in the autoclave.
filtering. Use: To determine fermentation of xylose.
(3) Add 1.0% peptone but no NaCI as the Reference: Fred and Peterson (1920 p. 540).
extract contains 50.0% NaCl.
697. Ventre's Basal Yeast Ash Peptone
Solution
Use: Meat infusion substitute for the
preparation of special media, Endoagar, Constituents
lactose litmus agar, etc. Do not heat 1. Water 1000.0 cc.
over 100C. or a brown color and a pre- 2. Glucose 170.0 g.
cipitate will develop. 3. Malt dust extract 2.0
Reference: Ickert (1918 p. 186). 4. Peptone (malto peptone).. 0.5
5. Yeast ashes 0.5 g.
695. Ickert's Yeast Infusion Peptone (NH4)2HP04
6. 1.0 g.
Solution
7. K0CO3 1.85 g.
1. Water 1000.0 cc. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
2. Pressed yeast 30.0 g. (2) Add one of the added nutrients and
3. Peptone 10.0 g. 1.85g. of K.COsto (1).
4. NaCI 5.0 g. Sterilization: Sterilize by heating in the
Preparation: autoclave at 120C., by filtration thru a
(1) Suspend 30.0 g. (40.0 g. when pre- candle or by treating with H..SO4 (250 mg.
paring agar medium) of pressed yeast H2SO4 per liter) (details of method not
in 1 liter of water. given).
(2) Allow to infuse for one hour. Use: Cultivation of yeast.
(3) Boil for 2 hours in the steamer. Added nutrients and variants:
(4) Clarify by the addition of 5.0 cc. of (a) The author added one of the fol-
ferric oxychlorate while hot, and filter lowing:
twice thru a double filter. tartaric acid 4.0 g.
(5) Add 0.5% NaCI and 1.0% peptone. malic acid 5.0 g.
Sterilization: Not specified. citric acid 0.5 g.
Use: Meat substitute for the
infusion (b) Used the basic solution without the
preparation of special media, Endo, lit- addition of other materials.
mus lactose agar, etc. Do not heat over Reference: Ventre (1914 p. 198).
100 C. or a brown color and a precipi-
698. Henneberg's Glucose Yeast Infusion
tate will form.
Solution
Reference: Ickert (1918 p. 186).
Constituents:
696. Fred and Peterson's Yeast Infusion
1. Yeast infusion (1.0%) 1000.0 cc.
Peptone Solution
2. Peptone (1.0%) 10.0 g.
Constituents: 3. Glucose (5.0%) 50.0 g.
1. Fresh yeast water extract. 1000.0 cc. Preparation:
2. K2HPO4 5.0 g. (1) Prepare a 1.0% yeast infusion
3. Difco peptone 5.0 g. (method not given).
4. Xylose 20.0 g. (2) Dissolve 2 and 3 in (1).
5. Brom cresol purple Sterilization: Not specified.
196 CULTURE MEDIA FOR CULTIVATIOX OF MICROORGANISMS
Use: Cultivation of lactic acid bacteria. Added nutrients: The author added 1.0%
Reference: Henneberg (1903 p. 8). of one of the following materials:
lactose mannitol
699. Dietrich's Glycerol Yeast Infusion
raffinose inulin
Peptone Solution maltose salicin
Constituents: glucose sucrose
1. Water 1000.0 cc. Reference: Thjotta (1921 p. 770).
2. Peptone (Witte) (1.0%) .... 10.0 g.
701. Wiegert's Fungus Infusion Peptone
3. Yeast (dry 1.0%) 10.0 g.
Solution
4. Glycerol 3.0% 30.0 g.
(1) Add 1.0% dry yeast to a 1.0% Witte 1. Water 1000.0 cc.
peptone solution. 2. Fungus 250.0 g.
(2) Boil 2 or 3 times in the autoclave. 3. Peptone 10.0 g.
(3) Add powdered egg white or dry serum 4. NaCl 5.0 g.

to clarify. Preparation
(4) Add 3.0% glycerol. (1) Chop 250.0 g. of fungus into small
(5) If the medium is not clear allow to pieces and place in a steamer for
stand for several days and decant. several hours.
Sterilization: Method not given. (2) Evaporate the fluid from the fungus
Use: Cultivation of tubercle bacilli and to a thick dark brown extract.
preparation of tuberculin. (3) Add 2.0% of (2), 1.0% peptone
1.0 or
Reference: Dietrich (1921 p. 406). and 0.5% NaCl to water.

700. Thjotta's Basal Bacterial Infusion Use: The fungus extract is substituted for
Peptone Solution meat extract.
Constituents: Reference: Wiegert (1922-23 p. 110).
1. Peptone water 1000.0 cc.
702. Will and Wanderscheck's Sulphur
2. Bacterial extract 40.0 cc.
Wort Solution
3. Andrade indicator
(1) Exact composition of peptone water 1. Beer Wort 1000.0 cc.
not given. 2. Peptone (Witte) 15.0 g.
(2) Add andrades indicator (amount not 3. Sulphur 3.0 g.
given). Preparation: (1) Add 1.5% Witte's peptone

(3) Dissolve 1.0% of one of the added and 0.3% sulphur to sterile beer wort con-
nutrients in (2). tained in Pasteur flasks.
(4) Distribute in 5.0 cc. lots. Sterilization: Method not given.
(5) Suspend a 24 hour growth of B. pro- Use: Study of hydrogen sulphide produc-
teus on agar plates, in normal saline tion by yeast. Authors reported that the
solution using 1.0 cc. for each plate. addition of peptone to beer wort con-
(6) Collect in a sterile centrifuge tube. taining sulphur did not increase the H2S
(7) Boil 5 minutes, centrifuge and pipette
production.
off the supernatent fluid. (Reaction Reference: Will and Wanderscheck (1906
pH = 7.6.) p. 308).
(8) Add 0.2 cc. of (7) to each tube of (4).

703. Gottheil's Mannitol Wort Solution
Use: To study bacterial nutrition. All Constituents:
sugars supported the growth of Bacillus 1. Water 1000.0 cc.
influenzae. Acid was produced (redness 2. Peptone 5.0 g.
of medium) only with glucose. Filtering 3. Beerwort (dry) 15.0 g.
the extract thru a Berkefeld filter did 4. Mannitol 10.0 g.
not change its value for promoting Preparation: (1) Dissolve 2, 3 and 4 in 1.
growth. Sterilization: Not specified.

CULTURE MEDIA FOR CULTIVATION OF MICRO ORGAXISMS 197
Use: General culture medium for organ- 707. Otabe's Wheat Infusion Peptone
isms found in the soil, on roots and Solution
rhizomes. Constituents:
Reference: Gottheil (1901 p. 432). 1. Distilled water 1600.0 cc.
2. Wheat 1.0 pound
704. de Kruyff's Malt Extract Peptone 3. Diastase 0.5 g.
Solution 4. Peptone 10.0 g.
2. Malt extract (Merck) 7.0 g. (1) Roast wheat (with or without husks)
3. Maltose 10.0 g. in an iron pan until it becomes
4. Glucose 10.0 g. brown.
5. Peptone 0.5 g. (2) Put 1 pound of (1) into 1600.0 cc. of
6. Asparagin 0.5 g. distilled water. Do not wash the
Preparation wheat.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. (3) Boil in a Koch boiler for 30 minutes.
(2) After sterilization add H3PO4 so that (4) Strain thru a clean cloth.
100.0 cc. of the medium will neu- (5) Make up to 1000.0 cc. with distilled
tralize 3.0 cc. of N/1 NaOH. water if the volume is below this
Method not given.
Sterilization: quantity.
Use: Enrichment of yeast Saccharomyces (6) Add 0.5 g. of taka-diastase or ordi-
javanicus. nary diastase and shake the flask
Reference: de Kruyff (1908 p. 617). well.
(7) Keep at 30 to 40C. for 30 minutes.
705. Zikes' Wort Peptone Solution (8) Filter. The filtrate should be quite
transparent with a yellowish color,
Constituents:
nearly the same as meat broth. It
1. Wort 1000.0 cc.
has a sweet smell and of a slightly
2. Glucose (5.0%) 50.0 g.
alkaline reaction.
3. Peptone (1.0%) 10.0 g.
(9) Add 5.0 g. NaCl and 10.0 g. peptone
Preparation: (1) Dissolve 5.0% glucose and
albumin.
1.0% peptone in wort.
(10) Boil and filter.
(11) To prepare wheat agar add 15.0 g.
Use: Cultivation of Apiculatus yeast,
(in winter) and 20.0 g. (in summer) of
Torula alba, Torula Molischiana, MycO'
agar to the filtrate of (10). Prepare
derma cerevisiae, Blastoderma salmoni-
almost as in the same manner as in
color.
the preparation of meat agar (de-
tails not given).
706. Peklo's Wort Peptone Solution
Use: Substitute for meat infusion medium.
Constituents: May be used as a basis for special media.
1. Distilled water 200.0 cc. Reference: Otabe (1919 p. 576).
2. Beer wort 800.0 cc.
3. K2HPO4 708. Ayers and Mudge's Cabbage Infusion
15.0 g.
4. K2CO3 Peptone Solution
2.0 g.
5. MgS04 1.8 g. Constituent^
6. Peptone (Witte 0.5%) 5.0 g. 1. Water 1000.0 cc.
Preparation 2. Peptone (Difco) 20.0 g.
(1) Mix 1 and 2. 3. Cabbage 100.0 g.
(2) Dissolve 3, 4, 5 and 6 in 1. Preparation
Sterilization: Not specified. (1) Soak 100.0 g. finely minced cabbage in
Use: Cultivation of plant actinomyces. 300.0 cc. distilled water in ice box for
Reference: Peklo (1910 p. 551). 48 hours.
(2) Steam 30 minutes and filter. Preparation: (1) Dissolve 5.0% glucose and
(3) Distribute 2.0% Difco peptone solu- 1.0% peptone inmust.
tion into a series of flasks in 50.0 cc. Sterilization: Not specified.
quantities. Use: Cultivation Torula alba,
of yeasts,
(4) Add varying amounts of filtrate of Torula Molischiana, Mycoderma cerevisiae,
(2) 1.0 cc, 5.0 cc. and 50.0 cc. Blastoderma salmonicolor.
(5) Make each flask to 100.0 cc. volume Reference: Zikes (1911 p. 147).
with water.
(6) Adjust to pH = 7.2. 711. Henneberg's Prune Infusion Peptone
Steam and filter. Solution
(7)
(8) Tube. Constituents
Sterilization: Method not given. 1. Prune infusion (1.0%) 1000.0 cc.
Use: To study promoting sub-
growth 2. Peptone (1.0%) 10.0 g.
stances for streptococci. Author re- 3. Glucose (5.0%) 50.0 g.
ported that the more cabbage extract Preparation
added, the better the growth. (1) Method of preparation of 1.0% prune
Reference: Ayers and Mudge (1922 p. 455). infusion not given.
709. Kaufmann's Jequirity Seed Infusion Sterilization: Not specified.
Peptone Solution Use: Cultivation of lactic acid bacteria.
Constituents:
1. Water 1000.0 cc. 712. Miquel's Wood Ash Peptone Solution
2. Jequirity seeds. 100.0 g. (Besson)
4. NaCl 5.0 g. 1. Water 1000.0 cc.
(1) Grind 100.0 g. Jequirity seeds in a 3. Peptone (Chapoteaut) 20.0 g.
mortar. 4. Wood ashes 0.1 g.
(2) The peeled or shelled seeds now weigh Preparation
about 80.0 g. (1) Dissolve 2 and 3 in 1 by heating.
(3) Add to 1000.0 cc. of water. (2) Add 0.1 g. of wood ashes to (1).
(4) Boil in a steam sterilizer for about (3) Boil.
2 hours. (4) Filter thru paper.
(5) Cool and filter.
(5) Add tartaric acid until the solution is
(6) Dissolve peptone in (5). neutral to litmus.
(7) Add 8 drops of concentrated soda (6) Boil 5 minutes.
solution to each 100.0 cc. of (6). (7) Filter.
(8) Distribute into test tubes. (8) Make up to 1000.0 cc.
Sterilization: Sterilize in the usual manner (9) Tube.
(Method not given). Sterilization: Sterilize at 113C.
Use: General culture medium. Bacillus
pyocyaneues and many other organisms Reference: Besson (1920 p. 30).
grew very well.
Variants: The author gave the following SUBGROUP I-C. SECTION 12
variants: Liquid media containing peptone (or

(a)Addition of 5.0 g. NaCl. other commercial digest) and at least one
(b) Addition of 60.0 g. glycerol. other organic nitrogen source of animal
Reference: Kaufmann (1891 p. 68). origin exclusive of extracts or infusions.
Ai.* Animal cells or tissues employed.
710. Zilces' Must Peptone Solution
Bi.* Red blood cells used.
Constituents: Rivers and Kohn's Blood Cell Pep-
1. Must 1000.0 cc. tone Solution 713
2. Glucose (5.0%) 50.0 g.
3. Peptone (1.0%) 10.0 g. 'See As, As, Bj, B3 and B4 next page.
Wordley's Blood Cell Peptone Solu- Kan-Ichiro Morishima's Nutrose

tion 714 Peptone Solution 740
B2. Brain tissue used. Elser and Huntoon's Basal Nutrose
Hall's Brain Peptone Solution 715 Solution 741
B3. Muscular tissue (skeletal) used. Loeffler's Malachite Green Nutrose
Hall and Peterson's Meat Mash Pep- Solution 742
tone Solution 716 Seliber's Basal Casein Peptone Solu-
Kitasato and Weyl's Chopped Beef tion 743
Peptone Medium 717 Albus and Holm's Milk Yeast Me-
B4. Other animal tissues used. dium (Medium Y) 744
Jablon and Pease's Liver Peptone B2. E.xcretions used.
Medium 718 Heller's Urine Peptone Solution.... 745
Deycke's Albumin Peptone Solution. 719 Piorkowski's Urine Peptone Solution. 746
Harvey's Egg Peptone Solution 720 Khouvin and Delaunay's Cellulose
Weissenbach's Albumin Peptone Fecal Infusion Solution 747
Solution 721
Sherman and Albus' Basal Yeast Fat 713. Rivers and Kohn's Blood Cell Peptone
Peptone Solution 722 Solution
Weiss and Wilkes-Weiss' Whole Egg
Constituents:
Solution 723
A2. Animal fluids employed. Peptone (Fairchilds) 20.0 g.
2.
Bi. Whole blood used.
3. NaCl 5.0 g.
Kelser's Blood Infusion Peptone 4. Red blood cells 10.0 cc.
Solution 724
Preparation
Wien's Blood Peptone Solution
(1) Dissolve by boiling 2 and 3 in 1.
(Klimmer) 725
Adjust to pH = 7.4.
(2)
B2. Serum used. Add 10.0 cc. of washed blood cells
(3)
Kligler's Peptone Serum Blood Me- and heat to 95C. (Time not given.)
dium 726
thru paper.
(4) Filter
Hiss' Basal Serum Peptone Solution. 727 After sterilization tube or flask in
(5)
B3. Other serous fluids used.
sterile containers and incubate to test
Emile-Weil's Pleuritic Serum Pep-
sterility.
tone Solution 728 Mandler
Sterilization: Filter thru a filter
Hiss' Basal Ascitic Fluid Peptone
to sterilize.
Solution (Park, Williams and
Use: Cultivation of influenza bacillus caus-
Krumwiede) 729
ing meningitis.
B4. Bile used.
Variants: Added some KNO3, amount not
Abbott's Bile Peptone Solution 730
given.
Stitt's Bile Peptone Solution 731
Reference: Rivers and Kohn (1921 p. 479).
Boudeille's Bile Peptone Solution. . . 732
Jackson and Melia'a Bile Peptone Wordley's Blood Cell Peptone Solution
714.
Solution 733
Olszewski and Kohler's Bile Peptone Constituents:
Solution 734 1. Peptone water 100.0 cc.
Muer and Harris' Brilliant Green 2. Red blood cells (Human). . . . 2.0 cc.
Lactose Peptone Solution 735 Preparation
Conradi's Bile Peptone Solution 736 (1) Exact composition of peptone water
A3. Animal secretions, excretions or their not given.
derivatives employed. (2) Distribute in 5.0 lots.
Bi. Milk or its derivatives used. (3) To each tube of (2) add 0.1 cc. of
Vierling's Whey Peptone Solution. . 737 centrifuged human red cells.
Park, Williams and Krumwiede's (4) Shake well.
Whey Peptone Solution 738 Sterilization: Not specified.
Cunningham's Peptone Whey Solu- Use: To study haemolysis by streptococci.
tion 739 Reference: Wordley (1921 p. 66).
715. Hall's Brain Peptone Solution 3. Peptone 25.0 g.
Constituents
4. NaCl 10.0 g.
Preparation
(1) Boil 2.5 liters of water with 1250 kg.
2. Sheep brain 1000.0 cc.
finely chopped peptone
beef, 25.0 g.
3. Peptone 40.0 g.
and 10.0 g. NaCl streaming steam.
in
4. Dextrose. ^ . . 20.0 g.
Preparation (2) Make slightly alkaline by the addi-
tion of soda.
(1) Boil sheep brains with an equal
Sterilization: Digest in the steamer until
volume of distilled water.
the meat extract is germ free.
(2) Decant water (save) and press brains
thru a potato ricer.
Use: Tetanin production. When the me-
dium has cooled following digestion,
(3) Add 2.0% peptone and 0.1% glucose
inoculate and replace the air in the con-
to the water decanted from (2).
tainer by hydrogen.
(4) Mix (3) and the brain tissue after it
Reference: Kitasato and Weyl (1890 p. 405),
has been passed thru a potato ricer.
(5) Tube by punching thru the filling 718. Jablon and Pease's Liver Peptone
funnel with a glass rod, filling tubes Medium
about half full.
Constituents:
Sterilization : Sterilize intermittently in the
Arnold sterilizer. Five daily runs of
2. Peptone 10.0 g.
30 minutes each are recommended.
Use: To enrich anaerobes. Growth indi-
3. NaCl 5.0 g.
4. Liver (rabbit, beef or human)
cated by turbidity and often gas pro-
Preparation
duction.
(1) Dissolve 2 and 3 in 1 by boiling
Reference: Hall (1920 p. 579).
30 minutes.
716. Hall and Peterson's Meat Mash (2) Neutralize to phenolphthalein.
Peptone Medium (3) Add 20.0 cc. of a normal NaOH solu-
Constituents
tion. The reaction must be a minus 2.
(4) Autoclave for 15 minutes at 115.
1. Water 1000.0 cc.
(5) Filter.
2. Ground meat 1.0 lb.
NaCl 5.0 g. (6) Tube in 10.0 cc. lots.
3.
4. Peptone (Bacto) 20.0 g. (7) Add approximately 1.0 g. of human,

beef or rabbit liver to each tube.
Preparation
(8) Incubate 3 days to test sterility.
Add 2 to (1). (9) The final reaction should be neutral

(2)
or slightly alkaline.
(3) Adjust to pH = 7.2 by the addition
Sterilization: Autoclave for 15 minutes at
of NaOH.
115C.
(4) Tube.
Arnold (stream- Use: Enrichment medium for streptococci
and anaerobes, from war wounds, Bacillus
ing steam) on 3 successive days.
Welchii, Bacillus oedematiens, B. sporo-
Use: Study of toxin production in vitro.
genes, B. putrificus and B. tetani.
Author reported that the addition of 1.0%
References: Jablons and Pease (1918 p.
glucose did not aid toxin production.
1073), Harvey (1921-22 p. 97).
The marble seal was used to secure
better anaerobic conditions.
719. Deycke's Albumin Peptone Solution
Variants The author added 10.0 g. glucose.
:
Reference: Hall and Peterson (1923 p. 327). Constituents

1. Water 1000.0 cc.
717. Kitasato and Weyl's Chopped Beef 2. Alkaline albumin 25.0 g.
Peptone Medium 3. NaCl 10.0 g.
Constituents 4. Peptone 10.0 g.
2. Beef 1250.0 g. (1) Dissolve 2, 3 and 4 in 1.
201
CULTURE. MEDIA FOR CULTIVATION OF MICROORGANISMS
Add 0.5 cc. of a 10.0% soda solution

(2) Neutralize by adding HCl drop by (6)
to each 100.0 cc. of (5).
drop, using litmus paper as an
(7) Autoclave (6) at
115 for 15 minutes.
indicator.
Add 2.0% of a soda solution contain- (8) Filter while hot.
(3)
Mix equal volumes of (4) and (8).
ing 2 parts water to one part soda. (9)
Filter. (10) Tube in 5.0 cc. lots.

(4)
Method not given. Sterilization: Autoclave the tubes at 110C.
Sterilization:
Enrichment medium for cholera for 15 minutes.
Use:
vibrio. Use: Cultivation of streptococci from
Reference: Deycke (1893 p. 245). wounds.
Reference: Weissenbach (1918 p. 491).
720. Harvey's Egg Peptone Solution
722. Sherman and Albus' Basal Yeast Fat
Constituents: Peptone Solution
NaCl 5.0 g. Constituents:
2.
Water 1000.0 cc.
3. Peptone 10.0 to 20.0 g. 1.
2. Peptone (1.0%) 10.0 g.

4_ Egg 100.0 cc.
3. Yeast (dry) (1.0%) 10.0 g.
5. Na2C03 (anhydrous
4. Butterfat (1.0%) 10.0 g.
6.5%soln.) 200.0 cc.
5. Agar (0.1%) 1-0 g-
Preparation
whole eggs with Preparation
(1) Mix the contents of
(1) Dissolve 2, 3, 4, 5 and 1.0%, of one of
an equal volume of water. (100.0 cc.
egg, 100.0 cc. distilled water.) the added nutrients in 1.
(2) Add an equal volume

of 6.5% an- (2) Adjust to pH from 6.5 to 7.0.
hydrous NaaCOs solution to (1). Sterilization: Not specified.
Store sterile (2) as a stock solution. Use: Cultivation of lactobacilli. The au-
(3)
Dissolve 2 and 3 in 1 liter of distilled thors report that the 0.1% agar is not
(4)
water. essential but seems to favor the growth
(5) Mix 1 volume of (2) with 9 volumes of some organisms.

of (4). Added nutrients: The authors added 1.0%
Sterilization: Sterilize (2) and (5) by heat- of any desired fermentable carbohydrate.
ing for one hour at 100C. Variants: Lard and mineral oil were sub-
Use: Enrichment of V. cholera. stituted for butterfat.
Reference: Harvey (1921-22 p. 86). Reference: Sherman and Albus (1922 p. 17).
721. Weissenbach's Albumin Peptone 723. Weiss and Wilkes-Weiss' Egg Solution
Solution Constituents:
1. Water 200.0 cc.
Constituents:
Peptone (Difco) (1.0%) 2.0 g.
1. Distilled water 2.
Sodium phosphate (0.2%) ... 0.4 g.
2. Peptone (Chapoteaut) 4.0 g. 3.
Agar (0.2%) 0.4 g.
3. NaCl 0.5 g. 4.
Eggs 2
4. Glucose 0.2 g. 5.
5. Egg white Preparation

Preparation (1) Remove the contents of two fresh
(1) Dissolve 2 and 3 in 100.0 cc. dis- eggs with sterile precautions.
tilled water. (2) Dissolve 2, 3 and 4 in 1.
(2) Make slightly alkaline to litmus. (3) Emulsify (1) in (2).
(3) Autoclave at 120C. for 15 minutes. (4) Adjust to pH = 7.8 and bring slowly
(4) Filter and add 0.2 g. glucose to the
to a boil, stirring frequently.
filtrate. (5) Tube in spirochete tubes.
Sterilization: Sterilize in the Arnold
for
(5) Add slowly one volume of egg white
sterile
to three volumes distilled water. 30 minutes and seal with 1 cm. of
Shake but do not cause the mixture yellow petrolatum while the tubes are
to foam. still hot.
Use: Cultivation of Spirochaeta pallida. ing the water, clot serum mixture
Reference: Weiss and Wilkes-Weiss (1924 (see (4) above) at 50C. for 20 minutes
p. 222). and then boiling 10 minutes instead of
5, boiled 5 minutes after the addition
724. Kelser's Blood Infusion Peptone
of the glacial acetic acid (see (9)
Solution
above), steamed the peptone NaCl
Constituents mixture (see (11) above) for 45
1. Distilled water 1000.0 cc. minutes instead of heating only until
2. Beef blood 500.0 g. solution was complete and adjusted
3. Peptone 15.0 g. the reaction to a definite pH value,
4. NaCl 7.5 g! faintly alkaline to litmus or 1.0%
Preparation acid to phenolphthalein. After ad-
(1) Allow beef blood to clot in covered justing the reaction the medium was
vessel. Let stand in refrigerator to steamed
for 30 minutes, filtered while
allow serum to separate from clot. hot thru wet thick filter paper and
(2) Remove clot and chop finely in tubed.
chopping machine. References: Kelser (1916 pp. 615, 616)^
(3) Mix serum and ground clot. Weigh. Harvey (1921-22 pp. 69, 76).
(4) Add two volumes of distilled water.
(5) Boil gently for 5 minutes. 725. Wiens' Blood Peptone Solution
(6) Filter thru cheese cloth. Take resi-
(Klimmer)
due and put thru fruit press to ex- Constituents:
tract as much fluid as possible, 1. Water 1000.0 cc.
lining presswith towel or heavy 2. Peptone (10.0%) 100.0 g.
cloth so that pulp does not pass thru. 3. Glucose (1.0%) 10.0 g.
(7) Discard the residue. 4. Blood.. 100.0 cc.
(8) Boil fluid, skimming off coagulated Preparation
proteins. (1) Prepare a 10.0% peptone solution in
(9) Add concentrated CH3COOH (0.5 cc. water.
per L) to cause flocculation and con- (2) Make slightly alkaline.
tinue to boil 5 minutes. (3) Dissolve 1.0% glucose in (2).
(10) Filter first thru absorbent cotton (4) Tube in 10.0 cc. quantities.
and two filter papers. (5) Add 1.0 cc. of blood to each tube.
(11) Ascertain volume and add 0.5% Sterilization: Not specified.
NaCl, and 1.0% peptone. Heat to Use: Cultivation of pneumococci.
get into solution. Reference: Klimmer (1923 p. 221).
(12) Neutralize with NaOH.
Sterilization: Sterilize in autoclave i hour 726. Kligler's Peptone Serum Blood
under 12 pounds pressure. Medium
Use: Nutrient medium for pathogenic Constituents
forms. The author reported that glucose 1. Saline 200.0 cc.
aided the growth of some organisms. 2. Serum (horse or rabbit) 100.0 cc.
Variants 3. Blood
(a) Kelser added 0.25% glucose. 4. Peptone (1.0%) 3.0 g.
(b) Harvey prepared a medium, boiling Preparation:
the water, clot and serum mixture (1) Dilute horse or rabbit serum 1:2 with
(see (5) above) for 10 minutes instead saline.
of 5, specified glacial acetic acid, (2) Adjust to pH 7.0.
steamed the peptone NaCl mixture (3) Add 1.0% peptone (1.0 cc. of a 10.0%
(see (11) above) for 45 minutes, and solution per 10.0 cc. medium).
adjusted the reaction to a definite (4) Distribute in 3 to 4.0 cc. lots in Was-
pH value or faintly alkaline to litmus, sermann tubes.
or 1.0% acid to phenolphthalein. (5) Add 0.1 cc. of blood. (For initial
(c) Harvey prepared a medium by heat- cultures, add infected blood. For
203
Add2.0 cc. of (6) to each tube of

subcultures add fresh normal rabbit (7)
under aseptic conditions,
(4)
blood.)
Cover with a layer of parafHn 1.5 cm. (c) Watabiki added 5.0 g. NaCl, used
(6)
10.0 g. Witte's peptone and prepared
high.
Not specified. the medium as follows:
Sterilization:
Dissolve 10.0 g. Witte's peptone,
Use: Cultivation of Spironema duttoni. (1)
5.0 g. NaCl in a liter of distilled
Incubate for 24 hours at 37C., and then
water.
at room temperature.
p. 215). (2) Boil for 30 minutes and filter.
Reference: Kligler (1922
(3) Add 15.0 g. of one of the
following
727. Hiss' Basal Serum Peptone Solution materials dissolved in a little water
(Buerger et al.) and 20.0 cc. of a 5.0% litmus solu-
Constituents tion to (2).
1000.0 cc. mannitol dulcitol

1. Distilled water
500.0 cc. maltose inulin
2. Serum (Beef)
glucose lactose
3. Peptone 30.0 g.
dextrin levulose
4. Litmus (Kahlbaum's)
sucrose galactose
Preparation:
(4) Distribute in 5.0 cc.
quantities in
(1) Mixa liter of distilled water and
test tubes.
500.0 cc. of beef serum.
minutes. (5) Sterilize for 30 minutes on each of
(2) Flask and steam for 10
Dissolve 30.0 g. of peptone in a little 3 successive days.
(3)
by heating (6) Add 5.0 cc. of fresh horse serum
water (100 to 150.0 cc.)
over a small flame. heated to 55 to 60 for 40 minutes to

and cool. each tube.
(4) Filter (3)
Cool and add to (3). (7) Incubate for 24 hours to test
(5) (2)
(6) Add one of the added nutrients and sterility.
sufficient Kahlbaum's litmus to give (d) Park, Williams and Krumwiede pre-
the desired color. pared the medium as follows:
Sterilization: Sterilize for 15 minutes on (1) Dilute serum with 2 or 3 times its
volume of distilled water.
each of 3 or 4 successive days in streaming
the Arnold.
(2) Sterilize (1) in
steam.
Use: To study fermentation ability of (3) Prepare a 10.0% solution of pep-
pneumococci, gonococci, streptococci, etc. tone.
(4) Sterilize (3), method not given.
(a) Buerger added 1.0% inulin. (5) Prepare a 10.0 or 20.0% solution
Ruediger added 5.0 g. NaCl, used of any desired carbon source or
(b)
20.0 g. Witte's peptone and prepared
fermentable material.
the medium as follows: (6) Heat (5) in small containers in the
Witte's Arnold sterilizer for 30 minutes on
(1) Dissolve 5.0 g. NaCl, 20.0 g.
each of 3 successive days. When
peptone and 2.0% inulin or other
carbohydrate in a liter of water. using inulin, sterilize in the auto-
Add 20.0 cc. litmus. clave.
(2)
Tube in 2.0 cc. lots. (7) Add, under aseptic conditions, suffi-
(3)
1 .0% peptone
cient (4) to (2) to give a
(4) Sterilize in the autoclave if using
inulin; using other carbohydrates
concentration.
sterilize intermittently. (8) Add, under aseptic conditions, suffi-
Collect beef serum without special cient (6) to (7) to give a 1.0% con-
(5)
precautions and dilute with an centration of carbon source. (Gen-
equal volume of water. erally 5.0% glycerol is employed
Pass (5) thru a Berkefeld filter^ instead of 1.0%.) In routine work

(6)
flask in 50.0 to 100.0 cc. quantities with glucose, lactose, sucrose, man-
nitol and dulcitol it is generally
and heat at 65C. for 30 minutes on
sufficient to add the sugar to the
each of 2 successive days.
medium and sterilize in the Arnold Use: Enrichment of typhoid bacilli.

for 30 minutes on each of 3 succes- Reference: Abbott (1921 p. 521).
sive days,
(e) Park, Williams and Krumwiede also 731. Stitt's Bile Peptone Solution
suggested the following method of Constituents
preparation: 1- Bile^ 1000.0 cc.
(1) Dissolve 20.0 g. of peptone and 2. Peptone (1.0%) 10.0 g.
20.0 g. of inulin or other carbo- Preparation
hydrate in a liter of water. (1) Secure ox bile from the abattoir or
(2) Add sufficient litmus to give the human bile from patients undergoing
desired color. bladder drainage in hospitals.
(3) Tube. (2) Dissolve 1.0% peptone in (1).
(4) Sterilize inautoclave when
the (3) Tube in 10.0 cc. quantities.
using inulin, and on each of 3 suc- Sterilization: Method not given.
cessive days when using any other Use: Enrichment of typhoid bacilli.
source of carbon. Reference: Stitt (1923 p. 47).
(5) When add an equal volume of
cool
serum to each tube.
sterile 732. Boudeille's Bile Peptone Solution
References: Buerger (1905 p. 524), Rue- Constituents
diger (1906 p. 756), Watabiki (1909 p. 366), 1. Water 1000.0 cc.
Park, Williams and Krumwiede (1924 2. Peptone 60.0 g.
p. 124). 3. Glucose
4. Bile
728. Emile-Weil's Pleuritic Serum Peptone
Preparation
Solution
Constituents (2) Adjustment of reaction not given.
1. Water 1000.0 cc. (3) Distribute in 5.0 cc. lots in tubes.
2. NaCl 8.0 g. (4) Prepare a 5.0% glucose solution.
3. Peptone 20.0 g. (5) Add 2.0 cc. of (4) to each tube of (3).
4. Glucose 6.0 g. (6) Add various amounts of bile to each
5. Pleuritic serum (human) . . 500.0 cc. tube. (0.0 to 4.0 cc. Add water to
Preparation other tubes to have an equal volume
(1) Dissolve 2, 3 and 4 in 1. in each tube.)
(2) Mix one part of human pleuritic serum Sterilization: Sterilize in the autoclave.
to 2 parts (1). Use To study effect of bile on colon bacilli.
:
Sterilization: Not specified. The presence of bile inhibited the fer-

Use: Cultivation of Bacillus leprae. mentation of glucose by the colon bacilli.
Reference: Emile-Weil (1905 p. 798). Enrichment medium for typhoid bacilli,
differentiation between streptococci, en-
729. Hiss' Basal Ascitic Fluid Peptone
terococcus and diplococcus.
Solution (Park, Williams and
Variants:
Krumwiede)
(a)Tribondeau and Dubreuil and Harvey
Same as medium Variant (e) except
727, autoclaved a mixture of 1.0% glucose
ascitic fluid is substituted for serum. and 1.0% peptone in beef bile at 120
for 20 minutes. The solution was
730. Abbott's Bile Peptone Solution
filtered thru wet filter paper while
Constituents: hot, tubed with fermentation tubes
1. Water 1000.0 cc. and sterilized at 115 for 20 minutes.
2. Bile (ox, dried) 100.0 g. This medium was used as an enrich-
3. Peptone 10.0 g. ment medium for the typhoid bacilli.
Preparation Paratyphoid bacilli produced gas
(1) Dissolve 2 and 3 in 1. while typhoid bacilli did not.
(2) Tube. (b) Weissenbach used the following solu-
Sterilization: Not specified. tion to differentiate between strepto-
enterococci and diplococci. (1922 p. 35), Park, Williams and Krum-

cocci,
wiede (1924 p. 132), Digestive Ferments
Enterococci and diplococci clouded
the medium while streptococci Co. (1925 p. 13).
showed no growth: 734. Olszewski and Kohler's Bile Peptone

1. Water 1000.0 cc.
Solution
2. Peptone 40.0 g.
NaCl 5.0 g. Constituents:
3.
4. Glucose 2.0 g. 1. Water 12,000.0 cc.
250.0 cc. 2. Peptone 75.0 g.

5. Bile (beef)
References: Boudeille (1912 p. 783), Tri- 3. NaCl 50.0 g.
4. Bile 500.0 cc.
bondeau and Dubreuil (1918 p. 131),
Litmus solution 600.0 cc.
Weissenbach (1918 p. 560), Harvey (1921- 5.
6. Lactose 75.0 g.
22 p. 89).
Preparation
733. Jackson and Melia's Bile Peptone (1) Dissolve 75.0 g. peptone and 50.0 g.
Solution NaCl in 500.0 cc. of bile by boiling.
Constituents (2) Filter.
1. Bile (sterile fresh ox) 1000.0 cc. (3) Dissolve 75.0 g. lactose in 600.0 cc.
Peptone 100 g- litmus solution prepared according to
2.
3. Lactose 10.0 g. Kubel and Tiemann with Kahlbaum's
Preparation litmus (method or reference not
given).
Boil (3) for 15 minutes.
(2) Tube in fermentation tubes.
(4)
Sterilization: Method of sterilization of ox (5) Filter and add NaOH or soda solu-
tion to neutralize if necessary.
bile or final medium not given.
Mix sterile (2) and sterile (5) before
Use Enrichment medium in water analysis.
:
(6)
Intestinal forms produced gas. use.
Variants: (7) Tube.

Sterilization: Sterilize (2) and (5) sepa-
(a) Obst specified that the bile should
rately and mix. Sterilize the mixture.
never be used after one week follow-
ing collection. Method of sterilization not given.
(b) 20.0 to 50.0 g. of dried
Bunker used Use Water analysis. Presumptive test
:
for
bile in a liter of water instead of B. coli. Add 10 parts of the water under
1000.0 cc. of fresh bile. investigation to 1 part of the medium.
(c) Ball specified the use of a 1.0% bile

B. coli turned the medium red, other
solution instead of fresh bile. forms being inhibited.
used a mixture of 500.0 cc. of Reference: Olszewski and Kohler (1922
(d) Ritter
water and 500.0 cc. ox bile instead of p. 307).
fresh ox bile. Harris' Brilliant Green

735. Muer and
(e) Tanner and Heinemann specified that
Lactose Peptone Solution
the final medium be sterilized in the
Arnold sterilizer on each of 3 succes- Constituents
sive days.
dehy- Oxgall (dried) 50.0 g.
(f) Bacto Lactose Peptone Bile, 2.
Peptone 10.0 g.
drated, a preparation of 50 parts
is 3.
Lactose 10-0 g.
Bacto ox Bile, 10 parts Bacto pep- 4.
Brilliant green 0.1 g.
tone and 10 parts Bacto lactose. 5.
Dissolve 70.0 g. of this preparation in Preparation

Heat 1 liter of distilled water in a
a liter of distilled water. Sterilize (1)
in the usual manner. double boiler until the water in the

References: Jackson and Melia (1909 outer vessel boils.
p. 194), Obst (1916 p. 76), Bunker (1916 (2) Add 50.0 g. of dried ox gall and 10.0 g.
peptone to stirring until solution
pp. 85-86), Ball (1919 p. 81), Ritter (1919
(1),
complete.
p. 609), Tanner (1919 p. 59), Heinemann
is
(3) Boil for one hour. in 200.0 cc. distilledwater by heat-

(4) Remove from the flame and add 10.0 g. ing on a water bath. (Milk powder
of powdered lactose. is prepared by evaporating skim
(5) Filter thru cotton flannel until clear. milk to 5 its volume in a vacuum,
(6) To each liter of the filtrate add 10.0 cc. powdered, and pass the powder thru
of a 1.0% solution of brilliant green. a hot stream of air to dry.)
(7) Tube. (2) Add 4.0 cc. of an 18.0% CaClo solu-
Sterilization: Sterilize in the autoclave for tion to (1) and heat in the steamer
15 minutes at 15 pounds pressure. for 40 minutes to precipitate the
Use: Presumptive test for B. coli in water casein.
analysis. Author reported that B. coli (3) Add 2.0 cc. N/1 soda solution and
produced gas in this medium. Other heat for 25 more minutes.
forms were inhibited, especially B. welchii (4) Filter thru a folded filter, pressing
and other anaerobes. out the whey by means of cloth.
References: Muer and Harris (1920 p. 875), (5) If not clear filter thru moistened
Levine (1921 p. 112), Park, Williams and filter paper shreds using suction.
Krumwiede (1924 p. 132). The whey will not become bright
and clear unless it is allowed to
736. Conradi's Bile Peptone Solution
stand in the ice box for 3 to 5 days.
Constituents: (6) Add 150.0 g. distilled water and
1. Bile (fresh beef) 900.0 cc. 150.0 cc. physiological salt solution
2. Peptone (Witte) 100.0 g. making the volume to 500.0 cc.
3.Glycerol 100.0 g. (7) Dissolve 0.02 or 0.03 g. Witte pep-
Preparation tone in (6).
(1) Mix 1, 2 and 3. (8) Sterilize for 30 minutes. A tur-
(2) Place the unfiltered sterile (1) in 2.0 budity is formed which settles out
or 3.0 cc. quantities in sterile glass by standing in the ice box over
tubes 9.0 cm. high and 18 mm. in night. Filtering is unsatisfactory.
diameter with rubber stoppers. (9) Add 20.0 to 30.0 cc. sterile Kahl-
Sterilization: Sterilize (1) in streaming baum's litmus tincture to (8) by
steam for 2 hours. Heat (2) at 100C. means of a sterile pipette.
for 30 minutes. (10) Standardize the tint by the addition
Use: Enrichment and isolation of typhoid of N/10 soda solution or lactic acid.
bacilli from patient's blood. About 0.5 The medium will become somewhat
to 2.0 cc. of the patient's blood is added bluer by sterilization so a rather red
to each tube of medium. tinge is to be desired.
Variants: Roddy and Stitt used 2.0% (11) Prepare the tubes in which the whey
peptone. is to be distributed by adding about
References: Conradi (1906 p. 58), (1906 10 mg. CaCOs to each tube and heat-
p. 1655), Roddy (1917 p. 42), Harvey ing for 1 hour, dry heat at 120 to
(1921-22 p. 89), Stitt (1923 p. 47), Klim- 140C. The tubes should be of Jena
mer (1923 p. 214). glass or tubes that have been used
a great deal. The CaCOa tends to
737. Vierling's Whey Peptone Solution
neutralize the acids formed from
Constituents: galactose and glucose which have
1. Distilled water. . . . 350.0 cc. come from hydrolysed lactose. It
2. Skim milk powder. 30.0 g. does not injure the reddening or the
3. Physiological salt whey. The tubes may be used with-
solution 150.0 cc. out the CaCOa.
4. Peptone 0.02 to 0.03 g. (12) Distribute into 5.0 cc. lots into
5. Kahlbaum's litmus sterile test tubes.
solution 20.0 to 30.0 cc. Sterilization: Sterilize for 5 minutes
Preparation (method not given).
(1) Dissolve 30.0 g. of skim milk powder Use: General culture medium.
Reference: Vierling (1922 p. 94), Klimmer Sterilization: Sterilize intermittently in
(1923 p. 209). steam.

Use Bacteriologic examination of milk and
:
738. Park, Williams and Krumwiede's dairy products.

Whey Peptone Solution Reference: Cunningham (1924 p. 101).
Constituents Morishima's Nutrose

740. Kan-Ichiro
1. Whey 1000.0 cc.
Peptone Solution
2. Peptone (2.0%) 20.0 g.
3. NaCl (0.5%).. 5.0 g. Constituents:
Preparation: 1. Water 1000.0 cc.
unpasterized 2. Nutrose 2.5 g.
(1) Allow 3.0 quarts of
milk to stand over night. 3. Peptone 10.0 g.
NaCl.'. 5.0 g.
(2) Remove the cream the next morning. 4.
Heat the milk over a flame and stir Preparation:

(3)
until ready to boil. (1) Dissolve 2 in 1.
(4) Add 6.0 cc. of glacial acetic acid (2) Inoculate with a culture of B. coli.
(0.2%) to coagulate the casein. (3) Incubate for 24 hours at 37C.
(5) Strain thru cheese cloth. (4) Autoclave.

Add 2.0% peptone and 0.5% NaCl. (5) Filter.
(6)
(7) Boil, stirring to dissolve the pep- (6) Dissolve 3 and 4 in the filtrate.
tone and salt. (7) Adjust to pH = 7.0 to 7.1.

Sterilization: Autoclave at 15 pounds for
(8) Adjust the reaction to 0.5% acid to
phenolphthalein. 15 minutes.
(9) Cool to 45C.

Use Cultivation of typhoid bacilli.
:
(10) Add the whites of 3 eggs to clear. Reference: Kan-Ichiro Morishima (1921
(11) Filter thru cotton and paper. p. 277).
Sterilization: Sterilize in the autoclave at

741. Elser and Hun toon's Basal Nutrose
15 pounds for 30 minutes.
Solution
Reference Park, Williams and Krumwiede
:
Constituents:
(1924 p. 134). 1. Distilled water 1000.0 cc.
2. Nutrose 10.0 g.
739. Cunningham's Peptone Whey Solution 3. Peptone (Witte) 10.0 g.
1. Whey 1000.0 cc. 5. Litmus solution
2. Peptone (1.0%) 10.0 g. (Merck's) 5.0 to 7.5 cc.
3. NaCl (0.5%) 5.0 g. Preparation

4. Andrades indicator (1.0%) . 10.0 cc. (1) Dissolve 2, 3 and 4 in 1.
Preparation (2) Add 5.0 to 7.5 cc. of a watery solution

(1) Warm 2 liters of clean, fresh skim of Merck's highly sensitized litmus.
milk in a large pot at 37C. (3) Prepare 100.0 cc. of a 10.0% solution
(2) Add sufficient rennet to curdle. of one of the added nutrients in dis-
Allow to settle for 10 minutes. tilled water.
(3)
(4) Break the curd into large pieces by (4) Mix sterile (2) with sterile (3).
means of a stirring rod. (5) Tube in sterile tubes.

(5) Heat to 80C. to contract the clot (6) Incubate for 3 days to detect acci-
and to express the whey. dental contamination.
Strain thru a cheese cloth. Sterilization: Sterilize (2) in the usual
(6)
manner (method not given). Sterilize
(7) Add 1.0% peptone and 0.5% NaCl.
(8) Steam
for 30 minutes. (3) at 100C. for 10 minutes.
Neutralize to turmeric paper.
(9) Use: To study fermentation by meningo-
(10) Steam for one hour. cocci. Other investigators used similar
(11) Filter thru paper until clear. media for the cultivation of diphtheria
(12) Add 1.0% Andrades indicator. bacilli.

Added nutrients (1) Dissolve the peptone and glucose

(a) The author added 1.0% of one of the (0.0 or 1.0%) in 800.0 cc. of dis-
following: tilled water.
glucose sucrose (2) Add 10.6 cc. of normal KOH.
galactose mannitol (3) Dissolve 10.0 g. nutrose in 200.0 cc.
levulose dulcitol of hot distilled water and add the
lactose inulin lactose.
maltose dextrin (4) Add (3) to (2).
The following solutions have been (5) Flask in 100.0 cc. quantities.
described as Thiel's Nutrose solu- (6) Sterilize on each of 3 successive days
tions and were used to study fer- for 10 minutes in streaming steam.
mentation ability of diphtheria and (7) Add 3.0 cc. of a 2.0% solution of
pseudo diphtheria bacilli. 120 Hochst malachite green to each
(b) Roux and Rochaix used the basic 100.0 cc. lot.
solution with 50.0 cc. of Kahlbaum's (8)Tube in 3.0 or 4.0 cc. quantities,
litmus solution instead of 5.0 to 7.5 cc. (c) Klimmer prepared the medium as in
Merck's without any additions. variant (b) above, but omitted the
(c) V. Przewaski used the same basic lactose (using 1.0% glucose) and only
solution with 5.0 cc. of Kahlbaum's added 1.0 cc. of the 2.0% malachite
litmus solution and added 20.0 cc. green per 100.0 cc. of medium instead
of a 1.0% crystalline soda solution. of 3.0 cc.
To this basic solution he added 1.0% References: Loeffler (1906 pp. 289-295),
of one of the following: Klimmer (1923 p. 213).
glucose inulin
lactose mannitol 743. Seliber's Basal Casein Peptone
levulose dulcitol Solution
mannose Constituents:
References: Elser and Huntoon (1909 1. Distilled water 1000.0 cc.
p. 404), Roux and Rochaix (1911 p. 247), 2. K2HPO4 1.0 g.
V. Przewaski (1912 p. 13), Klimmer (1923 3. MgS04 0.3 g.
p. 222). 4. NaCl 0.1 g.
5. CaCl2 0.1 g.
742. Loeffler's Malachite Green Nutrose
6. Peptone 10.0 g.
Solution
7. Casein 5.0 g.
1. Distilled water 1000.0 cc. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
2. Peptone (2.0%).... 20.0 g. (2) Neutralize (indicator not specified)
3. Nutrose (1.0%).... 10.0 g. and add a slight excess of NaOH.
4. Lactose (5.0%) .... 50.0 g. (3) Add 5.0 g. casein to (2).
5. Malachite green (4) Heat on a salt water bath, shaking
(2.0% solution) .... 30.0 to 100.0 cc. thoroughly to obtain a homogenous
Preparation mixture of casein.
(1) Dissolve 2, 3 and 4 in 1. (5) Filter or decant sterile (4).
(2) Add 10.6 cc. of normal NaOH and (6) Dissolve 1.0% of any desired carbo-
cool to about 50C. hydrate, alcohol, etc. in (5).
(3) Add from 30.0 to 100.0 cc. of a 2.0% (7) Tube.

malachite green solution. Sterilization: Sterilize (4) at 115C. for
Sterilization: Not given. 15 minutes. Sterilize the media after
Use: Enrichment of colon typhoid group. being tubed in the steamer.
Variants Use: Detection of fermentation by coagu-
(a) The author added 1.0% glucose. lation of casein.
(b) Klimmer prepared the medium as Variants: The author added a trace of
follows FeCl3.
Added nutrients: The author added 1.0% (6) Filter.
of any carbohydrate, alcohol, etc. (7) Tube.

References: Seliber (1914 p. 640), Harvey Sterilization: Sterilize in streaming steam.
(1921-22 p. 96). Use: General culture medium. The author
reported growth of a number of molds,
744. Albus and Holm's Milk Yeast Medium saprophytic and parasitic bacteria. Sug-
(Medium Y) gested as a substitute for beef infusion.
Variants
Constituents:
(a) The color and some inhibitory sub-
1. Water 1000.0 cc.
stances may be removed by animal
2. Yeast (1.0%) 10.0 g.
charcoal.
3. Peptone (1.0%) 10.0 g.
(b) Various carbon sources may be added
4. Lactose (1.0%) 10.0 g.
if desired.
5. Milk (Skim) 10.0 cc.
Reference: Heller (1890 p. 893).
6. Brom cresol purple (5.0%
alcoholic solution) 0.5 cc. 746. Piorkowski's Urine Peptone Solution
7. Sodium recinolate
Constituents:
Preparation:
1. Urine 1000.0 cc.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
2. Peptone 5.0 g.
(2) Divide into four lots and to three add
Preparation:
various quantities of sodium recino-
(1) Dissolve 5.0 g. of peptone in 100.0 cc.
late, keeping one lot as a control.
urine and distribute in 100.0 cc. lots.
The surface tension was determined
(2) Steam for 15 minutes in a steamer.
by the weight drop method following
(3) Filter.
sterilization.
(4) Distribute into 10.0 cc. lots.
minutes
Sterilization: Sterilize for 10 to 15
Use: To determine the effect of surface
on each of 2 days using the fractional
tension on growth of Lactobacillus bul-
method.
garicus and Lactobacillus acidophilus.
Use: Differentiation of Bact. coli and
The authors reported that L. bulgaricus
Bacillus typhi abdomin.
was inhibited at a surface tension lower
Reference: Piorkowski (1896 p. 687).
than 40 dynes, while L. acidophilus ex-
hibited good growth in the same medium, 747. Khouvine-Delaunay's Cellulose Fecal
depressed to 36 dynes. Infusion Solution
Variants: The authors used sodium tauro-
Constituents:
cholate and sodium glycocholate instead
1. Water 850.0 cc.
of sodium recinolate to lower the surface
2. Fecal infusion 250.0 cc.
tension.
3. Cellulose (Berzelius paper). 10.0 g.
Reference: Albus and Holm (1926 p. 14).
4. Peptone (pancreatic) 1.0 g.
5. NaCl 1.0 g.
745. Heller's Urine Peptone Solution
6. K2HPO4 10 g.
Constituents: 7. CaCOs 2.0 g.

1. Urine., 1000.0 cc. Preparation
2. Peptone 10.0 g. (1) Thoroughly mix 2 parts fecal ma-
3. NaCl 5.0 g. terial with 8 to 10 parts distilled
Preparation water.
(1) Secure human urine as near average (2) Filter thru a Laurent filter.
specific gravity as possible. (3) Sterilize (2) for 15 minutes at 110C.
(2) Make weakly alkaline with soda. (4) Allow the precipitate from (3) to
End point indicated by precipitation settle and use the clear supernatant
of salts. fluid.
(3) Filter. (5) Dissolve4, 5, 6 and 7 in 850.0 cc. of
(4) Add peptone and NaCl. waterand250.0cc.of (4). Add 10.0 g.
(5) Boil. of cellulose (Berzelius paper).

Sterilization: Final sterilization not given. Ci. Infusions specified.

Use: Cultivation of an anaerobic cellulose Wolf's Basal Casein Digest Infusion
digester from the human intestine. Broth (Kahn) 765
Reference: Khouvine and Delaunay (1922 Torrey and Buckell's Basal Ascitic
p. 922). Fluid Infusion Broth 766
Cj. Extracts specified.
SUBGROUP I-C. SECTION 13 Symmers and Wilson's Basal Ascitic
Fluid Extract Broth 767
Liquid media or basal solutions contain- Holman's Basal Serum Extract Broth. 768
ing peptone (or other commercial digest), Ca. Whether infusions or extracts em-
and an extract or infusion animal origin.
of ployed, not specified.
(Plant derivatives may also be present.) Di. All additional organic material of
Ai.* Employed as basal solutions with the known chemical composition.
addition of other materials. MacConkey's Basal Bile Salt Iodide
Bi. Not containing additional organic Bouillon 769
materials. Klimenko's Basal Glycerol Bouillon. 770
Ci. Infusions specified. D}. Additional organic material of un-
Committee A. P. H. A. (1899) Basal known chemical composition present.
Infusion Broth 748 El. Ascitic fluid used.
Heinemann's Basal Sugar Free Infu- Cantani's Basal Ascitic Fluid Bouil-
sion Broth 749 lon 771
Hopkins and Lang's Basal Veal Infu- Buerger's Basal Ascitic Fluid Bouil-
sion Broth 750 lon 772
Harvey's Basal Carbonate Infusion Akatsu's Basal Ascitic Fluid Bouil-
Broth 751 lon 773
Davis and Ferry's Basal Infusion El. Serum used.
Broth 752 Hiss' Basal Serum Bouillon 774
Cj. Extracts specified. Holman's Basal Serum Bouillon 775
Grimberg's Basal Carbonate Extract Ai. Employed as complete media.
Broth 753 Bi.* Not containing additional organic
Committee A. P. H. A. (1917) Basal materials.
Extract Broth 754 Ci. Prepared dehydrated media.
Percival's Basal Extract Broth 755 Ragit's Dehydrated Broth 776
Rogers, Clark and Evans' Basal Ex- Bacto Dehydrated Broth 777
tract Broth 756 Harvey and Iyengar's Dehydrated
Stern's Basal Fuchsin Sulphite Broth 757 .
Broth 778
Winslow, Rothberg and Parsons' Cj. Media prepared from constituents.
Basal Extract Broth 758 Di.* Infusions specified.
Elser and Huntoon's Basal Nahrstoff Ei.f Beef infusions employed.
Heyden Broth 759 Fi. Containing peptone.
Stutzer's Basal Nitrate Extract Dunham's Infusion Broth 779
Broth 760 Jensen's Nitrate Infusion Broth 780
Bacto (Dehydrated) Basal Extract Davis and Ferry's Nitrate Infusion
Broth 761 Broth 781
Cj. Whether infusion or extract employed Loeffler's Infusion Broth (Roux and
not specified. Rochaix) 782
Thoinot and Masselin's Basal Bouil- Zielleczky's Phenolphthalein Infu-
lon 762 sion Broth 783
Klecki's Basal Bouillon 763 Muller's Indicator Infusion Broth.. 784
Tanner's Basal Carbonate Bouillon. 764
.
F2. Not containing peptone.
Bj. Containing additional organic ma- Robinson and Rettger's Infusion
terials. Opsine Solution 785
* See next page for B2 and D2

See next column for Aj t See next page for Ej to E5.
Ej. Veal infusion employed. E2. Nitrates not added.

Warden, Connell and Holly's Veal Makgill's Indicator Bouillon 815
Infusion Broth 786 Thoinot's Arsenious Acid Bouillon.. 816
Schoenholz and Meyer's Veal Infu- Omeliansky's Dilute Bouillon 817
sion Broth 787 Muller and Malvoz's Iodine Bouillon. 818
Bunker's Veal Infusion Poly Peptide Fremlin's Ammonium Sulphate
Solution 788 Bouillon 819
E3. Infusions of animal organs employed. Stitt's Carbonate Bouillon 820
Krasnow's et al. Sugar Free Veal Dalimier and Lancereau's Opsine
Broth 789 Bouillon 821
Cesaris-Demel's Liver Infusion Broth. 790 Frieber's Trypsinized Bouillon
Owen, Martin & Pitt's Trypsin Beef (Comm. S. A. B.) 822
Tea 791 B2. Containing additional organic material.
Kligler's Heart Infusion Broth 792 Ci. Infusions specified.
Harvey's Heart Infusion Broth 793 Di.* Veal Infusions employed.

Park, Williams and Krumwiede's El. Additional constituents of known chem-
Heart Infusion Broth 794 ical composition.
Wroblewski's Suprarenal Capsule In- Wilcox's Glucose Veal Infusion Broth. 823
fusion Broth 795 Park, Williams and Krumwiede's
Harvey's Organ Infusion Broth 796 Glycerol Veal Infusion Broth 824
Kligler's Blood Clot Infusion Broth. 797 E2. Additional constituents of unknown
E4. Fish Infusion employed. chemical composition present.

Hunter's Fish Infusion Broth 798 Reeser's Potato Veal Infusion Broth. 825
Harvey's Fish Infusion Broth 799 Mellon's Serum Veal Infusion Broth. 826
Albert & Kelman's Blood Veal Infu-
Ee. Infusions other than Ei to E4 employed.
sion Broth 827
Richardson's Mucosa Infusion Broth 800 .
Boyer's Bone Infusion Broth 801 Thjotta & Gunderson's Blood Veal
Infusion Solution 828
Ball's Animal Infusion Broth 802
Robinson and Meader's Liver Veal
D2. Extracts specified.
Infusion Broth 829
El. Containing nitrates.
Starin and Dack's Casein Digest Veal
Stutzer's Nitrate Extract Broth.... 803
Infusion Broth 830
Davis and Ferry's Nitrate Bouillon
Bacto Veal Infusion Medium (De-
Extract Broth 804
hydrated) 831
Ej. Not containing nitrates.
Bruschettinis' Blood Egg Broth
Fi. Sodium chloride added. (Klimmer) 832
Debrand's Extract Broth 805
D2.t Liver infusion employed.
Barman and Rettger's Extract Pro- Jackson and Muer's Glucose Liver
teose Solution 806
Infusion 833
Guth's Selenium Extract Broth 807
834
Haslam's Brain Liver Infusion Broth.
Herman and Rettger's Salt Extract D3. Heart infusion employed.
Broth 808
Mueller's Heart Infusion Aminoid
Herman and Rettger's Trypsinized Solution 835
Extract Broth 809
Mueller's Heart Infusion Peptone
Olszewski and Kohler's Trypsinized Solution 836
Extract Broth 810 Huntoon's Hormone Heart Infusion
Bacto Cooledge Broth (Dehydrated) . 811 Broth 837
Fj. Sodium chloride not added. MacNoughton's Blood Infusion Broth. 838
Heinemann's Bouillon 812 D4. Beef (meat) infusions employed.
Wyant's Bouillon Cube Broth 813 El. All additional constituents of known
D3. Whether infusions or extracts employed composition.
not specified.
El. Nitrates added. * See next page for C2
Heinemann's Nitrate Broth 814 t See D3 to Ds.
Fi. Containing additional organic nitrogen. Fj. Animal fluids used.
Ficker and Hoffmann's Caffeine In- Wade and Manalang's Blood Infusion
fusion Broth 839 Broth 866
Ayers and Rupp's Hippurate Infusion Harvey's Ascitic Fluid Infusion Broth. 867
Broth 840 Stryker's Serum Infusion Broth 868
MacConkey's Bile Salt Infusion Foster's Serum Infusion Broth 869
Broth (Heinemann) 841 Beach and Hasting's Serum Infusion
Fz. Not containing additional organic and Extract Broth 870
nitrogen. Harvey's Blood Infusion Broth 871
F4. Other materials of unknown chemical
Gi. Carbohydrates added.
Smith's Glucose Infusion Broth 841a composition used.
Mueller's Meat Infusion Broth 842 Park, Williams and Krumwiede's
Robinson and Rettger's Glucose In- Potato Infusion Broth 872
fusion Broth 843 Lubinski's Potato Infusion Broth .873. .
Bronstein & Grunblatt's Indicator Jurewitsch's Potato Infusion Broth. 874 .
Glucose Infusion Broth 844 Olitsky and Gates' Bacteria Infusion

Broth 875
Ayers, Rupp and Johnson's Glucose
Infusion Broth 845 Ogata's Porphyra Infusion Broth 876
Ayers, Rupp and Mudge's Glucose Park, Williams & Krumwiede's

Infusion Broth 846 Stomach Digest Infusion Broth... 877
Torrey's Brilliant Green Infusion Hitchen's Yeast Autolysate Blood
847 Solution 878
Broth
Torrey's Acetic Acid Infusion Broth. 848 Hitchen's Semisolid Glucose Agar
(Mulsow) 879
Sherman's Lactose Infusion Broth. 849 .
Harvey's Starch Infusion Broth.... 850 Menten & Manning's Lactose Bile In-
fusion Solution 880
Loeffler's Malachite Green Infusion
Broth : 851 Hitchen's Glucose Agar Infusion
Duval and Lewis' Inulin Bouillon. 852
. . .
Solution (Mulsow) 881
Kreidler's Trypsinized Broth 882
Gi. Carbohydrates not added.
Bulir's Mannitol Infusion Broth 853 Ds. Infusions other than above employed.
Richardson's Ctocinoma Infusion
Harvey's Ferric Tartrate Infusion
Broth 854 Broth 883
Harvey's Lead Acetate Infusion Siebert's Horse Meat Infusion Broth . 884
855 Peklo's Potato Horse Meat Infusion

Broth
Broth 885
Omeliansky's Formate Infusion
856 Hida's Horse Meat Infusion Broth. 886 .
Broth
Jordan's Phenol Infusion Broth.... 857
Khouvine's Fecal Infusion Broth... 887
Cutler's Blood Clot Infusion Broth
E2. Chemical composition of at least one
(Stitt) 888
of the additional constituents not known.
Cj.* Extracts specified.
Fi. Egg used.
Di.* All additional constituents of known
Harvey's Egg Infusion Broth 858
chemical composition.
Olitsky and Kligler's Egg White In-
El. Containing additional organic nitrogen.
fusion Broth 859
Percival's Urea Extract Broth 889
Weiss & Wilkes-Weiss' Egg "Hor-
Stutzer's Dinitro Benzol Extract
mone" Broth 860
Broth 890
F2. Animal tissue used.
Harrison and Vanderleck's Aesculin
Kohman's Brain Infusion Medium . . 861
Extract Broth 891
Kreidler's Glucose Brain Broth 862
Sawin's Glycocholate Extract Broth. 892
Kligler's Heart Infusion Solutions. . 863
Olszewski and Kohler's Trypsinized
Havens & Taylor's Kidney & Blood Bile Salt Extract Broth 893
Infusion Broth 864 Frieber's Tryptophane Extract Broth. 894
Robertson's Cooked Meat Medium
(Torrey) 865 * See next page for C3 and Dj.
E2. Not containing additional organic Baruchello's Caffeine Bouillon 919

nitrogen. Picker's Crystal Violet Caffeine
Fi. Carbohydrates added. Bouillon (Bezangon) 920
Gi. Containing monosaccharides. M tiller's Lactose Ferro-cyanide
Bachmann's Indicator Extract Broth. 895 Bouillon 921
Rogers, Clark and Evans' Glucose E2. Not containing additional organic
Extract Broth 896 nitrogen.
Waksman and Joffe's Glucose Extract Fi. Monosaccharides employed.
Broth 897 Frost's Glucose Bouillon 922
Whipple's Glucose Extract Broth 898 Riva's Glucose Bouillon 923
Johnson's Glucose Phenol Extract MacConkey's Iodide Glucose Bouil-
Broth 899 lon 924
G2. Containing disaccharides. Kitasato's Formate Glucose Bouillon
Weisser's Sucrose Extract Broth. . . . 900 (Tanner) 925
Koegel's Lactose Extract Broth 901 Heymann's Acetic Acid Glucose
Gottheil's Sucrose Extract Broth. . . . 902 Bouillon (Finkelstein) 926
G3. Containing polysaccharides. Savage's Neutral Red Glucose Bouil-
Gordon's Starch Extract Broth 903 lon 927
La Garde's Dextrin Peptone Solu- DeKorti's Oleic Acid Glucose Bouil-
tion 904 lon 928
F2. Carbohydrates not added; alcohols or Plimmer's Tartaric Acid Infusion
acids used. Broth 929
Banning's Glycerol Extract Broth.. 905 F2. Disaccharides employed.
Jacobson's Mannitol Extract Broth. 906 Gage's Lactose Bouillon 930
Harrison and Vanderleck's Citrate Hall and Ellefson's Gentian Violet
Aesculin Extract Broth 907 Lactose Broth 931
D2. One or more of the additional constit- Jouan's Citrate Lactose Bouillon
uents of unknown chemical composition. (Besson) 932
El. Unknown added constituents of plant F3. Polysaccharides employed.
origin present.
Wollman's Starch Bouillon 933
De Gaetano's Potato Extract Broth Holzel's Glycogen Bouillon 934
(Kamen) 908
F4. Carbohydrates not added. Alcohols,
Albus and Holm's Medium X 909
organic acids, etc. employed.
E2. Unknown added constituents of animal
Heinemann's Glycerol Bouillon 935
origin present.
Kendall, Day and Walker's Mannitol
Flint's Serum Extract Broth 910
Bouillon 936
Grace and Highberger's Ascitic Fluid
Giltner's Adonitol Bouillon 937
Extract Broth 911
Vincent's Phenol Bouillon 938
Olszewski and Kohler's Bile Extract
Ds. Chemical composition of at least one
Broth 912
of the additional constituents not defi-
Rosenow's Brain Bacto Broth
nitely known.
(Haden) 913
Hoffman and Fischer's Nutrose Ex- El. Unknown constituents of plant origin
only.
tract Broth (Heinemann) 914
Keim's Honey Extract Broth 915 Thjotta and Avery's Yeast Bouillon . . 939
Ataki & Akimato's Sodium Citrate Thjotta and Avery's Tomato Bouil-
Blood Extract Solution 916 lon 940
Dimitroff's Egg Sea Water Medium. 917 .
Thjotta's Pea or Bean Bouillon 941
C3. Whether extract or infusion employed Thjotta and Avery's Potato Bouil-
not specified. lon 942
Di. All additional constituents of known Thjotta's Bacterial Emulsion Bouil-
chemical composition. lon 943
El. Containing additional organic nitrogen. Hitchens' Basal Sugar-Free Agar
Lohnis' Urea Bouillon 918 Solution 944
Hegner and Becker's Blood Agar Thjotta and Avery's Yeast Ascitic
Solution 945 Fluid Bouillon 972
E2.* Unknown constituents of animal origin Thjotta and Avery's Yeast Serum
only. Bouillon 973
Fi. Animal tissue or cells employed. Thjotta and Avery's Yeast Blood
Gi. Blood cells added. Bouillon 974
Thjotta and Avery's Blood Cell Thjotta and Avery's Yeast Blood Cell
Bouillon 946 Bouillon 975
Gj. Heart, kidney or other tissue added. Thjotta and Avery's Yeast Hemo-
Smith, Brown and Walker's Tissue globin Bouillon 976
Bouillon 947 Savini and Savini-Castano's Bacteria
Orr's Glucose Heart Bouillon 948 Blood Bouillon 977
Park, Williams and Krumwiede's
Meat Medium 949 748. Committee A. P. H. A. (1899) Basal
Kligler's Tissue Infusion Bouillon.. 950 Infusion Broth
G3. Eggs or derivatives added.
Capaldi's Egg Yolk Bouillon 951 Constituents:
Oberstadt's Egg Albumin Bouillon. . 952 1. Distilled water 1000.0 cc.
Egg Albumin Bouillon. 953
Lipschiitz's . 2. Meat 500.0 g.
Kahn's Casein Digest Egg Albumin 3. Peptone (1.0%) 10.0 g.
Bouillon 954 4. NaCl (0.5%) 5.0 g.
Besredka and Jupille's Egg Bouillon Preparation

(Besson) 955 (1) Macerate one part finely chopped
Robertson's Alkaline Egg Bouillon. . 956 lean meat with 2 parts distilled water
Piorkowski's Alkaline Egg Albumin in the ice box for 18 hours, stirring
Bouillon 957 occasionally.
F2.'* Animal fluids employed. (2) Strain, cold, thru a fine cloth.
Gi. Blood added. (3) Add 1.0% peptone and 0.5% NaCl to
Hibler's Blood Bouillon 958 the filtrate. Heat until solution is
Dieudonne's Alkaline Blood Bouillon. 959 complete.
Orcutt and Howe's Fat Blood Bouil- (4) Add NaOH until the reaction is
lon 960 slightly alkaline (practically neutral)

Fildes' Pepsinized Blood Bouillon. . . 961 to phenolphthalein.
G2. Serum added. (5) Heat on a water bath for 30 minutes
Dunham's Glucose Serum Bouillon. . 962 and boil for 5 minutes over a free
Shmamine's Liver Serum Bouillon. . . 963 flame.
Besson's Serum Bouillon 964 (6) Filter while hot thru paper or cotton
G3. Ascitic fluid added. and cloth.
Veillon's Ascitic Fluid Bouillon 965 (7) Add N/1 HCl to the flltrate to obtain
Kahn's Casein Digest Ascitic Fluid the desired reaction (+1.5).
Bouillon 966 (8) Add 1.0% of one of the added nu-
Lyall's Carbonate Ascitic Fluid trients.
Bouillon 967 Sterilization: Sterilize on each of 3 suc-

G4. Bile or other fluids added. cessive days at 100C.
Roddy's Bile Bouillon 968 Use: Used as a culture medium to deter-
F3. Animal secretions or excretions em- mine the fermentation of various sugars,
ployed. alcohols, etc. by bacteria.
Mayer's Mucin Bouillon 969 Added nutrients and variants:
Schloffer's Urine Bouillon 970 (a) The authors added 1.0% of any de-
Kligler's Nasal Secretion Bouillon.. 971 sired sugar.
E3. Containing both plant and animal con- (b) Committee A. P. H. A. (1905) pre-
stituents of unknown chemical compared the medium as follows:

position. (1) Infuse 500.0 g. finely chopped lean
meat with 1000.0 cc. water for
* See E3 and F3. 24 hours in refrigerator.
(2) Make up volume. (5) Throw on a thick cloth and press

(3) Strain thru cotton flannel. the meat free from juice.
(4) Add 10.0 g. Witte's peptone. (6) Filter the juice thru moistened
(5) Dissolve by heating on water paper.
bath. (7) Add 10.0 g. Chapoteaut or De-
(6) Heat over steam bath 30 minutes. fresne peptone, 5.0 g. NaCl and
(7) Restore volume. about 1.0 g. of sodium phosphate.
(8) Adjust reaction to +1 with phenol- (8) Boil stirring constantly until solu-
phthalein. tion is complete.
(9) Boil 2 minutes over free flame. (9) Neutralize or make slightly alka-
(10) Restore volume. line to litmus by the addition of
(11) Filter thru absorbent cotton and soda solution.
cotton flannel until clear. (10) Heat at 115 to 117C. for 5 minutes.
(12) Add 1.0% of any sugar to (11). (11) Filter until clear.
Jja. case of adding glucose the (12) Make up to 1000.0 cc. by the addi-
muscle sugar must be removed by tion of distilled water.
fermentation with B. coli. (13) Add 2.0 to 4.0% of one of the
(13) Sterilize in streaming steam. following: glucose, raffinose, lac-
(c) Percival prepared the medium as tose, galactose, mannitol, dulcitol,
follows: maltose, levulose or glycerol.
(1) Chop one pound of lean fat-free (14) Distribute as desired.
beef with a knife or mincing (15) Sterilize at 110 to 115C. for
machine. 20 minutes.
(2) Place (1) in a porcelain dish or (e) Harvey prepared the meat infusion
glass beaker and add 1 liter of peptone solution (see Dunham's
water. Meat Infusion Peptone Solution,
(3) Allow to soak over night in a cool variant (bb) 779) and added 1.0%
place. glucose, 1.0% lactose, 2.0% starch,
(4) Strain thru muslin, and boil for 1.0% mannitol or 5.0% glycerol.
one hour. Harvey also added 5.0 cc. of a 1.0%
(5) Filter thru filter paper into a large neutral red solution to meat infusion
flask. peptone solution and added 0.5% of
(6) Make the volume up to 1 liter. any desired sugar.
(7) Add 10.0 g. Witte's peptone and (f) Stitt prepared the infusion broth as
5.0 g. NaCl to (6). in variant (hh) 779, or extract broth
Neutralize to phenolphthalein.
(8) variant (o) 689, and added 1.0 to
Steam for 15 minutes.
(9) 2.0% of any desired carbohydrate,
(10) Neutralize again and adjust the alcohol, etc.
reaction to +10. (g) Park, Williams and Krumwiede pre-
(11) Filter into sterile flasks or test pared infusion broth as in variant
tubes. (11) 779, and proceeded as follows:
(12) Add 2.0% of lactose, 2.0% glucose (1) Adjust the reaction of meat ex-
or0.3%NaNO3. tract of the infusion to pH = 7.0
(d) Besson prepared the medium as (slightly alkaline to litmus).
follows: (2) To each liter add a broth culture
(1) Remove all fat and tendons from of B. coli or one of its allies.
beef and chop into small pieces. (3) Incubate for 48 hours.
(2) Allow 500.0 g. of (1) to macerate (4) Sterilize in the autoclave.
with 1000.0 cc. of cold water for (5) Test again for the production of
6 hours, or if one wishes to remove acid or gas before adding carbo-
the sugar 12 hours at 37C. hydrates.
(3) Place in an enamelled pot and (6) Sterilize (5). (Method not given.)
bring slowly to a boil. (7) Prepare 10.0 or 20.0% solutions
(4) Boil for ten minutes. of the carbohydrates, alcohols, etc.
(8) Heat (7) in small containers in the Sterilization: Method of sterilization not
Arnold sterilizer for 30 minutes on given. Sterilize in large Erlenmeyer
3 successive days. (Sterilize the flasks before filtering.
inulin solution in the autoclave.) Use: To study the fermentation of sugars,
(9) Add sufficient of (8) to sterile (6) alcohols, etc.

to give a 1.0% concentration of Added nutrients and variants:
sugar (under aseptic conditions), (a) The author added 1.0% of one of the
generally 5.0% glycerin is used following to the basic solution.
instead of 1.0%. glucose inulin
(10) In routine work with glucose, lac- lactose mannitol
tose, sucrose, mannitol and dul- sucrose salicin
citol it is generally sufficient to raffinose
add the sugar to the medium and (b) Besemer did not specify the prepara-
sterilize in the30 Arnold for tion or composition of meat infusion
minutes on each of 3 successive bouillon, and adjusted the reaction
days. to -hO.3 Fuller's scale. He added
They also used an extract broth, see 1.0% of one of the following:
variant (Q) 779. fructose amylose
References: Committee A. P. H. A. (1899 glucose dextrin
p. 77), (1905 p. 107), Percival (1920 p. 56), galactose inulin
Besson (1920 p. 31), Harvey (1921-22 mannose glycerol
pp. 87, 112), Stitt (1923 p. 34), Park, sorbose erythritol
Williams and Krumwiede (1924 p. 123). arabinose adonitol
xylose dulcitol
749. Heinemann's Basal Sugar Free rhamnose mannitol
Infusion Broth lactose sorbitol
Constituents sucrose amygdalin
1. Water 1000.0 cc. raffinose maltose
2. Chopped beef 1.0 lb. (c) Kan Ichiro Morishima, prepared
3. Peptone (Witte) 10.0 g. media containing indicators as fol-
4. NaCl 5.0 g. lows:

5. CaCl2 6.0 g. (1) Prepare meat infusion broth
Preparation (method not given).
(1) Chop beef fine and soak with water (2) Inoculate with B. coli, incubate
over night. for 24 hours, autoclave and filter.
(2) Add a culture of B. coli and incubate (3) Add 1.0% peptone and 0.5% NaCl.
the mixture at 37 for 24 hours. (4) Sterilize at 15 pounds for 15
(3) Then heat to 60 C. for two hours. minutes.

(4) Raise to boiling point and boil (5) To broth add sterile sugar solu-
30 minutes. tion in concentration of 1.0% sugar
(5) Press out meat, and the remaining solution prepared by dissolving
meat mixed again with water and sugar in sterile water and heating
pressed a second time. in autoclave for 10 minutes at
(6) Mix both fluids and bring to 10 pounds.
1000.0 cc. volume. (6) Add 5.0 cc. sterile litmus or 5.0 cc.
(7) Dissolve 3 and 4 in boiling (6) of 2.0% phenol red and 1.2 cc. of
(8) Add 5 and boil until the reaction is decolorized 1.0% aqueous solution
about 0.3 acid to phenolphthalein. of china blue per 100.0 cc. of broth.
(Requires from 30 to 60 minutes.) Adjust to pH = 7.0-7.1 by means
(7)
(9) Filter sterile (8) while boiling hot, of phenol red.

under aseptic conditions. (d) Giltner prepared a neutral red me-
(10) Dissolve one of the added nutrients dium as follows:
in (9). (1)Soak one pound of lean finely
(11) Distribute small sterile Erlen-

in chopped beef in 1000.0 cc. of dis-
meyer flasks or sterile Nessler tubes. tilled water over night (24 hours).
(2) Strain out the meat juice and 750. Hopkins and Lang's Basal Veal
make up to 1000.0 cc. with dis- Infusion Broth
tilled water.
Constituents:
(3) Add a 24 hour broth culture of 1. Veal Infusion 1000.0 cc.
B. coli to (2).
(4) Incubate at 37C. for 12 to 16 XaCl
3. 5.0 g.

hours no longer. Preparation
(5) Mix 1.0% (10.0 g.) peptone into a
(1) Preparation of veal infusion not
thin paste with a little water and
specified.
add to (4). Dissolve 2 and 3 in (1),
(2)
(6) Heat in the autoclave for 20
Adjust the reaction to -f-0.3.
(3)
minutes or in the steamer for
(4) Dissolve 1.0% of one of the added
one hour.
nutrients in (3).
(7) Neutralize to phenolphthalein.
(8) Boil over a free flame for 3 to
Use: To study fermentation.
5 minutes.
Variants: The author used 2.0% Witte's
(9) Add 1.0% glucose (or any other
peptone instead of 1.0%.
desired sugar) and 10.0 cc. of a
0.5% solution of neutral red and of one of the following:
stir until the sugar is dissolved.
glucose mannitol
(10) Filter until clear.
lactose inulin
(11) Distribute into fermentation
sucrose soluble starch
tubes.
salicin arabinose
(12) Sterilize in the autoclave or in
raffinose
flowing steam.
Reference: Hopkins and Lang (1914 p. 72).
(e) Baker prepared a Brom thymol blue
medium as follows: 751. Harvey's Basal Carbonate Infusion
(1) Digest meat and water for two Broth
hours. Cook. Filter thru ab-
Constituents:
sorbent cotton and sterilize in
1. Water 1000.0 cc.
autoclave at 18 pounds for 20
2. Beef 500.0 g.
minutes.
3. Peptone 10.0 g.
(2) Inoculate cold broth with Bad.
4. NaCl 5.0 g.
saccharolyte (Rivas), incubate at
5. CaCOa 5.0 to 20.0 g.
37C. for 48 hours.
Preparation
(3) Sterilize in Arnold for 20 minutes.
(1) See Dunham's Meat Infusion Pep-
(4) Add peptone and NaCl. tone Solution variant (bb) 750 for
(5) Adjust to pH = 7.0 with brom- preparation of broth from water,
thymol blue. peptone and beef.
(6) Steam for 20 minutes and readjust (2) Dissolve 1.0% of one of the added
reaction. nutrients in (1).
(7) Filter medium. (3) Add from 5.0 g. to 20.0 g. CaCOj to
(8) Add 12.0 cc. per liter medium of (2).
0.2% alcoholic solution of brom- Sterilization: Method not given.
thymol blue. Tube. Use: To study fermentation.
(9) Sterilize at 18 pounds for 20 Added nutrients: The author added 10%.
minutes. of any desired carbohydrate, alcohol,
(10) Add sterile sugar solution under etc.
aseptic conditions. Reference: Harvey (1921-22 p. 107).
References: Heinemann (1915 p. 221),

752. Davis and Ferry's Basal Infusion Broth
Besemer (1917 p. 179), Kan-Ichiro Mori-
shima (1921 p. 277), Baker (1922 pp. 301, Constituents:
302), Giltner (1921 p. 380). 1. Water 500.0 cc.
2. Peptone 100 g. Added nutrients: The author added 3.0%

3. Beef infusion 500.0 cc. ofany desired fermentable sugar.
Preparation Reference: Grimbert (1895 p. 840).
(1) Composition of beef infusion not
Author also used a 1.0% 754. Committee A. P. H. A. (1917) Basal
given.
Liebig's beef extract Extract Broth
solution of
instead of infusion. Constituents:
(2) Prepare 500.0 cc. of a 2.0% peptone 1. Distilled water 1000.0 cc.
solution. 2. Beef extract 3.0 g.
(3) Mix (1) and (2). 3. Peptone 5.0 g.
(4) Dissolve one of the combinationg Preparation:

given under added nutrients in (3). (1) Add 3.0 g. of beef extract and 5.0 g.
(5) Steam 15 minutes and check the of peptone to a liter of distilled

reaction. water.
(6) Distribute as desired. (2) Heat slowly on a steam bath to at
Sterilization: Sterilize at 115 for 20 least 65C.
minutes. (3) Neutralize to phenolphthalein.
Use: Cultivation of Bad. diphtheriae and (4) Cool to 25C. and filter thru paper
toxin production. until clear.
Added nutrients: The authors added one (5) Add 1.0% of one of the added nu-
of the following materials or combina- trients.
tions of materials. Sterilization: Sterilize in the autoclave at
(a) xanthine 0.05 g, 15 pounds (120C.) for 15 minutes after
hypoxanthine 0.05 g, the pressure reaches 15 pounds.
(b) glucose amine hydrochloride. 2.0 g Use: To study fermentation of carbohy-
(c) sodium asparaginate 1.5 g drates, alcohols, etc., by bacteria.
(d) creatin 0.2 g Added nutrients and variants:
creatinin 0.15 g (a) The committee added 1.0% of any
(e) cystine 0.5 g desired carbohydrate.
(f) glutaminic acid hydro- (b) Mudge specified the use of Liebig's
chloride 2.5 g meat and used 1.0% Witte's
extract,
(g) glycocoll 0.75 g peptone. He neutralized to phenol-

(h) histidine dichloride 0.5 g phthalein, added 1.0% of any desired
(i) leucine 30.0 g, carbohydrate, tubed and sterilized
(j) tryptophane for 15, 30, 60 or 120 minutes in the au-
(k) tyrosine toclave, or 15 minutes in the Arnold
Reference: Davis and Ferry (1919 pp. 235, on each of 3 successive days.
236). (c) Committee S. A. B. used the same
constituents as Committee A. P.H. A.
753. Grimbert's Basal Carbonate Extract They specified that the medium might
Broth
be clarified by the addition of egg
Constituents: and adjusted to pH = 6.6 to 7.4.
1. Water 1000.0 cc. One per cent of any desired carbo-
2. Peptone 3.0 g. hydrate was added.
3. Meat extract (Liebig's) .... 2.0 g. (d) Tanner and Ball added 1.0% of any
4. CaC03 10.0 g. desired carbohydrate, alcohol, etc.,

Preparation: to variant (b) 695.
(1) Dissolve 2 and 3 in 1. (e) Committee A. P. H. A. (1920) ad-
(2) Dissolve 3.0% of one of the added justed to a faint pink using phenol
nutrients in (1). red as an indicator or neutralize to
(3) AddtheCaCOs to (2). phenolphthalein if the reaction does
Sterilization: Method not given. not come between neutral and +1.0.
Use: To study fermentation. Author used The committee used 0.5% carbo-
Friedlander's pneumobacillus. hydrate instead of 1.0% as in 1917.
Sterilize at 15 pounds for 15 minutes Use: To study fermentation of carbo-

after the pressure reaches 15 pounds, hydrates, alcohols, etc.
provided the total time of exposure Added nutrients and variants:
to heat is not more than 30 minutes. (a) The author added 1.0% of one of the
Otherwise sterilize a 10.0% watery following:
solution of any desired carbohydrate lactose dulcitol
at 100C. for 1.5 hours, and add to sucrose adonitol
sterilenutrient broth in sufficient glucose
quantity to give 0.5% carbohydrate. (b) Harvey prepared the medium as
Tube and sterilize this mixture at follows:
100C. for 30 minutes. It is per- (1) Add the whites of 2 eggs to 1000.0 cc.
missible to add sufficient sterile water.
carbohydrate solution to each tube (2) Add to the mixture by degrees to
of sterile neutral broth by means of make a suspension: Lemco 3 to
a sterile pipette. Incubate these 5.0 g., peptone 10.0 g., sodium
tubes for 24 hours to test sterility. chloride 5.0 g.
(f) Park, Williams and Krumwiede (1924) (3) Steam or boil 45 minutes.
added 2.0% litmus solution (reagent (4) Filter while hot thru well-wetted,
of highest purity) or 1.0% Kahl- thick filter paper, or thru 2 layers
baum's azolitmin to Committee of absorbent cotton wool.
A. P. H. A. (1920) broth. They ad- (5) Bring the volume up to 1000.0 cc.
justed the reaction to neutral to by the addition of water.

phenolphthalein (pH about 8.0) and (6) Estimate and adjust to a definite
sterilized for 15 minutes at 15 pounds pH value or faintly alkaline to
pressure after the addition of 0.5% litmus or 1.0% acid to phenol-
of any desired carbohydrate. The phthalein.
tubes were cooled rapidly. (7) Add 1.0% of any desired carbo-
(g) Committee A. P. H. A. (1925). Same hydrate, alcohol, etc.
medium as Committee A. P. H. A. (8) Sterilize in the autoclave or flowing
(1920) except that the reaction is steam.
adjusted from pH 6.2 to pH 7.0. References: Percival (1920 p. 308), Harvey
References: Mudge (1917 p. 406), Com- (1921-22 pp. 68, 108).
mittee A. P. H. A. (1917 p. 85), Com-
756. Rogers, Clark and Evans' Basal
mittee S. A. B. (1918 p. 116), Ball (1919
Extract Broth
p. 77),Tanner (1919 p. 44), Committee
A. P. H. A. (1920 p. 95), Levine (1921 Constituents:
p. 110),Park, Williams and Krumwiede 1. Water 1000.0 cc.
(1924 p. 131), Committee A. P. H. A. 2. Peptone 10.0 g.
(1925 p. 97). 3. Beef extract 4.0 g.

4. Na2HP04 5.0 g.
755. Percival's Basal Extract Broth Preparation: (1) Dissolve 2, 3, 4 and 1.0%
Constituents: of one of the added nutrients in 1.
2. Meat extract (Lemco) 10.0 g. Use: To determine fermentation of carbo-
3. Peptone 10.0 g. hydrates, alcohols, etc. by bacteria.
4. Sodium bicarbonate 1-0 g. Added nutrients and variants:
5. Litmus (Kubel-Tiemann) (a) The authors added 1.0% of one of the
Preparation following materials to the ba.sic
(1) Dissolve 2, 3, 4 and one of the added solution:

nutrients in 1. glucose inulin
(2) Add sufficient boiled Kubel-Tie- sucrose mannitol

mann's litmus solution to give a violet lactose glycerol
tinge. raffinose adonitol
Sterilization: Method not given. starch dulcitol
(b) Rogers, Clark and Davis used 5.0 g. (5) Prepare a fresh 10.0% watery sodium
K2HPO4 instead of 5.0 g. Na2HP04. sulfite solution.
They added 1.0% of one of the (6) Prepare a 0.025% watery chrysoidine
following materials: solution.
glucose rafhnose (7) To each 100.0 cc. of sterile (3) add
levulose starch 5 to 6 drops alcoholic fuchsin, 2.0 cc.
galactose inulin sodium sulfite solution, 0.5 to 1.0 cc.
adonitol mannitol chrysoidine solution and one of the
sucrose glycerol added nutrients.
lactose salicin (8) Distribute into tubes.
dulcitol Sterilization: Sterilize (3); method not
(c) Evans used 5.0 g. K2HPO4 instead given. Sterilize the tubed medium;
of 5.0 g. Na2HP04 and added 1.0% method not given.
of any desired carbohydrate, alco- Use: Differentiation of the colon-typhoid
hol, etc. group.
(d) Sherman and Albus used 3.0 g. beef Added nutrients: The author added 1.0%
extract instead of 4.0 g. and 5.0 g. of one of the following:
K2HPO4 instead of 5.0 g. Na2HP04. glucose xylose
They added 1.0% of one of the levulose arabinose
following test materials: galactose mannitol
glucose inulin maltose glycerol
galactose starch sucrose
levulose glycerol Reference: Stern (1916 p. 483).
maltose mannitol
lactose salicin
758. Winslow, Rothberg and Parsons Basal
sucrose dextrin Extract Broth
raffinose Constituents
They omitted the K2HPO4 when glu- 1. Water 1000.0 cc.
cose, galactose, levulose and maltose 2. Beef extract (Bacto) 3.0 g..
were added. 3. Peptone (Bacto) 5.0 g.
References: Rogers, Clark and Evans 4. Brom cresol purple (0.04%
(1914 p. Ill), Rogers, Clark and Davis alcoholic solution 27.0 cc.
(1914 p. 421), Evans (1916 p. 445), Sher- 5. Carbohydrate 5-0 g.
man and Albus (1918 p. 162). Preparation
757. Stern's Basal Fuchsin Sulphite Broth
(2) Add 27.0 cc. of a 0.04% alcoholic solu-
Constituents tion of brom cresol purple.
1. Water 1000.0 cc. (3) pH = 6.7 to 6.8.
2. Peptone 20.0 g. (4) Add 0.5% of one of the nutrients to
3. Meat extract 10.0 g. sterile (3).
4. NaCl 5.0 g. Sterilization: Sterilize at 15 pounds for 15
5. Fuchsin (10.0% alcoholic solution) minutes.
6. Na2S04 (10.0% water solution) Use: To study fermentation of carbohy-
7. Chrysoidine (0.25% solution) drates, alcohols, etc., by bacteria.
Preparation Author used streptococci.
(1) Dissolve 2, 3 and 4 in 1. Added nutrients: The author added 0.5%
(2) Adjust the reaction so that it is basic ofany desired carbohydrate, alcohol, etc.
to phenolphthalein. Reference: Winslow, Rothberg and Par-
(3) Boil and filter. sons (1923 p. 151).
(4) Prepare a saturated alcoholic fuchsin
solution. Add about 10.0 g. fuchsin
759. Elser and Huntoon's Basal NShrstofE
to 100.0 g. alcohol and allow to stand Heyden Broth
in the thermostat for 24 hours. Filter Constituents
and place in dropping glass. 1. Distilled water 1000.0 cc.
2. Peptone (Witte) 10.0 g. 2. Beef extract (Bacto) 3.0 g.

3. Niihrstoff Heyden 10.0 g. 3. Peptone (Bacto) 5.0 g.
5. Litmus solution (1) These basic media are prepared in
(Merck) 5.0 to 7.0 cc. dehydrated form and contain 5 parts
Preparation (0.5%) of one of the added nutrients.
(1) Dissolve 2, 3 and 4 in 1. Dissolve 13.0 g. of the dehydrated
(2) Add 5.0 to 7.5 cc. of a watery solution medium in 1000.0 cc. of distilled
of Merck's highly sensitized litmus. water.
(3) Prepare 100.0 cc. of a 10.0% solution (2) pH = 6.6 + if sterilized for 20 minutes
of one of the added nutrients in dis- at 15 pounds pressure.
tilled water. Sterilization Sterilize in the usual manner.
:
(4) Mix sterile (2) and sterile (3). Use: General culture medium and with the
(5) Tube. addition of carbohydrates to study fer-
(6) Incubate for 3 days to detect acci- mentation. This medium conforms to
dental contamination. the "Standard Methods 1920" formula.
Sterilization: Sterilize (2) in the usual Added nutrients:
manner (method not given). Sterilize (a) Digestive Ferments Co. prepare de-
(3) at 100C. for 10 minutes. hydrated glucose and lactose broth.
Use: To study fermentation of carbo- (b) Digestive Ferments Co. prepare au
hydrates, alcohols, etc. by bacteria. The Andrade Basic Medium to which they
author used meningococci. add 0.5% sucrose, lactose, mannitol,
Added nutrients: The authors added 1.0% maltose and glucose. This dehy-
of one of the following: drated medium is the same as the one
glucose maltose given above except that 0.025 g.
sucrose mannitol (0.0025%) of Difco Andrade Indicator
galactose dulcitol is added.
levulose inulin Reference: Digestive Ferments Co. (1925

lactose dextrin pp. 10, 15, 16).
Reference: Elser and Huntoon (1909
p. 404).
762. Thoinot and Masselin's Basal Bouillon
Constituents:
760. Stutzer's Basal Nitrate Extract Broth
1. Bouillon 1000.0 cc.
Constituents Preparation: (1) Add one of the nutrients
1. Water 1000.0 cc. to a liter of sterile peptone bouillon.
2. Meat extract 5.0 g. Sterilization: Method not given.
3. Peptone 5.0 g. Use: To study fermentation of carbohy-
4. K2HPO4 1.0 g. drates, alcohols, etc. by bacteria.
5. KNOj 2.5 to 3.0 g. Added nutrients and variants:
Preparation: (1) Dissolve 2, 3, 4, 5 and one (a) The authors added from 3.0 to 4.0%
of the added nutrients in 1. of glucose, lactose or glycerol to the
Sterilization: Method not given. basic solution.
Use: To study denitrification. Author re- (b) Roux and Rochaix added 1.0 to 2.0%
ported that when glucose is used as a glucose, 2.0% lactose or 1.0 to 10.0%
carbon source, no decomposition of KNO3 glycerol to peptone bouillon. They
takes place. If the salt of an organic acid then added 10.0 cc. of a 5.0% neutral
(lactic acid) is used as a carbon source the red solution or sufficient litmus to
KNO3 is decomposed. give a color.
Added nutrients: The author added 10.0 g. (c) Douglas, Fleming and Colebrook
of glucose, or sodium lactate. added a piece of one of the following
Reference: Stutzer (1901 p. 82). to bouillon.
potato charcoal
761. Bacto (Dehydrated) Basal ExtractBroth
bran cork
Constituents: carrot sand
1. Water 1000.0 cc. cabbage card
grape blotting paper Preparation

asbestos wool khaki cloth (1) Dissolve one of the added nutrients
cotton wool rusty nail in peptone bouillon.
lint capillary tube (2) Add 2.0% of CaCOa to (1).
sponge Sterilization: Method not given.
They studied the c ultivation of wound Use: To study fermentation of carbohy-
anaerobes, B. perfringens Bacillus drates, alcohols, etc., by bacteria.
malignant oedema. The authors re- Variants: Dopter and Sacquepee added a
ported that the tube containing the small piece of CaCOs to each tube.
blotting paper showed no growth and Added nutrients:
that growth took place first in the (a) The author added 1 .0% of any desired
interstices of the porous material. carbohydrate, alcohol, etc., to the
Anaerobic conditions were probably basic solution.
produced in these places. (b) Dopter and Sacquepee added 2.0%
(d) Roddy added 0.5 to 2.0% of any of any desired carbohydrate, alcohol,
desired carbohydrate, alcohol, etc., etc., to the basic solution.
to bouillon. (Added 6.0% glycerol.) References: Tanner (1919 p. 47), Dopter
(e) Dopter and Sacquepê added 2.0% and Sacquepee (1921 p. 119).
of any desired carbohydrate, alcohol,
etc., to a liter of sugar free peptone
765. Wolf's Basal Casein Digest Infusion
bouillon. Broth (Kahn)
References: Thoinot and Masselin (1902 Constituents:
p. 24), Roux and Rochaix (1911 p. 109), 1. Water 1000.0 cc.
Douglas, Fleming and Colebrook (1917 2. Beef infusion
p. 530), Roddy (1917 p. 41), Dopter and 3. Casein 200.0 g.
Sacquepee (1921 p. 118). Preparation:
(1) Add 20.0 g. anhydrous NajCOs to
763. Klecki's Basal Bouillon 1 tap water in a 2 liter fiask.
liter of
(2) Boil, and place on a water bath.

Constituents:
(3) Add 200.0 g. casein by dusting it in
gradually, shaking from time to time
2. Peptone 10.0 g.
to avoid lumps.
3. NaCl 5.0 g.
(4) Allow the mixture to cool and add
Preparation
3.0 g. pancreatin and 15.0 cc. chloro-
(1) Preparation or composition of bouil-
form.
lon not given.
(5) Incubate for 5 days at 38C. shaking
vigorously each day to break up any
nutrients in (1).
crusts that form.
(3) Distribute in Pasteur fermentation
(6) Add 3.0 g. more of pancreatin and
tubes.
incubate for 10 days longer.
(7) At the end of the 15 days, add
Use: To study fermentation by Bacillus
400.0 cc. N/1 HCl.
saccharobutyricus. Author reported
(8) Steam for 30 minutes and filter.
that butyric acid odor was present. Fer-
(9) Add 120.0 cc. N/1 NaOH and adjust
mentation lasts much longer in the lac-
the reaction to pH = 7.2.
tose bouillon than in the lactate medium.
(10) Prepare beef infusion broth in the
Fermentation starts after 13 to 16 hours.
usual manner (details not given).
(11) Adjust (10) to pH = 7.0.
of lactose or 100.0 g. of calcium lactate.
(12) Add 2.0% of (9) to (11).
Reference: Klecki (1896 p. 254).
(13) Seed with a strain of B. coli which
actively ferments dextrose.
764. Tanner's Basal Carbonate Bouillon Incubate for 24 hours.
(14)
Constituents: (15) Heat in the sterilizer for 45 minutes
(1) Bouillon 1000.0 cc. and adjust to pH 7.2 by adding
(2) CaC03 (2.0%) 20.0 g. N/1 NaOH.
(16) Heat in the Arnold for 15 minutes (7) Prepare 12.0% solutions of one of the
and filter. added nutrients.
(17) Tube in 9.0 cc. quantities. (8) Add 0.5 cc. of sterile (7) to each tube
(18) Prepare 10.0% solutions of one of of (6).
the added nutrients in the sugar free (9) Incubate 3 days at 37C. to determine
broth (16). sterility.
(19) Add 1.0 cc. of sterile (18) to each Sterilization: Sterilize (5) in the autoclave.
tube of (17).
sterile Sterilize (8) by heating in flowing steam
(20) Cap the tubes with a vaseline cap. at 100C. for 12 minutes.
(21) Heat in the Arnold for 10 minutes. Use: To study fermentation of carbohy-
Sterilization: Sterilize (17) in the auto- drates and alcohols by bacteria. Author
clave. Sterilize (18) by heating in the used gonococci.
Arnold at 100C. for 10 minutes. Added nutrients The authors added 12.0
: g.
Use: To study the fermentation of carbo- of one of the following materials:

hydrates, alcohols, etc., by bacteria. glucose maltose
Kahn used the spore bearing anaerobes. galactose levulose
Inoculation was made thru the liquid Reference: Torrey and Buckell (1922
vaseline by means of a Pasteur pipette, p. 142).
and immediately immersing the tubes in

cold water.
767. Symmers and Wilson's Basal Ascitic
Fluid Extract Broth
of one of the following: Constituents:
glucose levulose 1. Water 1000.0 cc.
galactose maltose 2. Meat extract (Lemco) 1-0 g.
sucrose lactose 3. NaHCOa 1.0 g.
raffinose mannose 4. Peptone 1-0 g.
xylose arabinose 5. Litmus (10.0% soln.) 100.0 cc.
starch inulin 6. Ascitic fluid 100.0 cc.
dextrin salicin Preparation
mannitol melezitose (1) Dissolve 2, 3, 4 and 5 in 1.
glycerol inositol (2) Dissolve 1.0% of one of the added

Reference: Kahn (1922 p. 166). nutrients to sterile (1).
(3) Add 10.0 cc. of sterile ascitic fluid to
766. Torrey and Buckell's Basal Ascitic each 100.0 cc. of sterile (2).
Fluid Infusion Broth Tube in sterile tubes.
(4)
Constituents: (5) Incubate at 37C. to test sterility.
1. Beef infusion 1000.0 cc. Sterilization: Sterilize (1) in the autoclave.

2. Peptone 10.0 g. Sterilize (2) by steaming on each of 3
3. NaCl 5.0 g. successive days for 10 minutes. When

4. Ascitic fluid 200.0 cc. using levulose, arabinose and xylose heat
Preparation over the free flame instead of the steamer.
(1) Method of preparation or exact com- Use: To study fermentation of carbohy-
position of beef infusion not given. drates, alcohols, etc., by bacteria. Au-
(2) Inoculate (1) with B. coli and incu- thors used meningococci.
bate 24 hours. Filter. Added nutrients and variants:
(3) To filtrate add 2 and 3 and dissolve. (a) The authors added 1.0% of any de-
(4) Adjust reaction to pH = 7.0. sired carbohydrate, alcohol, etc., to
(5) Tube in 5.0 cc. lots. the basic solution.

(6) To each tube of sterile (5) add 1.0 cc. (b) Rosenow and Towne obtained initial
sterile ascitic fluid. The fluid stored growth of the causative agent of
in the ice box for a long period to poliomyelitis, pleomorphic strepto-
hydrolyze the fermentable materials cocci, using a medium prepared as
that it might contain. follows:
(1) Prepare an extract broth using Added nutrients and variants:

and peptone.
Liebig's extract (a) The author added 4.0 g. of any de-
(2) Titrate and adjust reaction of (1) sired carbohydrate, alcohol, etc.
to 0.6-0.8 acid to phenolphthalein. (b) The author prepared a similar me-
(3) Add 2.0% glucose, if desired, and dium in the following manner:
10.0% ascitic fluid. (1) Collect beef blood from abattoir
(4) Distribute in six ounce nursing in sterile quart jars.
bottles containing 150.0 cc. of fluid (2) Allow blood to clot in the cool
in a column 4.5 cc. wide and 8.75 cc. room of the abattoir for about 15
high. minutes.
Fresh sterile pieces of rabbit kidney
(5) (3) Take to laboratory and allow to
may be added, if desired. stand over night in a cool place.
(6) Method of sterilization not given. (4) Obtain clear serum by centrifu-
References: Symmers and Wilson (1909 gation.
p. 9), Rosenow and Towne (1917 p. 176). (5) Add 100.0 cc. of serum to 300.0 cc.
distilled water.
768. Holman's Basal Serum Extract Broth (6) Sterilize for 15 to 20 minutes in
Constituents flowing steam on 3 successive days.
1. Distilled water 500.0 cc. (7) Dissolve 40.0 g. Witte's peptone,
2. Beef serum 100.0 cc. 12.0 g. Liebig's meat extract and
3. Peptone (Witte) 4.0 g. 20.0 g. NaCl in 1000.0 cc. distilled
4. Meat extract (Liebig's) 1.2 g. water.

5. NaCl 2.0 g. (8) Titrate while hot to neutral to
6. Andrade's Indicator 4.0 cc. phenolphthalein.
Preparation :
(9) Add 4.0 g. of any desired carbo-
hydrate and 4.0 cc. of Andrade's
(1) Collect beef blood from abattoir in
sterile quart jars. indicator per 100.0 cc.
(2) Allow blood to clot in a cool room of

(10) Sterilize in flowing steam for 15 to
the abattoir for about 15 minutes. 20 minutes on 3 successive days.
(3) Take to laboratory and allow to

(11)The remaining method of prepara-
stand over night in a cool place. is somewhat ambiguous.
tion
(4) Obtain clear serum by centrifuga-

"The sterile serum water was then
tion. mi.xed with this quadruple strength
Add 100.0 cc. of serum to 300.0 cc. carbohydrate broth, and the medium,
(5)
distilled water. consisting of 1 part serum to 4 parts
(6) Dissolve 3, 4 and 5 in 200.0 cc. of dis- 1%

carbohydrate broth, was tubed
tilled water. into sterile tubes by means of a
Adjust to +1.2% acid. sterile tubing-funnel, or by use of a
(7)
Add 4.0 cc. of Andrade's indicator sterile syphon."
(8)
and 4.0 g. of one of the nutrients Reference: Holman (1914 pp. 210, 211).
listed below.
769. MacConkey's Basal Bile Salt Iodide
(9) To cool sterile (8) add 500.0 cc. of
Bouillon
sterile (5).
(10) Tube by means of a sterile tubing Constituents:
funnel or by means of a sterile 1. Nutrient bouillon 100.0 g.
syphon. 2. Sodium taurocholate (Com-
(11) Incubate several days to insure mercial) 0.5 g.
sterility. 3. KI 0.5 g.
Sterilization : Sterilize (5) by filtering thru Preparation
a Berkefeld filter. Sterilize (8) in flowing (1) Preparation of nutrient bouillon not
steam 15 to 20 minutes on each of 3 suc- given.
cessive days. (2) Dissolve 2. 3 and one of the added
Use: To study fermentation of carbohy- nutrients in (1).
drates, alcohols, etc. by bacteria. The (3) Tube in fermentation tubes.

author used streptococci. Sterilization: Sterilize in a steamer on 2
successive days for 10 minutes, taking Use: Cultivation of highly parasitic and
care not to overheat. pathogenic bacteria.
Use: Enrichment of lactose fermenters. Added nutrients and variants:
The author reported that the addition of (a) The author mixed glycerol with equal
KI may tend to delay the anaerobic parts of urine, pus, milk, egg white or
growth while not interfere with aerobic other albuminous fluids.
multiplication. (b) The author used a mixture of equal
Added nutrients: The author added 0.5 g. parts of ascitic fluid and glycerol
glucose, 1.0 g. sucrose or 0.5 g. lactose. instead of ascitic fluid in step (5)
Reference: MacConkey (1905 p. 338). above.
Reference: Cantani (1910 p. 472).
770. Klimenko's Basal Glycerol Bouillon
772. Buerger's Basal Ascitic Fluid Bouillon
Constituents:
1. Peptone bouillon 1000.0 cc. Constituents:
2. Glycerol (1 .0%) 10.0 g. 1. Bouillon sugar free 1000.0 cc.
Preparation 2. Ascitic fluid 333.0 cc.
(1) Preparation of peptone bouillon not 3. Litmus
given. Preparation:
(2) Add (1.0%) glycerol to (1). (1) Preparation of sugar free bouillon
(3) Mix equal parts of (2) and one of the not given.
added nutrients. (2) Add litmus solution to (1) until a
Sterilization: Not specified. desirable color is obtained.
Use: Cultivation of whooping cough bacil- (3) Add I volume of sterile ascitic fluid to
lus. Author reported that the organism sterile (2).
grows first on the surface and then on the (4) Add sterilewatery concentrated solu-
bottom of the tube. tions of one of the added nutrients to
Added nutrients: The author mixed equal (3) until a 1.0% strength is obtained.
parts of one of the following and the (5) Tube into sterile tubes.
basic solution: (6) Incubate in incubator to test sterility.
ascitic fluid Sterilization: Method of sterilization of (2)
blood serum not given. Sterilize the solution of
defibrinated blood added nutrients by heating on each of 3
Reference: Klimenko (1909 p. 312). successive days.
Use: To determine the fermentation of
771. Cantani's Basal Ascitic Fluid Bouillon
carbohydrates, alcohols, etc., by bac-
Constituents: teria. Buerger used streptococci.
1. Bouillon Added nutrients and variants:
2. Ascitic fiuid (a) The author added 1.0% of one of the
3. Glycerol following materials:
Preparation arabinose dulcitol
(1) Mix equal parts of glycerol and one of rhamnose sucrose
the added nutrients. Storing the glucose lactose
material with the glycerol tends to levulose maltos
sterilize the material. galactose dextrin
(2) After some time test the sterility mannitol inulin
of (1). (b) Elser and Huntoon prepared a sim-
(3) Exact composition of bouillon not ilar medium using one part ascitic
given. fluid with two parts bouillon. The
(4) Tube (3). procedure was as follows:
(5) Add 6 parts of sterile ascitic fluid to (1) Mix two parts sterile broth (exact
1 part (2). method of preparation or composi-
(6) Add 0.5 to 0.75 cc. of (5) to each tion not given) with one part sterile
sterile tube of (4). ascitic fluid. Method of steriliza-
Sterilization: Method not specified. tion not specified.
(2) Add 3.0 to 4.5 cc. of a watery solu- 774. Hiss' Basal Serum Bouillon
tion of Merck's highly sensitized Constituents:
litmus.
1. Bouillon (sugar free) 1000.0 cc.
(3) Prepare a 10.0% solution of one of 2. Serum (beef) 500.0 cc.
the following in distilled water: Preparation
glucose, galactose, levulose, lac-
(1) Method of preparation of sugar free
tose, maltose, sucrose, mannitol,
broth not given.
dulcitol, inulin and dextrin. Adjust (1) to 1.0% acid to phenol-
(2)
(4) Sterilize (3) at 100C. for 10
phthalein (neutral to litmus).
minutes. Mix 1 part sterile beef serum with 2
(3)
(5) Mix (2) and (4). parts sterile (2).
(6) Tube in sterile tubes. Dissolve one of the added nutrients
(4)
Incubate for 3 days to detect acci-
(7) in (3).
dental contamination. Sterilization: Sterilize at 65-68C. for one
Fermentation by the meningococci hour on 6 consecutive days.
was studied in these media. Use: Differentiation of streptococci and
References: Buerger (1907 p. 430), Elser pneumococci. Pneumococci fermented
and Huntoon (1909 p. 404).
starch. Streptococci did not. Strepto-
cocci and pneumococci fermented lac-
773. Akatsu's Basal Ascitic Fluid Bouillon saccharose and dextrin, produced
tose,
Constituents acid and formed a yellowish white
1. Bouillon 1000.0 cc. coagulum.
2. Ascitic fluid 1000.0 cc. Added nutrients and variants:
3. Tissue (a) The author added 1.0% sucrose, 1.0%
Preparation lactose, 1.0% glucose, 1.0% dextrin
(1) Composition or method of prepara- or 0.66% starch.
tion of bouillon not given. (b) Koch and Pokschischewsky prepared
(2) Mix equal parts of bouillon and similar media for the differentiation
ascitic fluid and tube in 10.0 cc. lots. of Streptococcus longus seu. erysipe-
(3) Add to each tube of (2) a piece of latos and Streptococcus equi. They
fresh tissue. reported that Streptococcus equi gave
(4) Add 1.0% of one of the added nu- a flocculent flaky sediment leaving a
trients to each tube. clear bouillon. Streptococcus longus
Sterilization: Not specified. seu erysipelatos gave a uniform tur-
Use : To study the fermentation of carbohy- bidity to the medium. (Medium
drates, alcohols, etc., by bacteria. containing no sugars). The media
Akatsu used spirochetes, Treponema pal- were prepared as follows:
lidum, Treponema calligyrum, Trepo- (1) Mix one part sterile horse serum
nema microdentium, Treponema mucosum and two parts ordinary bouillon.
and Spirochaeta refringens. Medium was (2) Sterilize on 3 successive days at
covered with a layer of sterile paraffin 60C. for one hour.
after inoculation and incubated at 36C. (3) Prepare a 10.0% solution of one of
Added nutrients: The author added 1.0% the following:
of one of the following materials: sorbitol maltose
amygdalin galactose dulcitol lactose
arabinose glycogen mannitol glucose
beerwort glucose levulose mannose
dextrin inulin galactose raffinose
mannitol lactose sucrose
raffinose levulose (4) Sterilize on 3 successive days for
sucrose maltose 2 or 3 minutes in streaming steam.
starch (5) Add 1.0 cc. of (4) to 9.0 cc. of (2),
Reference: Akatsu (1917 p. 376). under aseptic conditions.
The medium was also employed with- Use: This solution is equivalent to a 1.0%
out the addition of carbohydrates, peptone broth.
alcohols, etc. Variants: Lohnis prepared the medium as
(c) Klimmer added 0.5 to 1.0% of any follows
desired carbohydrate, alcohol, etc., (1) Heat 22.0 g. of Ragit bouillon in a
and mixed one part
to bouillon, of liter of tap water in the steam steri-
serum with one to twenty parts of an hour.
lizer for
the sugar bouillon. (2) Filter thru filter paper.
References: Hiss (1901-05 p. 323), Koch (3) Add concentrated soda solution until
and Pokschischewsky (1913 p. 10), Klim- red litmus paper is turned slightly
mer (1923 p. 200). blue.
(4) Heat for 30 to 45 minutes in the steam
775. Holman's Basal Serum Bouillon sterilizer.
(5) Filter.
Constituents:
(6) Sterilization not specified.
1. Water 400.0 cc.
References: Marx (1910 p. 361), Lohnis
2. Bouillon, double strength. . 400.0 cc.
(1913 p. 14).
3. Andrade's Indicator 80.0 cc.
4. Beef serum 200.0 cc. 777. Bacto Dehydrated Broth
Preparation
Constituents:
(1) Preparation of double strength bouil-
lon not given.
2. Bacto bouillon (0.8%) 8.0 g.
(2) Adjust (1) to +1.2.
Preparation
(3) Add 200.0 cc. of water to 400.0 cc. of
Dissolve (Prepared
(1) 2 in 1. accord-
(1).
ing to directions given on the bottle).
(2) Initial pH = 7.1. (If sterilized at
nutrients in 80.0 cc. of Andrade's
15 pounds pressure for 20 minutes
indicator and add to (3).
pH = 6.5+.)
manner.
Sterilization: Sterilize in the usual
beef serum.
(6) Mix 400.0 cc. of sterile (5) and 600.0 cc.
References: Brown (1921 p. 562), Digestive
sterile (3).
Ferments Co. (1925 p. 10).
(7) Tube into sterile test tubes.
(8) Incubate 2 days to determine sterility. 778. Harvey and Iyengar's Dehydrated
Sterilization: Sterilize (4) by heating in Broth
flowing steam on each of 3 successive days.
Constituents: 1. Trypsinized mutton in-
Sterilize (5) by filtering thru a Berke-
fusion broth.
feld filter.
Preparation
Use: To study fermentation of carbo-
(1) Preparation of tryptic digest of
hydrates, alcohols, by bacteria.
etc.,
mutton infusion broth not given.
The author used streptococci. The pro- Adjust topH = 8.0.
(2) (1)
duction of acid coagulates the serum.
(3) Cut up agar fibre into small pieces.
Added nutrients and variants: The author
(4) Add (3) to (2) (6.0% by weight).
added 8.0 g. of any desired carbohydrate, Autoclave at 120 for an hour to
(5)
alcohol, etc. to the basic solution.
melt the agar.
References: Holman (1916 p. 385), Stitt
(6) Filter thru cotton, wool and muslin
(1923 p. 36). into a tin receptacle.
(7) Cut the agar out of the receptacle
776. Ragit's Dehydrated Broth
and into slices.
Constituents: (8) Pass slices thru meat mincing ma-
1. Water 1000.0 cc. chine with a finely perforated outlet
2. Ragit Bouillon (Marck) .... 22.0 g. disc.
Preparation: (1) Dissolve 2 in 1. (9) Spread minced nutrient agar on
Sterilization: Not specified. metal or other type of trays.
(10) Dry in hot air oven or any other 5.0 g. NaCl and the whites of two
convenient way. eggs, beaten up in 2 or 3 volumes
(11) Store powder in sterile glass stop- of water.
pered bottle. (6) Boil under a gas flame for 15
(12) Add 6.0% by weight (11) to cold minutes.
water. (7) Adjust to faint alkalinity, using
(13) Extract (12) at room temperature phenolphthalein as an indicator,
for 2 hours. and the end point being a faint
(14) Filter. red color.
Sterilization: Method of sterilization of the (8) Pour into an iron kettle, add
not given.
filtrate 100.0 cc. distilled water, boil
Use: General culture medium. strongly for 5 minutes and filter.
Reference: Harvey and Iyengar (1921-22 (9) Distribute.

p. 366). (10) Sterilization not specified.
(b) Frothingham (1895).
779. Dunham's Infusion Broth Add one pound of finely chopped
(1)
Constituents lean meat to 1000.0 cc. of water
1. Water 1000.0 cc. and allow to stand from 12 to 24
2. Beef 500.0 g. hours in a cool place.
3. NaCl (0.5%) 5.0 g. (2) Strain thru a cheese cloth or a
4. Peptone (1.0%,) 10.0 g. coarse towel and squeeze in a
Preparation meat press or by twisting the ends
(1 Boil finely chopped beef with a double of the cloth until 1000.0 cc. of the
weight of water for 2 hours. meat juice is obtained. Makeup
(2) Filter. to 1000.0 cc. by the addition of
(3) Make slightly alkaline. water if necessary.
(4) Dissolve 0.5% NaCl, and 1.0% pep- (3) Dissolve 5.0 g. NaCl and 10.0 g.
tone in (3). dried peptone in (2).
Sterilization: Not specified. (4) Boil 15 minutes, either in steam

Use: Detection of cholera vibrio. Author or over a flame.
reported that cholera vibrio gave a red (5) Make the reaction slightly alka-
ring where the H2SO4 and medium met, line by the addition of a satu-
when concentrated H2SO4 was poured rated solution of Na2C03.
down the side of a culture tube. The (6) Boil from 45 minutes to an hour.
medium is used as a general culture Filter. The filtrate should be
(7)
medium. clear. If not, add the yolks of
Variants Numerous authors have prepared
:
2 eggs, mix rapidly and boil for
media from these constituents. The a quarter to a half hour. Filter.
method of preparation varied in a num- (8) Sterilization not specified.
ber of respects as indicated below, (c) Nicolle (1896).
(a) Schultz (1891).
(1) Infuse 500.0 g. of fresh finely
(1) Place 500.0 g. of the best quality water
chopped beef with 1 liter of
meat, without fat or tendons, in a
overnight at 10C. to 12C.
glass container fitted with a lid.
(2) Add 2.0% peptone and 0.5%, NaCl
(2) Pour 1300.0 cc. of distilled water
to (1).
over the meat.
(3) Boil, filter and make rather
(3) Store in a cool place until the
strongly alkaline.
next day.
(4) Heat for 10 minutes at 120C.
(4) Filter thru 4 thicknesses of
"Morly" and press the remaining (5) Filter and distribute in flasks in
meat to obtain as much fluid as the ratio of one liter medium to a
possible.
two liter flask.
Pour the filtrate into a kettle, (6) Sterilize for 15 minutes at 115C.
(5)
and add 10.0 g. peptone (siccum), (d) Stutzer and Hartieb (1897).
(1) Add 2 liters of water to 1000.0 g. obtain the desired reaction

of finely chopped lean meat. (+1.5).
(2) Heat in the steamer for 2 hours. (8) If the medium is clear distribute
(3) Pour off the liquid. in tubes or flasks. If not clear,
(4) Make (3) up to 3 liters. clarify by adding the white of one

(5) Neutralize by the addition of egg to the agar cooled to 50 to
soda, indicator not specified. 60 C. and then boiled vigorously.
(6) Filter. Filter.
(7) Sterilize (Method not given). (9) Sterilize either by fractional or
(8) Dissolve 2.0% peptone, 10.0 g. continuous method.
NaCl, and 10.0 cc. of a 10.0% (g) Migula (1901).
Na2C03 solution in (7). (1) Mix 500.0 g. of finely chopped lean
(e) Wurtz (1897). beef with 1 liter of water and
(1) Chop 500.0 g. of lean beef (or allow to stand in the ice box for
other meat) into fine pieces. 12 to 24 hours.
(2) Add a liter of water and place in (2) Press the liquid thru a towel and
the cold for 24 hours. make up the volume to 1 liter.
(3) Pass thru a sieve. (3) Boil in the steam cooker for 30
(4) Filter thru a linen towel and free minutes.
the meat from juice. (4) The infusion may be boiled for an
(5) Add sufficient water to make the hour before removing the meat
volume up to 1000.0 cc. and then filtered thru paper.
(6) Add 10.0 g. peptone and 5.0 g. If the liquid is still red, boil
NaCl. again for 15 minutes.
(7) Heat in the autoclave at 125 (5) Filter when cold to remove the
for 15 minutes. (30 minutes for fat.
2 liters.) (6) Add peptone and
10.0 g. Witte's
(8) Filter. 5.0 g.NaCl.
(9) Make slightly alkaline to litmus. (7) Neutralize by the addition of a
(10) Heat for 10 to 15 minutes at concentrated solution of NajCOj
125C. until litmus is colored violet.
(11) Allow to stand for 12 hours. (8) Add the desired amount of soda.
(12) Filter. Generally 10.0 cc. of 15.0% soda
(13) Distribute in sterile test tubes or solution is added per liter.
Erlenmeyer flasks. (9) Boil and filter.
(14) Sterilize at 115 for 15 minutes, (10) Distribute in tubes or flasks.

(f) Committee A. P. H. A. (1899). (11) Boil for one hour to sterilize,
(1) Macerate one part finely chopped (h) Thionot and Masselin (1902).
lean meat with 2 parts distilled (1) Macerate 500.0 g. of finely
water in the ice box for 18 to 24 chopped beef with a liter of water
hours, stirring occasionally. for about 12 hours.
(2) Strain, cold, thru a fine cloth. (2) Express the juice from the meat.
(3) Add 1.0% peptone and 0.5% NaCl (3) Boil the juice slowly for several
to the filtrate. Heat until solu- minutes.
tion is complete. (4) Filter on a wetted filter paper.
(4) Add NaOH until the reaction is (5) Add 10.0 g. peptone and 5.0 g.
slightly alkaline (practically neu- NaCl to the filtrate.
tral) to phenolphthalein. (6) Make slightly alkaline to litmus
(5) Heat on a water bath for 30 by the addition of soda.
minutes and boil for 5 minutes (7) Heat in the autoclave for 15
over a free flame. minutes at 115C.
(6) Filter while hot thru paper or (8) Filter at once.
cotton and cloth. (9) Distribute in tubes or flasks.
(7) Add N/1 HCl to the filtrate to (10) Sterilize at 115C. for 20 minutes.
(i) Smith (1902). (15) Sterilize for 20 minutes in a
(1) Soak 500.0 g. of finely chopped steam sterilizer on 3 consecutive

fresh fat free beef in 1000.0 cc. days or in the autoclave at 120^
of boiled water over night in the for 20 minutes.
ice box. (k) Frost (1903).
(2) Strain thru a piece of cheese (1) Remove all the fat and con-
cloth. Compress the pulp to nective tissue from 500.0 g. of

obtain a liter of fluid. Make up beef and mince or use hamburg
to a liter if necessary. steak.
(3) Heat between 50 and 60C. and (2) Add 1 liter of distilled water.
add 10.0 g. peptone and 5.0 g. (3) Place in a vessel for cooking and
NaCl. Stir constantly until solu- cook for 30 minutes at about
tion complete.
is 70C.
(4) Boil with constant stirring. (4) Filter thru paper and make up
(5) Filter thru paper. to one liter.
Reboil and filter again if the (5) Add 1.0% peptone (Witte) and
(6)
filtrate is turbid. 0.5% NaCl to (3).
(7) Cool to room temperature and The remainder of the preparation
make the volume to one liter. is identical with variant (j)
(8) Adjust the reaction. above, from step (5). These two
(9) Boil and filter. bouillons may be inoculated with
(10) Tube or flask. B. coll and incubated at 38 C.
(11 Sterilize by the fractional method for several hours to render them
in steam at 100C. or in the auto- sugar free.
clave at 120C. (I) Committee A. P. H. A. (1905).
(j) Frost (1903). (1) Infuse 500.0 g. finely chopped

(1) Remove the fat and connec-
all lean meat with 1000.0 cc. water
tive tissue from 500.0 g. of beef for 24 hours in refrigerator.
and mince (or use hamburg (2) Make up volume.
steak). (3) Strain thru cotton flannel.
(2) Add 1 liter of distilled water to (4) Add
10.0 g. Witte's peptone.
(1), shake thoroughly and set in (5) Dissolve by heating in water
the ice box for 12 to 24 hours. bath.

(3) Squeeze thru a cloth and add (6) Heat over steam bath 30 minutes.
enough distilled water to make (7) Restore volume.

1 liter. (8) Adjust reaction to +1 with
(4) Add 1.0% peptone (Witte) and phenolphthalein.
0.5% NaCl to (3). (9) Boil 2 minutes over free flame,
(5) Weigh the solution and vessel. stir.
(6) Heat to not above 60C. until (10) Restore volume.

the ingredients are in solution. (II) Filter thru absorbent cotton and
(7) Restore the loss in weight by the
cotton flannel until clear.
addition of distilled water. (12) Tube in 10.0 cc. lots.
(8) Neutralize to phenolphthalein (13) Sterilize autoclave or by inter-
by the addition normal NaOH.

of mittent method.
(9) Boil for 5 minutes and restore the (m) Heinemann (1905).
weight. (1) Add 500.0 g. lean minced beef,
(10) Test the reaction and readjust if free from fat and tissue as pos-
necessary. sible, to 1000.0 cc. tap water.

(11) Add 0.5 to 1.5% of normal HCl. (2) Place on ice for 24 hours.
(12) Heat until the precipitate ap- (3) To prepare a sugar free broth
pears flaky. add a pure culture of Bacillus coli
(13) Filter thru moistened filter paper. to (2) and incubate at 37 for
(14) Tube. 24 hours.

(4) Weigh the saucepan and cook for (5) Heat for 60 minutes in the steam
about 30 minutes. sterilizer.
(5) Strain thru cheese cloth, and (6) Filter thru filter paper.
press all the liquid out in a meat (7) Add concentrated soda solution
press. until red litmus paper is turned
(6) Replace the water lost by evap- slightly blue.
oration. (8) Heat for 30 to 45 minutes in the
(7) Dissolve 10.0 g. of Witte's pep- steam sterilizer.
tone in (6). (9) Filter.
(8) Adjust the reaction. (10) Sterilization not specified,

(9) Filter into flasks. (p) Sears (1916).
(10) Sterilize in the autoclave at 120 (1) Grind beef fine and obtain juice
for 10 minutes. in 1 liter of water (soaking or
(11) Tube (sterile tubes?). standing not specified).
(n) Committee A. P. H. A. (1909). (2) Dissolve 10.0 g. Witte's peptone
(1) Infuse 500.0 g. finely chopped lean and 5.0 g. NaCl in (1).
meat with 1000.0 cc. distilled (3) Adjust to neutral to phenol-
water for 24 hours in the ice box. phthalein.
(2) Make up the loss due to evapora- (4) Distribute in 2-500.0 cc. lots.
tion. (5) Sterilize at 15 pounds pressure
(3) Strain thru cotton flannel. (time not specified).
(4) Add 1.0% peptone and heat on (q) Roddy (1917).
the water bath until solution is (1) Mix 500.0 g. of minced lean beef
complete. or veal in 1000.0 cc. of water.
(5) Heat over boiling water or steam (2) Place in an ice box for 24 hours in
for 30 minutes. a shallow dish.
(6) Restore the loss due to evapo- (3) Skim the fat.
off
ration. (4) Filter thru linen.
(7) Add normal NaOH until the (5) Express the juice contained in
reaction is +1.0%. the meat.
(8) Boil 2 minutes over a free flame. (6) Boil for 2 hours.
Stir constantly. (7) Filter thru linen.
(9) Make up the loss due to evap- (8) Boil again for one hour.
oration. (9) Add water to make up the loss
(10) Filter thru absorbent cotton until due to evaporation.
clear. (10) Add 10.0 g. of peptone and 5.0 g.
(11) Retitrate and record the re- NaCl.
action. (11) Boil for 30 minutes.
(12) Tube in 10.0 cc. quantities. (12) Make up the loss due to evap-
(13) Sterilize (method not given), oration.
(o) Lohnis (1913). (13) Adjust the reaction.
(1) Mix one pound of lean unsalted (14) Filter while hot and again when
minced meat with one liter of cool.
water. (15) Sterilize in the autoclave,
(2) Allow to stand in a cool place for (r) Roddy (1917).
12 to 24 hours. Prepared as variant (q) above,
(3) Filter thru cloth, squeezing the step (1) thru (11).
meat free from juice until 1 liter (12) Inoculate with a culture of Bacil-
of juice is obtained. Make up to lus coll.
a liter by the addition of water, (13) Incubate for 24 hours at 37.
if necessary. (14) Boil for 30 minutes.
(4) Add 1.0% Witte's peptone and (15) Filter while hot and again when
0.5% NaCl. cool.
(16) Adjust the reaction. (9) Sterilize in the autoclave at 115

(17) Sterilize in the autoclave, for 15 minutes.
(s) Mcintosh and Smart (1918). (u) Percival (1920).
(1) Mince 1.0 pound of fat free meat (1) Chop one pound of lean, fat free
and macerate in 1000.0 cc. cold beef with a knife or mincing
water over night. Or the mix- machine.
ture may be macerated at 40C. (2) Place (1) in a porcelain dish or
for 30 minutes instead. glass beaker and add 1 liter of
(2) Boil for 15 minutes. water.

(3) Filter thru muslin and then (3) Allow to soak over night in a cool
paper. place.
(4) Add 10.0 g. peptone and 5.0 g. (4) Strain thru muslin, and boil for
NaCl. one hour.
(5) Make up to 1000.0 cc, dissolve (5) Filter thru filter paper into a
and steam for 45 minutes. large flask.
(6) Measure 10.0 cc. of the broth into (6) Make the volume up to one liter.
a porcelain evaporating dish, and (7) Add 10.0 g. Witte's peptone and
add 25.0 cc. of distilled water. 5.0 g. NaCl to (6).
(7) Prepare another dish in the same (8)Neutralize to phenolphthalein.
way as a control. (9) Steam for 15 minutes.
(8) Add 5 drops of a 0.5% alcoholic (10) Neutralize again and adjust the
solution of thymolphthalein to reaction to +10.
one dish. (11) Filter into sterile flasks or test
(9) Add N/10 NaOH to (8) until the tubes.

color changes from the yellow to (12) Sterilize on 3 successive days for
a bluish tint. 20 minutes each time.
Calculate and add the amount of (v) Giltner (1921).
(10)
normal NaOH that would be re- (1) Add 500.0 cc. of tap water to
quired to give the same reaction 500.0 g. of finely chopped fresh
to the remainder of the broth. lean beef in a 3.5 liter agate-ware
pail.
Add 10.0 cc. of N/1 HCl per liter (2) Mix thoroly and allow to stand
(12)
in a cool place (refrigerator) for
of filtrate. This gives a pH
= not more than 16 to 24 hours.
7.6.
(3) Strain the infusion thru a cheese
(13) Tube or flask and sterilize in the
cloth, thoroly pressing out all
autoclave for 20 minutes at 115C.
the juice.
(t) BezauQon (1920).
(4) Make up to 500.0 cc. by the addi-
(1) Soak 500.0 g. of finely chopped
tion of tap water if necessary.
lean beef (or other meat) in
(5) Place (4) in a sterile liter Erlen-
1000.0 cc. of water for 12 hours.
meyer flask.
(2) Collect the liquid from the meat
(6) Heat in the autoclave at 120C.
and add 10.0 g. peptone and 5.0 g.
for 30 minutes.
NaCl.
(7) Pour the contents of the flask into
(3) Autoclave for 10 minutes at an agate pail and add 500.0 oc.
115C. of tap water.
(4) Filter thru a wetted filter.
Add 1.0% Witte's peptone and
(8)
(5) Make slightly alkaline to litmus 0.5% NaCl.
by the addition of 10.0% soda (9) Thoroly mix 10.0 g. of egg albu-
tiolution. min with 100.0 cc. of tap water
(6) Autoclave for 15 minutes at and add to (8) or one well beaten
120C. egg white may be used.
(7) Filter. (10) Heat in the autoclave at 120C.
(8) Distribute as desired for one hour.
(11) Adjust the Reaction to +1.5% by (1) Prepare beef infusion broth from
the addition of NaOH or HCI. 1000.0 cc. water, 300.0 g. lean
(12) Filter thru the same paper until beef and Parke Davis
10.0 g.
the medium is bright and clear. & Co. peptone (Procedure not
(13) Distribute as desired. given).
(14) Sterilize in autoclave
the at (2) Initial pH = 5.0 to 9.0, changing
120C. for 30 minutes. every 0.4 interval N/10
using
(w) Dopter and Sacquepee (1921). or N/1 NaOH and N/10 or N/1
(1) Add 1000.0 cc. of water to 500.0 g. HCI.
of finely chopped fat and tendon (3) Sterilize at 15 pounds for 15
free beef. minutes.
(2) Allow to stand in the ice box for (z) Randall and Hall (1921).
12 hours, or heat at 50 to 55 for (1) Soak ground lean beef in water
30 minutes. over night and then boil 10
(3) Heat slowly to boiling. minutes.

(4) Boil slowly for 10 minutes stir- (2) Remove coagulated albumin by
ring constantly. straining under pressure and
(5) Press the liquid thru a clean paper filtration of the liquid.
cloth. (3) Add peptone (2.0%) and NaCl
(6) Add 20.0 g. peptone and 5.0 g. (0.5%).
NaCl 1000.0 g. of the filtrate (4) Adjust to any desired reaction.
and dissolve by shaking. (5) Method of sterilization not given,
(7) Filter thru a wetted filter paper. (aa) Ayers, Rupp and Mudge (1921).
(8) Make slightly alkaline to litmus (1) Method of preparation or compo-

by the addition of NaOH or sition of infusion broth not
NasCOa. given.
(9) Flask. (2) Dissolve 40.0 g. Bacto peptone
(10) Autoclave at 117 for 15 minutes. in (1).
(11) Filter thru filter paper. (3) pH = 7.5.

Adjust to
(12) Make up to 1000.0 cc. by the (4) Method of sterilization not given,
addition of distilled water. (bb) Harvey (1921-22).
(13) Tube or flask. (1) Mince finely fat-free beef.
(14) Sterilize at 115C. for 20 minutes, Note: Veal, chicken, ox or
(x) Abbott (1921). horse heart, horse flesh, rabbit

(1) Add 500.0 g. of chopped lean flesh, fish, blood, placenta, liver,
beef to 1 water and soak

liter of spleen, kidneys, brain, and vege-
for 24 hours, kept at ice box tem- table materials, such as yeast,
perature. wheat, etc., may serve to furnish
(2) Strain thru a coarse towel and the extract used as basis for the
press a liter of fluid is
until medium.
obtained. (2) Add 500.0 g. to 100.0 cc. distilled
(3) Add 10.0 g. (1.0%) dry peptone water or clear tap water.
and 5.0 g. (0.5%) NaCl. (3) Heat the mixture 20 minutes over
(4) Make slightly alkaline or neutral a free flame at a temperature not
(indicator not specified). exceeding 50C.
(5) Place in a porcelain lined sauce- Note: Or simply keep in a
pan, and boil over a flame. Boil cool place over night.
until all the albumin is coagulated (4) Skim off fat floating on the sur-
and the fluid portion is of a clear face.
pale straw color. (5) Raise the temperature to boiling
(6) Filter thru a folded paper. point.
(7) Sterilize by steam (method not (6) Boil 10 minutes.
given). (7) Pour the mixture on to a wet,
(y) Foster and Randall (1921). thick, clean cloth.

(8) Collect thefluid which drains (cc) Harvey (1921-22).

thru the cloth together with that Prepare the medium like variant
obtained by squeezing the meat (aa) (1) thru (15).
in the cloth. (16) Inoculate bouillon with B. coli.
(9) Filter the fluid collected thru (17) Incubate 24 hours.
well-wetted, thick filter paper. (18) Boil 20 minutes.
(10) Add to the filtrate: peptone (19) Make a paste or suspension of
sodium chloride 5.0 g.
10.0 g., about 15 g. purified talc with a
Note: The peptone should be little of the dead bouillon culture.
worked into a paste or suspension (20) Add the suspension to the culture.
by gradual addition of a little of (21) Filter and refilter thru filter
the meat extract before addition paper till clear.
to the filtrate. (22) Distribute into test tubes.
(11) Steam or boil 45 minutes. (23) Sterilize.
(12) Bring the volume up to 1000.0 cc. (dd) Harvey (1921-22).
by the addition of water. (1) Mince finely fat-free beef.
(13) Estimate and adjust reaction to a (2) Add 500.0 g. to 1000.0 cc. distilled
pH value or slightly alkaline to water or tap water.
litmus or 1.0% acid to phenol- (3) Keep in a cool place over night.
phthalein. (4) Skim off fat floating on the
(14) Steam 30 minutes. surface.
(15) Clarify if necessary, and filter (5) Pour the mixture on to a wet,
while hot thru well wetted, thick thick, clean cloth.
filter paper, or thru two layers of (6) Collect the fluid which drains
absorbent cotton wool. thru the cloth together with that
Note: If simple filtration thru obtained by squeezing the meat
thick paper alone is not sufficient in the cloth.
to give a clear medium, clearing (7) Bring the volume up to 1000.0 cc.
should be effected by means of by the addition of water.
white of egg or other clearing (8) Add peptone 10.0 g., sodium
agent. chloride 5.0 g.
(a) Beat up the white of one or two (9) Estimate and adjust the reaction
eggs along with the crushed to a definite pH or faintly alka-
shells in about 20.0 cc water.
. line to litmus or 1.0% acid to
Note: Raw meat juice, 15.0 cc. phenolphthalein.
per liter of medium may be sub- (10) Steam or boil 45 minutes.
stituted for white of egg. (11) Filter, while hot, thru well-
(b) Add to the medium little by wetted, thick filter paper, or thru
little before filtration and at a 2 layers of absorbent cotton
temperature not exceeding 60C. wool.
(c) Stir to mix. (12) Distribute the filtrate into flasks
(d) Steam for 30 minutes. or test tubes.
(e) Remove from steamer and shake (13) Sterilize(S9.5, 9.6 according to
up well to mix. Harvey).
(f) Steam again 15 minutes. (ee)Heinemann (1922).
(g) Filter in the steamer thru thick (1) Clean one pound of beef or veal
filter paper, or thru 2 layers of of adhering fat, etc., and grind in
absorbent cotton wool. a meat chopper.
(h) Refilter, if necessary, the first (2) Cover with a liter of water.
portion of the filtrate. (3) Digest over night at room tem-
(16) Distribute the filtrate into perature.
or test tubes. (4) Heat to 60C. and digest at this
(17) Sterilize. temperature for two hours.
(5) Boil for30 minutes. (5) Dissolve 2.0% Park Davis bac-
(6) Press the liquid from the meat teriological peptone and 0.5%
in a meat press. NaCl in (4).
(7) Mix the meat with more water, (6) Adjust to pH = 8.0.
and press out again. (7) Distribute in desired containers.
(8) Bring the volume of the com- (8) Pass steam thru the autoclave
bined liquids to 1 liter. for one hour after which slowly
(9) Dissolve 20.0 g. peptone and 5.0 g. raise the pressure to 10 pounds
NaCl in (8). (time about 30 minutes) and keep
(10) Adjust the reaction to 1.2% acid at this temperature for 30
with phenolphthalein as an indi- minutes.
cator. (hh) Stitt (1923).
(11) Filter until perfectly clear. (1) Cut up 500.0 g. fat-free meat in
(12) Sterilize in the autoclave. a sausage mill.
(ff) Pitfield (1922). (2) Pour 1000.0 cc. of water over (1).
(1) Cover 500.0 g. of finely cut fat (3) Keep in the ice chest over night.
free beef with 1000.0 cc. of water. (4) The following morning skim off
(2) Shake well and place on ice over the scum of fat by means of a
night. piece of absorbent cotton.
(3) Squeeze out the fluid by means of (5) Squeeze out the infusion thru a
a cloth and make up the volume strong muslin cloth, making the
to 1 liter. amount up to 1000.0 cc.
(4) Inoculate with a culture of the (6) Dissolve 1.0% Witte's peptone
colon bacillus. and 0.5% NaCl in (5). Mix the
(5) Allow to stand at room tempera- salt and peptone with a little of
ture over night. the infusion and prepare a paste
(6) Boil and add 10.0 g. Witte's pep- of themixture in a mortar before
tone and 5.0 g. NaCl. adding it to (5).
(7) Weigh the sauce pan and contents (7) Add sufficient normal NaOH to
and heat to 60C. make the reaction +1.0 to phenol-
(8) Make up the loss in weight by the phthalein.
addition of water. (8) Place in the inner chamber of a
(9) Neutralize to litmus. rice cooker and bring to boil.
(10) Boil 5 minutes. Boil for 20 minutes. It is neces-
(11) Make up the loss in weight by the sary to have NaCl or CaClj in
addition of water. the outer chamber. Do not stir
(12) Adjust the reaction as desired the medium.
(+0.5 to +1.5%). (9) Filter thru a wet filter paper.
(13) Filter. (10) Make up the volume to 1000.0 cc.
(14) Distribute into flasks or tubes. by the addition of distilled water.
(15) Sterilize(method not given). (11) Readjust or record the reaction.
(gg) Hartley (1922) used the following (12) Filter if the reaction is adjusted
medium for diphtheria toxin pro- in (11).
duction: (13) Sterilize in the autoclave at
(1) Soak 1 pound of lean finely 115C. for 15 minutes or in the
chopped horse meat in 1000.0 cc. Arnold on 3 successive days.
of tap water over night. (ii) Stitt (1923).
(2) Raise the temperature to 95C. Prepare the medium like the above
the following morning. variant (gg) (1) thru (12).
(3) Allow to cool slightly and filter (13) Inoculate with 5.0 cc. of a 24-hour
thru paper pulp. culture of the colon bacillus.
(4) Adjust the reaction of the filtrate (14) Incubate over night at 37C.
to pH = 8.0. (15) Boil in a sauce pan.
(16) Put 15.0 g. of purified talc (tal- (11) Heat in an autoclave.

cum purificatum U. S. P.) in a (12) Filter thru grey coarse filter paper
mortar. until clear.
(17) Add (15) to (16), stirring con- (13) Make up the volume to one liter
stantly. with distilled water.
(18) Filter thru paper until clear. (14) Tube in 8.0 cc. lots.
(19) Adjust the reaction to +1.0 to (15) Sterilize at 22.5 pounds pressure.
phenolphthalein. (11) Park, Williams and Krumwiede
(20) Sterilization not specified, (1924).
(jj) Klimmer (1923). (1) Soak 5.0 pounds of finely chopped
(1) Preparation of meat infusion not lean beef or veal in 5000.0 cc. tap
specified. water over night in an ice box or
(2) Add 10.0 g. peptone and 5.0 g. at room temperature.
NaCl to (1) and heat gently until (2) Strain thru cheese cloth.
solution is complete. Squeeze by twisting the cloth or
(3) Boil. use a meat press. This is meat
(4) Neutralize. juice.
(5) Neutralize to litmus and add (3) Add 1.0% peptone and 1.0% NaCl
7.0 cc. normal soda solution per to the filtrate.
liter. (4) Heat to 50C. to dissolve peptone
(6) Filter. and NaCl.
(7) Distribute as desired. (5) Determine the reaction and ad-
(8) Sterilize in the autoclave with just if necessary. (In this case
one heating or in streaming steam boil again.)
each on 3 successive days. (6) Filter thru cotton and filter paper
(kk) Cunningham (1924). in a glass funnel.
(1) Mince or chop into as small "Sterilize in tubes or flasks by
(7)
pieces as possible 1 pound of lean heating in an autoclave for an
unsalted meat, preferably rump hour at 15 pounds pressure."
steak or veal. The meat must be
(mm) Park, Williams and Krumwiede
fat-free.
(1924).
(2) Cover (1) with a liter of water in
(1) Soak 5.0 pounds of finely chopped
a large beaker.
lean beef or veal in 5000.0 cc. tap
(3) Allow to stand in a cool place for
water over night in an ice box or
24 hours.
at room temperature.
(4) Skim off the fat.
(2) Weigh the kettle used in (1) and
(5) Strain thru a piece of cheese
its contents.
cloth.
(3) Heat at 45C. for an hour.
(6) Press out as much liquid as
(4) Boil for 30 minutes.
possible from the meat.
(7) Make up to 1000.0 cc. by the addi- (5) Make up the loss by weight by
tion of water.
the addition of water.
Add 1.0% peptone and 0.5% (6) Strain thru cheese cloth.
(8)
NaCl. Squeeze by twisting the cloth or
Steam for an hour in a double use the meat press.
(9)
walled pot (one hour from the (7) This may be sterilized in flasks
time the water boils). and stored for future use. This
(10) Add normal NaOH solution until is meat infusion.
the liquid turns a piece of mois- (8) If used at once, add 1.0% peptone
tened turmeric paper dipped into and 0.5% NaCl.

it faintly but distinctly brown. (9) Heat to boiling.
The reaction is acid to phenol- (10) Determine the reaction and ad-
phthalein, pH varies from 7.7 to just if necessary (in this case boil
8.0. again).
(11) Filter thru cotton and filter paper Preparation:

in a glass funnel. (1) Prepare a meat infusion from 1 and 2
(12) Sterilize tubes or flasks by-
in (method not given).
heating in an autoclave for an (2) Dissolve 3, 4 and 5 in (1).
hour at 15 pounds pressure. (3) Adjust to a slight alkalinity with
(nn) Kreidler(1926). soda.
(1) Infuse 500.0 g. of finely chopped Sterilization: Method not given.
lean beef in 1000.0 cc. water in an Use: To study denitrification.
ice box over night. Variants
(2) Boil for 30 minutes over a free (a) Percival prepared a similar medium
flame. by adding 3.0 g. NaNOa to a liter of
(3) Filter thru gauze and then thru meat infusion (See Dunham's Meat
paper to remove the fat. Infusion Peptone Solution, variant
(4) Make up to 1000.0 cc. volume. (u) 634).
(5) Add 10.0 g. peptone (Difco) and (b) Harvey prepared a similar medium
5.0 g. NaCl. by adding 5.0 g. KXO3 to a liter of
(6) Boil to dissolve. meat infusion (see Dunham's Meat
(7) Adjust to pH = 7.8. Infusion Peptone Solution, variant
(8) Filter thru paper. (bb) 634).
(9) Sterilize in the Arnold using the Reference: Jensen (1898 p. 406), Percival
fractional method. (1920 p. 56), Harvey (1921-22 p. 67).
References: Dunham (1887 p. 338), Schultz
781. Davis and Ferry's Nitrate Infusion
(1891 p. 56), Frothingham (1895 p. 53),
Broth
Nicolle (1896 p. 333), Stutzer and Hartleb
(1897 p. 404), Wurtz (1899 p. 26), Com- Constituents
mittee A. P. H. A. (1899 p. 77), Migula 1. Beef infusion solution 500.0 cc.
(1901 p. 19), Thionot and Masselin (1902 2. 2.0% peptone solution 500.0 cc.
p. 19), Smith (1902 p. 80), Frost (1903 3. NaCl 4.0 g.
p. 6), Heinemann (1905 p. 27), Committee 4. K2HPO4 3.0 g.
A. P. H. A. (1905 p. 107), (1909 p. 285), 5. MgS04 0.4 g.
Smith (1905 p. 195), Lohnis (1913 p. 14), 6. KNO3 0.2 g.
Sears (1916 p. 113), Roddy (1917 p. 41), Preparation
Mcintosh and Smart (1919 p. 724), Per- (1) Preparation of beef infusion not
cival (1920 p. 56), Bezangon (1920 p. 108), given.
Giltner (1921 p. 29), Dopter and Sac- (2) Mix 1 and 2.
qu6pee (1921 p. 117), Abbott (1921 p. 124), (3) Dissolve 3, 4, 5 and 6 in (1).
Foster and Randall (1921 p. 151), Randall (4) Add enough N/1 NaOH to give final
and Hall (1921 p. 347), Ayers, Rupp and reaction pH = 8.0 to 8.2.
Mudge (1921 p. 258), Harvey (1921-22 (5) Steam 15 minutes and check reaction.
pp. 67, 68), Heinemann (1922 p. 31), Pit- (6) Distribute as desired.
field (1922 p. 13), Hartley and Hartley Sterilization: Sterilize at 115 for 20
(1922 p. 460), Stitt (1923 p. 30), Klimmer minutes.
(1923 p. 192), Frost (1923 p. 64), Cunning- Use: Cultivation of Bad. diphtheriae and
ham (1924 p. 11), Park, Williams and toxin production. Authors reported good
Krumwiede (1924 p. 96), Kreidler (1926 growth.
p. 190). Reference: Davis and Ferry (1919 p. 236).
780. Jensen's Nitrate Infusion Broth 782. LoefHer's Infusion Broth (Roux and
Rochaix)
Constituents:
2. Meat 500.0 g. 1. Distilled water 1000.0 cc.
3. NaCl 5.0 g. 2. Beef 500.0 g.
4. Peptone 10.0 g. 3. NaCl 5.0 g.
5. NaNOs 3.0 g. 4. Peptone 20.0 to 25.0 g.
5. Sodium or potassium (13) Distribute as desired.

phosphate 2.0 g. (14) Sterilize at 100 to 115"C. for 20
Preparation minutes.
(1) Chop 500.0 g. of fat and tendon free Reference Roux and Rochaix (1911
: p. 106),
beef into very small pieces. Besson (1920 p. 26).
(2) Add 1 liter of water and allow to
783. Zielleczky's Phenolphthalein Infusion
stand over night in the cold.
Broth
(3) Press the juice thru a linen cloth by
means meat press.
of a Constituents:
(4) Make up the volume to 1000.0 cc. 1. Water 1000.0 cc.
by the addition of water. 2. Beef... 500.0 g.
(5) Add NaCl, 2.0 g. sodium or
5.0 g. 3. Peptone 10.0 g.
potassium phosphate and 20 to 25 4. NaCl 5.0 g.
grams of peptone. 5. Phenolphthalein solution
(6) Boil for an hour. Preparation
(7) Filter thru a linen cloth and then (1) Prepare bouillon from 1, 2, 3 and 4.
thru paper. (Exact method not given.)
(8) Adjust the reaction slightly alkaline (2) Make alkaline with NasCO,.
to litmus by the addition of KHCO3 (3) Prepare a mixture of 50.0 cc. of water
solution, drop by drop. and 50.0 cc. of absolute alcohol.
(9) Boil for 15 minutes. (4) Dissolve 1.0 g. of phenolphthalein
(10) Allow to settle. Skim off the fat if in (3).
any be present. (5) Just before use add 1.0 cc. of (4) to
(11) Distribute as desired. 19.0 cc. of water.
Sterilization: Sterilize at 115 to 120C. for (6) Add 0.7 to 0.8 cc. of (5) to 5.0 cc. of
30 minutes or by the fractional method. bouillon.
Use: General culture medium. Sterilization: Not specified.
Variants: Besson prepared a similar me- Use: Differentiation of coli and typhoid
dium as follows: organisms. Coli decolorized the red
(1) Remove all fat and tendons from medium completely and more thoroly
beef and chop into small pieces. than did typhoid. Coli caused decolori-
(2) Allow 500.0 g. of (1) to macerate with zation after 5 to 7 hours.
1000.0 cc. of cold water for 6, or if one Reference: Zielleczky (1902 p. 153).
wishes to remove the sugar, 12 hours
784. Muller's Indicator Infusion Broth
at 37 C.
(3) Place in an enamelled pot and bring Constituents
slowly to a boil. 1. Meat Infusion 1000.0 cc.
(4) Boil for 10 minutes. 2. Peptone 10.0 g.
(5) Throw on a thick cloth and press the 3. NaCl 5.0 g.
meat free from juice. 4. Indicator
(6) Filter the juice thru moistened Preparation
paper. (1) Exact method of preparation of meat
(7) Add 10.0 g. of Chapoteaut's or De- infusion from fresh meat not given.
fresne's peptone, 5.0 g. NaCl and (2) Dissolve 2 and 3 in 1.
about 1.0 g. of sodium phosphate. (3) Make slightly alkaline.
(8) Boil stirring constantly until solu- (4) Add 10 drops (12 drops = 1.0 cc.) of
tion complete.
is a 1:100 solution of methylene blue,
(9) Neutralize or make slightly alkaline 10 drops of 1 100 solution of rosanalin,
:
to litmus by the addition of soda 12.0 cc. of a concentrated watery solu-

solution. tion of litmus or 10.0 cc. of a 2:100
(10) Heat at 115 to 117C. for 5 minutes. solution of indigo carmine.
(11) Filter until clear. Sterilization: Not specified.
(12) Make up to 1000.0 cc. by the addi- Use: To study the reduction ability of the
tion of distilled water. colon-typhoid group and other bacteria.
Variants : Harvey added one of the follow- (2) Dissolve 2 and 3 in 1.

ing combinations of dyes or indicators to (3) Adjust to pH = 7.9 by the addition
a liter of meat infusion peptone solution of XaOH.
prepared as indicated in variant (bb) 634. Sterilization: Sterilize in the autoclave.
(a) Cresol red (1.6% solution in 95.0% Use: Cultivation of C. diphtheriae and
alcohol) 1.0 cc. B. megatherium and to produce large
(b) Brom cresol purple (1.6% solution in quantities of toxin. Authors reported
95.0% alcohol) 4.0 cc. that a very high grade of toxin was
(c) Brom cresol purple, cresol red indi- produced.
cator solution 1.0 cc. Variants The following authors have sug-
:
(d) China blue 0.025 g. + rosolic acid or gested various methods of preparing this
its sodium salt 0.05 g. medium:
(e) China green (0.2% solution) 15.0 cc. (a) Dernby and David.
(f) Malachite green 1.0 g. (1) Immerse 6.0 g. of fresh finely
(g) China blue 0.025 g. + phenol red chopped veal in 12 liters of tap
(phenol sulphone phthalein) 0.01 g. water and add 70.0 g. of ordinary
Reference: Muller (1899 p. 805), Harvey distillers yeast.
(1921-22 pp. 88-92), Omelianski (1903 (2) Incubate at 36C. for 16 hours.
p. 4). (3) Press thru cheese cloth.
(4) Add 1.5% Witte's peptone and
785. Robinson and Rettger's Infusion
0.4% NaCl.
Opsine Solution Adjust to pH = 7.2 or 7.3.
(5)
Constituents: (6) Boil several minutes.
1. Beef Infusion 1000.0 cc. Filter while warm thru paper.
(7)
2. Opsine 20.0 g. Distribute in liter flasks.
(8)
Preparation (9) Sterilize at 110 for 20 minutes.
(1) Method of preparation or composi- (b) Dernby and Blanc.
tion of beef infusion not given. (1) Chop veal finely and allow to
(2) Dissolve 2 in (1). autolyzeat37for24hours. Then
(3) Adjust reaction faintly alkaline to boil and filter.
litmus. (2) Dissolve 2.5 g. XaCl and 5.0 g.
(4) Distribute in 20.0 cc. lots in 150.0 cc. peptone in (1).
Erlenmeyer flasks. (3) Adjust to desired pH with XaOH
Sterilization: Sterilize at 12 to 14 pounds and HCl.
pressure for 15 minutes. (4) Sterilize at 110 for 20 minutes.
Use: Production of diphtheria toxin by (c) Harvey.
B. diphtheriae. The author reported (1) Mince finely fat-free veal.
that the addition of 0.1% glucose had (2) Add 500.0 g. to 1000.0 cc. tap water.
little effect. M.L.D. = 0.035 cc. (3) Add an 18 hour culture of B. coli.
Variants (4) Incubate 20 hours at 22C.
(a) The author added 0.1% glucose with (5) Heat the mixture 2 hours at a
or without 0.5 g. XaCl. temperature not exceeding 50C.
(b) The author added 0.5 g. XaCl. (6) Skim off fat on the surface.
Reference: Robinson and Rettger (1917 (7) Raise to boiling point.
p. 367). (8) Boil 10 minutes.
(9) Pour the mixture onto a wet, thick,
786. Warden, Connell and Holly's Veal
clean cloth.
Infusion Broth
(10) Collect the fluid which drains thru
Constituents the cloth together with that ob-
1. Veal Infusion 1000.0 cc. tained by squeezing the meat in
2. Peptone (Difco proteose)... 20.0 g. the cloth.
3. NaCl 5.0 g. (11) Filter the fluid collected thru well
Preparation wetted, thick filter paper.
(1) Method of preparation of veal infu- (12) Add to the filtrate: peptone
sion not given. 20.0 g., sodium chloride 5.0 g.
(13) Bring the volume up to 1000.0 cc. Variants Omit the sodium and potassium
:
(14) Make the reaction 1.2% acid to phosphate.

phenolphthalein. Reference: Schoenholz and Meyer (1921
(15) Steam 30 minutes. p. 387).
(16) Filter while hot, thru well wetted,
thick filter paper. 788. Bunker's Veal Infusion Polypeptide
(17) Distribute the filtrate into test Solution
tubes or flasks.
Constituents
(18) Sterilize.
(d) Brown did not specify the method of
2. Lean veal 1.0 lb.
preparation of the veal infusion, but
3. NaCl 5.0 g.
specified the use of 10.0 g. of Fair-
4. Polypeptides 2.0 g.
child's, Witte's, Bacto peptone or
Preparation:
aminoids. The initial pH was be-
(1) Mix chopped veal and water in after-
tween 6, 7 and 7.
noon and warm to 40C. in large open
.
References: Warden, Connell and Holly

kettle.
(1921 p. 104), Dernby and David (1921
(2) Inoculate with B. coli.
p. 156), Dernby and Blanc (1921 p. 420),
(3) Allow to stand over night.
Harvey (1921-22 p. 113), Brown (1921
(4) Heat to coagulate proteins.
p. 561).
(5) Strain and place in flasks.
787. Schoenholz and Meyer's Veal Infusion (6) Heat in the autoclave at 20 pounds
Broth for I hour.
(7) Add NaCl and polypeptides and
Constituents
NaOH to give pH = 7.8.
1. Water 1000.0 cc.
(8) Heat in autoclave at 15 pounds for
2. Lean veal 500.0 g.
15 minutes.
3. Difco peptone 10.0 g.
(9) Filter thru bibulous filter or thick
4. Na2HP04
cloth.
5. KH2PO4
(10) Distribute in amber glass bottles.
Preparation
Sterilization : Sterilize with steam in usual
(1) Infuse 500.0 g. finely chopped veal in
manner.
a liter of water on ice over night.
Use: Study of production of diphtheria
(2) Boil 20 minutes and strain thru cheese
toxin.
cloth and allow to cool.
Reference: Bunker (1919 pp. 404, 405).
(3) Filter to remove fat.
(4) Add 3 to (3).
789. Krasnow's et al. Sugar Free Veal Broth
(5) Adjust reaction to pH = 7.4.
(6) Stabilize in Arnold for 30 minutes. Constituents:
(7) Filter and tube in 10.0 cc. lots. 1. Tap water 1000.0 cc.
(8) To each 10.0 cc. tube HCl or NaOH is 2. Bacto veal 75.0 g.
added to obtain the desired reaction 3. Peptone 10.0 g.
(varying from pH 4.3 to pH 9.0). 4. NaCl 5.0 g.
(9) Prepare 0.5M solutions of KH2PO4 Preparation
and NajHPO^ and mix in equal (1) Place 75.0 g. Bacto veal in 1000.0 cc.
volumes. tap water and heat in the Arnold
(10) Add 2.0 cc. of sterile (9) to each tube sterilizer for two hours.
of sterile (7). (2) Allow the coagulum thus formed to
Sterilization: Sterilize (7) in steam on each settle to the bottom of the container.
of 3 consecutive days. Method of steri- Allow to cool very slowly.
lization of NaOH, HCl or phosphate (3) Strain thru a wire sieve.
solutions not given. (4) Dissolve 3 and 4 in (3).
Use: To determine optimum pH values for (5) Adjust to pH = 7.9.
typhoid bacilli. Optimum growth oc- (6) Steam in the Arnold for 15 minutes.
curred between pH = 6.8 and 7.0 in a salt- (7) Filter.
free medium. Sterilization: Sterilize in the Arnold for
30 minutes on each of three successive num sponge. Glucose (1.0 or

days. 2.0%) may be added if desired.
Use: Cultivation of streptococci. (9) Boil the tubes for 10 minutes in the
Reference: Krasnow, Rivkin and Rosen- water bath or steamer.
berg (1926 p. 389). (10) Cool quickly and inoculate.
(11) "Hepin" a material from Much
790. Cesaris-Demel's Liver Infusion
and Romer, may replace the
Broth
platinum sponge. Boil the bouil-
Constituents lon and add 1 drop of the sterile
1. Water 1000.0 cc. Hepin by means of a small pipette.
2. Liver (calf) 250.0 g. (b) Kessler prepared the medium as fol-
3. Peptone 10.0 g. lows and used it as an enrichment
4. NaCl 5.0 g. medium for the typhoid bacilli from
5. Litmus the blood.
Preparation: (1) Chop 500.0 g. of beef liver into
(1) Chop fresh calf liver into small pieces. small pieces and add to 500.0 g.
(2) Add to 1000.0 cc. water, 250.0 g. of (1) water.
and infuse for 24 hours. (2) Extract (1) at about 50C. in a
(3) Press out the infusion and filter. steamer for 30 minutes.
(4) Boil the liquid for 1 hour at 100C. (3) Boil for 30 to 45 minutes.
(5) Filter and add 3 and 4. (4) Filter.
(6) Filter and neutralize with normal (5) Make up the filtrate to 500.0 cc.
soda solution (usually requires 3.0 cc. (6) Dissolve 5.0 g. peptone (1%) and
solution to obtain the proper 2.5 g.NaCl (0.5%) in (5).
reaction). (7) Neutralize with addition of NaOH
(7) Place in the autoclave for ^ hour at to litmus.
115C. (8) Boil 30 minutes in the steamer and
(8) Filter and add 20.0 cc. of neutral readjust the reaction.
litmus solution. (9) Distribute in 9.0 cc. lots in tubes.
(9) Tube in 10.0 cc. lots. (10) Sterilize on 3 successive days for
Sterilization: Sterilize in the autoclave. 30 minutes each.
Use: Cultivation of the colon-typhoid (c) Harvey prepared the medium as
group and other organisms. follows. He stated that other organs
Variants: This medium has been prepared may be similarly treated.
in a variety of manners. The following (1)Make a sterilized liver extract in
authors gave the various listed methods the same way as a meat extract
of preparation: with 1000.0 g. finely minced fresh
(a) Pfuhl cultivated anaerobes in a me- ox liver and 1000.0 cc. water.
dium prepared as follows: (2) Prepare a solution of peptone 2.0%
(1) Pass 500.0 g. of fresh beef liver thru and NaCl 1.0%.
a meat grinding machine and add (3) Sterilize the peptone solution.
to 1000.0 cc. of water. (4) Prepare while the solutions are
(2) Allow (1) to stand in the ice box for hot, and with sterile precautions:
1 to 2 hours. sterilized liver extract 1; sterilized
(3) Boil the mixture for 1 hour. peptone solution 1.
(4) Strain thru a straining cloth. (5) Distribute with sterile precautions
(5) Add peptone, NaCl and soda solu- into test tubes.
tion in the usual manner (amount (d) Goss et al. cultivated B. chauvoei, on
not given). a medium prepared as follows:
(6) Boil and filter. (1) Grind 500.0 g. beef liver and add
(7) Distribute into test tubes in 1000.0 cc. water.
10.0 cc. lots and sterilize in the (2) Cook (1) in flowing steam 1 hour.
autoclave (time not given). (3) Strain thru cheese cloth and
(8) To each tube add 1.0 g. of plati- cotton.
(4) Add peptone (1.0%) and NaCl feld candle (length of extraction not
(0.5%). given).
(5) Titrate to pH = 8.2. (5) Decant the ether extract, evaporate
(6) Autoclave at 15 pounds pressure nearly to dryness under suction, and
for 20 minutes. take up the residue in an amount of
(e) Stitt used the medium as a bile sub- saline equal to the original volume.
stitute. He used 500.0 g. of ground (6) Add 1.0 cc. of (4) or (5) or a mixture
beef liver, 1 liter of water and 1.0% of the two to 5.0 cc. of (1).
peptone. The procedure was the Sterilization: Method not given.
same as for Stitt's variant of Dun- Use : To show growth accessory substances
ham's Infusion Broth (see variant for pathogenic bacteria. Author reported
(gg) 665). that the saline extract contained most

References: Cesaris-Demel (1899 p. 532), growth accessory materials while the
Pfuhl (1907 p. 379), Kessler (1911 p. 604), ether extracts were practically devoid of
Harvey (1921-22 p. 69), Goss (1921 p. 615), them.
Stitt (i924p. 47). Reference: Kligler (1919 p. 41).
791. Owen, Martin and Pitts' Trypsin 793. Harvey's Heart Infusion Broth
Beef Tea
Constituents: Ox
heart Bouillon.
1.
1. Bouillon (Beef tea) (1) Prepare bouillon 1% acid to phenol-
2. Trypsin phthalein from ox heart which has
Preparation been "hung" 2 days.
(1) Add 10.0 cc. of trypsin (Liquor pan- (2) Sow with B. lactis aerogenes.
creatica. Digestive Ferments Co.) to (3) Incubate 48 hours.
90.0 cc. of sterile bouillon (Beef tea). (4) Steam 20 minutes.
(2) Distribute in 5.0 to 10.0 cc. lots in (5) Make reaction 1% acid to phenol-
sterile test tubes. phthalein.
Sterilization: Method of sterilization of (6) Sow with B. coll.
beef tea not given. (7) Incubate 48 hours.

Use: Enrichment medium for blood cul- (8) Steam 20 minutes.
tures. Authors report earlier develop- (9) Test for absence of sugar by cultiva-
ment of staphylococci, pneumococci, tion of B. coll or B. lactis aerogenes,
streptococci and typhoid bacilli than on using fermentation tubes.
"glucose beef tea." (10) Leave in a cool place for the growth
Reference: Owen, Martin and Pitts (1916 to sediment.
p. 198). (11) Filter the supernatant fluid thru
thick filter paper or thru a porcelain
792. Kligler's Heart Infusion Broth candle.
Constituents (12) Distribute into test tubes.
1. Peptone phosphate solution. Sterilization Method not given.
:
2. Beef heart. Use: Sugar free culture medium.

3. Saline solution. Reference: Harvey (1921-22 p. 113).
Preparation
794. Park, Willams and Krumwiede's
(1) Exact composition of peptone phos-
Heart Infusion Broth
phate solution not given.
(2) Weigh out 2 equal portions of beef Constituents:
heart. 1. Water (tap) 1000.0 cc.
(3) Suspend one portion of (2) in 10 2. Heart (Beef) 500.0 g.
volumes of saline solution and the 3. Peptone 15.0 g.
other in ten volumes of alcohol ether 4. NaCl 5.0 g.
(1:3). The alcohol is added first to 5. Egg 1
desiccate the clots and then the ether. Preparation

(4) Filter the saline extract thru Berke- (1) Add 500.0 cc. of tap water to 500.0 g.
chopped fat and tendon free beef Preparation

heart. (1) Mince finely lungs, liver, spleen, kid-
(2) Add 15.0 g. of peptone, 5.0 g. NaCI ney, testicles, thymus, placenta or
and one well beaten egg to (1). other organs.
(3) Heat in a water bath, double boiler (2) Proceed as Harvey's Variant of
or over the open flame, stirring con- Schultz's Beef Infusion Peptone Solu-
stantly until the color changes to tion (see variant (v) 750).
brown (at about 68 to 70C). Reference: Harvey (1921-22 p. 98).
(4) Strain thru a wire sieve or wire
gauze. Do not use cheese cloth, 797. Kligler's Blood Clot Infusion Broth
cotton or paper.
Remove all the serum from blood clots
(5) Heat 500.0 cc. of water to 70C.
by centrifugation and use blood clots in-
(6) Mix (5) and (4).
. stead of beef heart to prepare medium
(7) Adjust the reaction to +0.2 phenol-
(see 792.)
phthalein or to about pH = 7.4.
(8) Heat in an autoclave at 15 pounds 798. Hunter's Fish Infusion Broth
for 30 minutes.
Remove the kettle carefully and set Constituents:
(9)
aside for sedimentation to take place 1. Water 1000.0 cc.
or run in Sharpies centrifuge. 2. Salt-water trout 1000.0 cc,
(10) Decant, if allowed to settle. 3. Peptone 15.0 g.
(11) Melt. 4. Raw fish
(12) Tube. Preparation

Sterilization: Sterilize in an autoclave at (1) Add and 15.0 g. pep-
1000.0 cc. water
15 pounds for 30 minutes. tone to 1000.0 chopped salt-

g. finely
Use: Cultivation of meningococcus. water trout or weakfish from which

Reference: Park, Williams and Krumwiede skin and bones have been removed.
(1924 p. 126). (2) Heat in Arnold or in water both at
95-100C. for an hour.
795. Wroblewski's Suprarenal Capsule (3) Strain juice thru cheese cloth, filter
Infusion Broth thru cotton and adjust reaction to
Constituents neutral.
1. Water 1000.0 cc. (4) Heat in Arnold for 30 minutes at 100
2. Suprarenal capsule (ox) 500.0 g. and filter thru paper.
3. Peptone 15.0 g. (5) Tube in 10.0 cc. lots and add to each
4. NaCl 5.0 g. tube about 1.5 raw fish.
5. Soda If anaerobic cultures are desired,
(6)
Preparation cover the surface with layer of liquid

(1) Prepare bouillon in the usual manner
petrolatum.
using suprarenal capsule (ox) instead Sterilization: Sterilize in autoclave for 15
of fat free beef. minutes at 15 pounds.

(2) Dissolve 3, 4 and 5 in (1). Use To study the decomposition of fish by
:
Sterilization: Not specified. organisms resembling colon-aerogenes

Use: General culture medium. group. Author reported that some cul-
Reference: Wroblewski (1896 p. 529). tures produced foul odor and indol.
Reference: Hunter (1920 p. 543).
796. Harvey's Organ Infusion Broth
799. Harvey's Fish Infusion Broth
Constituents:
2. Organs (lungs, liver, spleen, 1. Water 1000.0 cc.
testicles, thymus, placenta 2. Fish flesh 500.0 g.
or other organs) 500.0 g. 3. NaCI 26.5 g.
3. Peptone 10.0 g. 4. KCI 0.75 g.
4. NaCI 5.0 g. 5. Peptone 5.0 g.
Preparation : (8) Neutralize again and boil for 20

(1) Mince finely the flesh of fish. minutes.
(2) Add 500.0 g. to the following solu- (9) Filter.
tion: sodium chloride 26.5 g.; po- (10) Tube.
tassium chloride 0.75 g.; magnesium Sterilization: Sterilize using the fractional
chloride 3.25 g. water 500.0 cc.
; method.
(3) Heat the mixture 20 minutes over a Use Attempt to cultivate bodies found in
:
free flame at a temperature not ex- carcinomatous tissue. Author reported

ceeding 50C. no growth.
(4) Raise the temperature to boiling Reference: Richardson (1900-01 p. 73).
point.
Boil 10 minutes.
801. Boyer's Bone Infusion Broth
(5)
(6) Pour the mixture on to a thick, clean Constituents :
cloth. 1. Water 900.0 cc.

(7) Collect the fluid which drains the 2. Bones beef ribs 500.0 g.
cloth together with that obtained by 3. HCl 100.0 cc.
squeezing the cloth. 4. Peptone 15.0 g.
(8) Filter the fluid collected thru well Preparation :
wetted, thick filter paper. (1) Grind beef bones to small pieces.
(9) Add peptone, 5.0 g. (2) Mix 500.0 g.of (1) with 900.0 g. water
(10) Steam 45 minutes. and 100.0 cc. normal HCl.
(11) Bring the volume up to 1000.0 cc. (3) Allow to stand for 12 to 24 hours.
by the addition of water. (4) Heat in the autoclave at 125 to
(12) Steam 30 minutes. 130C. for 30 minutes.
(13) Filter while hot, thru well-wetted, (5) Cool and then filter thru linen.
thick filter paper. (6) Add 15 parts of peptone to 1000
(14) Distribute into flasks or test tubes. parts of the liquid.
Sterilization: Sterilize in streaming steam. (7) Neutralize by the addition of a
Use: General culture medium. dilute soda solution. (Indicator not
Reference: Harvey (1921-22 p. 69). specified.)
(8) Heat in the autoclave at 125-130
800. Richardson's Mucosa Infusion Broth for 30 minutes.
Constituents : (9) Filter while hot.
1. Distilled water 500.0 cc. (10) Allow to stand for one day in the
2. Mucosa, hog intestine 250.0 g. cold.
3. Peptone 5.0 g. (11) Filter again.
4. NaCl 2.5 g. Sterilization: Sterilize at 120 (time not
Preparation : specified).
(1) Take the small hog in
intestine of a Use: General culture medium. Author
fresh without cutting,
condition, cultivated streptococci, Streptococcus
wash thoroly in running water, until pyogenes and other organisms.
the water runs perfectly clear. Reference: Boyer (1918 p. 230).
(2) Lay the intestine open and scrape
802. Ball's Animal Infusion Broth
off the mucosa with a glass slide
(100.0 g. of mucosa is easily obtained Use the flesh of guinea pigs as well as
from one hog). other experimental animals instead of beef
(3) To 250.0 cc. of the mucosa add in the preparation of Dunham's Infusion
500.0 cc. of distilled water. Broth (779).
(4) Boil for 30 minutes and cool. Reference: Ball (1919 p. 84).
(5) Boil again, neutralize and boil once
803. Stutzer's Nitrate Extract Broth
more.
(6) Filter. Constituents:
(7) Boil and 5.0 g. peptone and 2.5 g. 1. Water.
NaCl. 2. Peptone.
3. Meat extract. 805. Debrand's Extract Broth

4. KNO3. Constituents
Preparation 1. Water 1000.0 cc.
(1) Dissolve 2, 3 and 4 in 1. 2. NaCl 5.0 g.
(2) Reaction to be slightly alkaline. 3. Peptone (Chapoteaut) 10.0 g.
(3) Distribute in 10.0 cc. lots. 4. Meat extract (Liebig) 5.0 g.
Sterilization: Method not given. Preparation: (1) Dissolve 2, 3 and 4 in 1.
Use: To study denitrification. Not specified.
Sterilization:
Variants Use: Debrand used the medium for the
(a) von Caron used 6.0 g. Liebig's Meat enrichment of the tetanus bacillus. The
extract, 6.0 g. peptone and 2.0 g. medium was inoculated with soil that had
KNO3 per liter. been heated on each of 3 successive days
(b) Tanner used 3.0 g. meat
Liebig's at 100C. for 2 minutes. Other investi-
extract, 10.0 g. Witte's peptone and gators used the medium as a general
3.0 g. KNO3 per liter. culture medium.
(c) Committee S. A. B. used 0.1% KNO3, Variants
3.0 g. beef extract and 5.0 g. peptone (a) Smith (1902) did not specify the type
per liter. of peptone or meat extract used, and
(d) Percival used 3.0 sodium nitrate,
g. prepared the medium as follows
5.0 g. Lemco meat extract and 10.0 g. (1) Dissolve 2, 3 and 4 in 1 by heating
peptone per liter. gently and stirring with a glass
References Stutzer (1901
: p. 83), von Caron rod.
(1912 p. 70), Tanner (1919 p. 45), Com- (2) Boil for 5 to 10 minutes, stirring
mittee S. A. B. (1920 p. 128), Percival constantly.
(1920 p. 165). (3) Filter thru paper.
(4) Cool and make up to 1 liter.
804. Davis and Ferry's Nitrate Bouillon Adjust the reaction.
(5)
Extract Broth
(6) Boil.
1. Water 1000.0 cc. (8) Distribute
2. Peptone 20.0 g. (9) Sterilize by the fractional method
3. Meat extract (Liebig's) .... 10.0 g. in steam at 100C. or in the auto-
4. NaCl 4.0 g. clave at 120C.
5. K2HPO4 3.0 g. (b) Frost (1903) used 3.0 g. Liebig's beef
6. MgS04 0.4 g. extract, and 1.0% Witte's peptone
7. KNO3 0.2 g. and prepared the medium as follows:
Preparation (1)Weigh out 3.0 g. of beef extract
(1) Dissolve 2.0% peptone in 500.0 cc. such as Liebig's.
water. (2) Add a liter of distilled water.
(2) Dissolve 1.0% Liebig's meat extract (3) Place in a cooking vessel.
in 500.0 cc. water. (4) Add 1.0% peptone (Witte) and
(3) Mix (1) and (2). 0.5% NaCl to (3).
(4) Dissolve 4, 5, 6 and 7 in (3). (5) Weigh the solution and the vessel.
(5) Add sufficient normal NaOH to give a (6) Heat to not above 60C. until the
final pH = 8.0 to 8.2. ingredients are in solution.
(6) Steam 15 minutes. (7) Restore the loss in weight by the
(7) Check the reaction. addition of distilled water.
(8) Distribute as desired. (8) Neutralize to phenolphthalein by
Sterilization:Sterilize at 115 for 20 the addition of normal NaOH.
minutes. (9) Boil for 5 minutes and restore the
Use: Cultivation of Bad. diphtheriae and weight.
toxin production. Author reported fair (10) Test for reaction and readjust if
growth. necessary,
Reference: Davis and Ferry (1919 p. 236). (11) Add 0.5% to 1.5% of normal HCl.
(12) Heat until the precipitate appears Witte's peptone. The medium was
flaky. prepared as follows
(13) Filter thru moistened filter paper. (1) Make a paste of the peptone.
(14) Tube. (2) Dissolve (1) in 1.
(15) Sterilize for 20 minutes in a steam (3) Add 2 and 3 to (2).
sterilizer on 3 consecutive days or (4) Boil and stir for 30 minutes.
in the autoclave at 120 for 20 (5) Make up the loss due to evapora-
minutes. tion by the addition of water.
(c) Roux and Rochaix (1911) did not (6) Bring to the boiling point.
specify the type of peptone used. (7) Adjust the reaction as desired.
(d) Day and Baker (1912-13) specified (8) Filter when hot and again when
Lemco meat extract and Witte's pep- cool.
tone. They adjusted the reaction to (9) Sterilize in the autoclave.
+1 and cultivated an organism caus- (j) Brussoff (1918) cultivated sludge
ing ropiness in beer. forms on a medium containing 5.0 g.
(e) Lohnis (1913) used Witte's peptone NaCl, 10.0 g. Witte's peptone, and
and adjusted the reaction alkaline 10.0 g. Liebig's meat extract in a
to litmus. liter of tap water rich in potassium.
(f) Rideal and Walker (1913) used 20.0 g. (k) Foster and Randall (1921) studied the
Liebig'smeat extract, 20.0 g. Witte's changes in pH values of a medium
peptone and 10.0 g. NaCl. The me- due to autoclaving and standing.
dium was used for the growth of B. They used 5.0 g. NaCl, 10.0 g. Parke-
typhosus in the Rideal-Walker test Davis & Co. peptone, 3.0 g. of Liebig's
of disinfectants. beef extract, or 50.0 g. of Bacto beef
(1) Dissolve the constituents in water in a liter of distilled water.
by boiling for 30 minutes. (1) Harvey used 3.0 to 5.0 g. of Lemco
(2) Filter. meat extract and prepared the me-
(3) Neutralize to phenolphthalein. dium as follows:
(4) Add 15.0 cc. of N/1 HCl. This (1) Add the whites of 2 eggs to
gives a +1.5 reaction. 1000.0 cc. water.
(5) Make up to 1 liter. (2) Add to the mixture by degrees to
(6) Filter. make a suspension; Lemco 3 to
(7) Sterilize (method not given) in 5.0 g., peptone 10.0 g., sodium
500.0 cc. lots. chloride 5.0 g.
(8) Sterilize again on the next day. (3) Steam or boil 45 minutes.
(9) Tube in 5.0 cc. quantities into (4) Filter while hot thru well-wetted,
sterile test tubes. thick filter paper or thru 2 layers
(10) Plug with sterile cotton. of absorbent cotton wool.
(11) Place in the steam sterilizer for (5) Bring the volume up to 1000.0 cc.
about 30 minutes. by the addition of water.
(g) Brussoff (1916) used 5.0 g. of Merck's (6) Estimate and adjust to a definite
iron peptone and 2.5 g. NaCl. The pH value or faintly alkaline to
medium was used for the cultivation litmus or 1.0% acid to phenol-
of Ferribacterium duplex. A slight phthalein.
yellow membrane was formed after (7) Sterilize in streaming steam or in
5 or 10 days incubation. the autoclave.
(h) Berman and Rettger (1916) studied (m) Pitfield (1922) used 3.0 g. Liebig's
erepsin production by members of beef extract, 10.0 g. Witte's peptone
the colon-typhoid group using 2.5 g. and 5.0 g. of NaCl per liter. The
Liebig's meat extract and did not medium was prepared as follows:
specify the type of peptone (10.0 g.) (1) Dissolve the constituents in
used, 5.0 g. NaCl was also used, 1000.0 cc. of water.
(i) Roddy used 2.0 g. Liebig's
(1917) (2) Weigh the saucepan and contents
beef extract, 5.0 g. NaCl, and 10.0 g. and heat to 60C.
(3) Make up the loss in weight by the did not specify the type of peptone
addition of water. emploj'ed.
(4) Neutralize to litmus, (r) Cunningham (1924) prepared the
(5) Boil 5 minutes. medium as follows:
(6) Make up the loss in weight by the (1) Weigh out 10.0 g. peptone, 5.0 g.
addition of water. NaCl and 10.0 g. of Liebig's meat
(7) Adjust the reaction as desired extract. Weigh out the extract on
(+0.5 to +1.5%). a piece of paper.
thru paper.
(8) Filter (2) Place (1) into pot and add
(9) Distribute into flasks or tubes. 1000.0 cc. of tap water.
(10) Sterilize (method not given). (3) Steam for an hour in a double
(n) Dopter and Sacquepee (1921) did not walled pot (one hour from the time
specify the use of Liebig's meat ex- the water boils).
tract, nor they specify the
did (4) Add normal NaOH solution until
amount of NaCl or peptone used. the liquid turns a piece of mois-
(o) Stitt (1923) prepared the medium as tened tumeric paper dipped into
follows: it faintly but distinctly brown.
(1) Place 3.0 g. Liebig's meat extract, The reaction is acid to phenol-
10.0 g. peptone, and 5.0 g. NaCl phthalein, pH varies from 7.7
in a mortar. to 8.0.
(2) Dissolve the white of one or two (5) Heat in an autoclave.
eggs in 1000.0 cc. of water. (6) Filter thru gray coarse filter paper
(3) Add (2) little by little to (1) until until clear.
a brownish solution is obtained. (7) Make up the volume to 1 liter with
(4) Pour (3) into the inner compart- distilled water.
ment of a rice cooker. (8) Tube in 8.0 cc. lots.
(5) Apply heat to the outer compart- (9) Sterilize at 22.5 pounds pressure.
ment containing NaCl or CaCl2. References: Debrand (1900 p. 759), Smith
(6) Bring to a boil and boil for 15 to (1902 p. 82), Frost (1903 p. 6), Roux and
20 minutes. Do not stir. Rochaix (1911 p. 108), Day and Baker
(7) Place the inner compartment on (1912-13 p. 435), Lohnis (1913 p. 14),
a scales, and its counterpoise and Rideal and Walker (1913 p. 575), Brussoff
a one kilo weight on the other side. (1916 p. 552), Berman and Rettger (1916
Add water to the medium until p. 537), Roddy (1917 p. 41), Brussoff
the two arms balance. (1918 p. 205), Foster and Randall (1921
(8) Filter. pp. 152, 153), Pitfield (1922 p. 114), Dopter
(9) Sterilize in the autoclave at 115C. and Sacquepee (1921 p. 118), Stitt (1923
for 15 minutes or in the Arnold on p. 33), Manwaring, Boyd and Okami (1923
3 successive days. p. 307), Park, Williams and Krumwiede
(10) The reactionrarely exceeds +0.75 (1924 p. 97), Cunningham (1924 p. 11),
(from +0.6 to +0.9) and does not Besson (1920 p. 30), Harvey (1921-22
require adjusting. p. 68).
(p) Manwaring, Boyd and Okami (1923)
cultivated S. cholerae in a medium
806. Berman and Rettger's Extract Proteose
Solution
containing 10.0 g. peptone, 2.5 g.
beef extract, and 5.0 g. NaCl per Constituents
liter. The reaction was adjusted to 1. Water 1000.0 cc.
be identical to that of Locke's solu- 2. Proteose 2.5, 5.0, 8.0 g.
tion (0.015% NaHCOs). 3. Beef e.xtract 2.5 or 5.0 g.
(q) Park, Williams and Krumwiede (1924) 4. NaCl 5.0 g.
suggested the use of 2.0 to 5.0 g. Preparation
Armour or other commercial beef (1) Prepare proteose by salting out the
extracts in addition to Liebig's proteose from commercial peptone
product. They used 5.0 g. NaCl, but (details of the method not given).
(2) Dissolve 2.5, 5.0 or 8.0 g. of (1), 2.5 pany's amenoids instead of Witte's
or 5.0 g. of beef extract and 5.0 g. peptone.
NaCl ia a liter of water. Reference: Berman and Rettger (1918
Sterilization: Not specified. pp. 371-377).
Use: To study bacterial nutrition.
809. Berman and Rettger's Trypsinized
Reference: Berman and Rettger (1918
Extract Broth
pp. 377-380).
Constituents:
807. Guth's Selenium Extract Broth 1. Water 1000.0 cc.
2. Beef e.xtract 2.5 g.
1. Water 1000.0 cc.
4. Peptone (Witte)
2. Meat extract (Liebig) . 10.0 g.
Preparation
(1) Prepare peptone digest by digesting
4. NaCl 5.0 g.
at 45C. for several hours solutions
5. Sodium selenicate of Witte's peptone with commercial
(Merck) 1.0 to 2.5 g. Then sterilize and add ex-
trypsin.
Preparation :
tractand NaCl. The amount of pep-
tone was not specified.
(2) Adjust to neutral using litmus as an Tube in 10.0 cc. lots.
(2)
indicator. Sterilization: Method not given.
Dissolve 5.0 g. Merck's sodium Au-
(3) Use: To study bacterial nutrition.
selenicate in 50.0 cc. distilled water. thors reported that more nitrogen was
(4) Add normal HCl to (3) until the
available in this medium than in similar
reaction is neutral to litmus (requires undigested media.
about 20.0 cc). Reference: Berman and Rettger (1918
(5) Make (4) up to 100.0 cc.
pp. 383-384).
(6) Heat in the autoclave for about 15
minutes. 810. Olszewski and Kohler's Trypsinized
(7) Tube (1) in 10.0 cc. quantities. Extract Broth
(8) Add 0.3 cc. of (6) to each tube of (7)
Constituents :
1. Water 1000.0 cc.
Use: Enrichment of typhoid bacilli. Colon Peptone 10.0 g.
2.
bacilli are inhibited. Liebig's meat extract 5.0 g.
3.
Reference: Guth (1915-16 p. 490). 4. NaCl 5.0 g.
5. Physiological salt solution. 3000.0 g.
808. Berman and Rettger's Salt Extract Preparation :
Broth
(1) Prepare ordinary nutrient bouillon
Constituents using 10.0 g. peptone, 5.0 g. Liebig's

1. Water 1000.0 cc. meat extract and 5.0 g. NaCl.
2. Peptone (Witte) 5.0 g. (2) Make alkaline by the addition of
3. Liebig's beef extract 2.5 g. 7.0 cc. N/L soda solution after neu-
4. NaCl 5.0 g. tralizing to litmus.
5. K2HPO4 1.0 g. (3) Boil.
6. MgS04 0.2 g. (4) Cool to 40C. and add 0.2 g. trypsin,

7. CaCl2 0.1 g. 10.0 cc. chloroform and 5.0 cc. toluol.
Preparation : (5) Place in a glass stoppered flask and

(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. place in the incubator for 24 to 48
(2) Tube in 10.0 cc. lots. hours, shaking occasionally.
Sterilization: Not specified. (6) Filter thru a moistened folded filter
Use: To study bacterial nutrition. paper.
Variants The authors used Digestive Fer-
: (7) For use dilute one part of this stock
ments, Park Davis, Eimer and Amend's solution with 3 parts physiological
peptone or Arlington Chemical Com- salt solution.
sterilization: Not specified. Sterilization: Autoclave at 120 for 5

Use: Water analysis and indol production. minutes.
Author tested for indol by adding 10.0 Use: General culture medium.
drops of Ehrlich's reagent and 10.0 drops Variants
of a saturated watery solution of po- (a) Committee S. A. B. dissolved 3.0 g.
tassium persulphate to a tube after beef extract and 5.0 g. peptone in a
incubation. liter of distilled water. The medium
Variants: Frieber specified the use of may be clarified using egg white.
Griibler's trypsin and did not give the Adjust from pH = 6.6 to 7.4.
exact amounts of chloroform or toluol (b) Ball, Tanner, Levine (A. P. H. A.)
used. and Park, Williams and Krumwiede
References: Olszewski and Kohler (1923 used 3.0 g. beef extract and 5.0 g.
p. 5), Frieber (1921-22 p. 275). peptone and adjusted the reaction to
811. Bacto Cooledge Broth (Dehydrated)

+ 1, if the reaction does not fall be-
tween +0.5 and +1.0.
Constituents: (c) Brown used 3.0 or 5.0 g. of Liebig's
1. Water meat extract and 10.0 or 20.0 g. of
2. Beef extract (Bacto) 3.0 g. peptone or 10.0 g. of
Fairchild's
3. Peptone (Bacto) 10.0 g. Witte's peptone or 10.0 g. of Bacto
4. NaCl 5.0 g. peptone. Initial pH from 7.1 to 7.4.
5. Dibromthymolsulphoneph- References: Heinemann (1905 p. 25), Com-
thalein 0.032 g. mittee S. A. B. (1918 p. 115), Ball (1919
Preparation p. 76), Tanner (1919 p. 44), Levine (1921
(1) Dissolve 18.0 g. of Bacto Cooledge p. 109), Brown (1921 p. 561), Park, Wil-
Broth, (dehydrated) in 1000.0 cc. of liams and Krumwiede (1924 p. 131).
distilled water. The formula for the
broth is given above. 813. Wyant's Bouillon Cube Broth
(2) Reaction pH = 7.0.
Constituents:
Sterilization: Sterilize in the usual manner.
Use: General culture medium. 1. Water 1000.0 cc.
Reference: Digestive Ferments Co. (1925 2. Bouillon cubes 3.0 to 5.0 g.

3. Peptone 10.0 g.
p. 13).
Preparation
812. Heinemann's Bouillon (1) Dissolve 2, and 3 in boiling tap water.
Constituents (2) Cool liquid to coagulate fats.
Water (tap) (3) Filter cold.

1. 300.0 cc.
2. Peptone (4) Tube.
3.0 g.
3. Beef extract 1.0 g.
Sterilization: Sterilize at 15 pounds in
Preparation autoclave.
Use: Substitute for beef extract or meat in
(1) Weigh a saucepan and measure
300.0 cc. tap water into it.
nutrient media. A very satisfactory sub-
(2) Dissolve 3.0 g. of peptone in (1)
stitute for meat. Can also be used in
when hot.
special media omitting the peptone.
Make up the loss due to evaporation Reference: Wyant (1920 p. 189).
(3)
by adding water.
(4) Dissolve 1.0 g. of beef extract in (3). 814. Heinemann's Nitrate Broth
(5) Adjust the reaction to alkaline to Constituents
litmus or neutral to phenolphthalein 1. Bouillon 1000.0 cc.
and add 0.5% normal HCl. 2. KNO3 5.0 g.
(6) Distribute into Erlenmeyer flasks. Preparation: (1) Dissolve 2 in 1.
(7) Autoclave at 120 for 10 minutes. Sterilization: Not specified.

(8) Store for 24 hours in a cool place. Use: To study denitrification.
(9) Filter. Variants
(10) Tube. (a) Sewerin used 3.0, 6.0, 7.0, 8.0 or
NaNOa instead of 5.0 g. KNO3 typhoid had not changed color.

9.0
to bouillon.
Coli bacilli formed a greenish blue
(b) Lohnis added about 0.1% NaNOs to zone of about 1 cm. on the surface of
bouillon. the bouillon.
Percival added 3.0 g. NaNO, to (f) Signorelli added 0.5 cc. of a 1.0%,
(c)
bouillon. dahlia solution, 10.0 cc. of a 1.0%
References: Heinemann (1905 p. 131), erythrosin solution or 10.0 cc. of a

1.0% safranin solution to 100.0 cc.
Sewerin (1909 p. 349), Lohnis (1913 p. 98),
Percival (1920 p. 165). of bouillon. He

studied the adsorp-
tion of dye by the cholera vibrio.
815. Makgill's Indicator Bouillon He reported that the organisms de-
Constituents veloped slowly. After 24 to 36 hours
1000.0 cc. the colored mass of bacteria settled
1. Bouillon
2. Neutral red (saturated to the bottom and the medium was

aqueous solution) 10.0 cc. decolorized after a week. If the dye
Preparation: (1) Add 1.0% of a saturated was added after the culture had
solution of neutral red to nutrient developed the vibrio absorbed the
bouillon. dye. With erythrosin and safranin
Sterilization: Not specified. the bacteria were well colored, but
of Bacillus coli in water the medium was not decolorized.
Use: Detection
Bacillus coli gave a canary (g) Groenewege
added 10.0 drops of
analysis.
yellow color to the medium. Different methylene blue to 150.0 cc. of bouil-
lon. The medium was used to study
authors have added various indicators for
other special purposes. These will be the reduction of Phytobacter lyco-
indicated under variants.
persicum n. sp. the cause of tomato
rot. Partial reduction occurred.
Variants
Gordon reported that Streptococcus (h) Botez added 2.0 cc. of a 5.0%o methyl
(a)
brevis decolorized bouillon contain- violet solution to 100.0 cc. of bouillon.
ing 2.0 cc. of a 2.0%o watery solution He reported that B. typhosus grew
of neutral red per liter while Strepto-
well. Did not decolorize (reduce)
methyl violet. Para typhoid A
coccus longus did not decolorize the
medium. changed the color to a pale violet.
(b) Heinemann added 10.0% of a 1.0% Paratyphoid B completely decolorized
the medium after 48 hours. B. coli
litmus solution to nutrient bouillon.
completely decolorized the medium
(c) Calandra added 4 drops of a 1.0%
of Brilliant cresyl after 48 hours. B. fecal alcaligenes
aqueous solution
blue to 10.0 cc. bouillon. He re- did not reduce methyl violet. B.
ported that typhoid bacilli caused no Shiga did not grow. Cholera vibrio
change. B. coli after 24 hours gave grew slowly and did not change the
color after 10 days. B. pyocyaneus
a blue colored layer about i cm. high
on the upper surface. After 48 hours reduced methyl violet,
Tanner added 2.0 cc. of 1.0%o neutral
the blue color persisted in the upper (i)
layer. The remainder of the tube red solution to 1000.0 cc. of bouillon
was decolorized. containing 1.0% peptone and 0.5%
(d) Calandra added 3 drops of a 1.0% NaCl.
congo red solution to 10.0 cc. of (j) Heinemann added 2.0 to 3.0 cc. of a
bouillon. reported that typhoid
He 2.0%, solution of Hochst's 120" mala-
caused no change in color after 24 chite green to bouillon, alkaline to
hours. After 24 hours, B. coli gave litmus.

References: Makgill (1901 p. 431), Gordon
a strawberry red color. Both ty-
phoid and B. coli were red after (1904 p. 271), Heinemann
(1905 p. 128),
48 hours. Calandra (1910 pp. 570, 571), Signorelli

(e) Calandra added 3 drops of a 1.0% (1912 p. 472), Groenewege (1913 p. 26),
alkali blue to 10.0 cc. of bouil- Botez (1915 p. 489), Tanner (1919 p. 46),
Kuhne
lon. He reported that after 48 hours Heinemann (1922 p. 35).
816. Thoinot's Arsenious Acid Bouillon parts of ordinary bouillon. Method

of preparation of bouillon not given.
Constituents
Mix well.
(2) Distribute in 90.0 cc. lots in 150 or
2. Arsenious acid 0.1 g.
200.0 cc. flasks.
Preparation
(3) Make 50.0 g. of crystalline sodium
(1) Prepare peptone bouillon.
hyposulphite up to 100.0 cc. by the
(2) Add 0.01% arsenious acid to (1).
addition of water.
(4) Mix thoroly in a mortar 20.0 g. KI
Use Differentiation between colon bacillus
:
and 25.0 g. iodine, and add enough

and the Eberth bacillus. Author re-
water to make 100.0 cc.
ported that the Eberth bacillus did not
(5) To each 90.0 cc. of sterile (2) add
grow in a medium containing 0.01%
10.0 cc. of sterile (3) and 2.0 cc. of
arsenious acid. Coli bacillus grew well
sterile (4). Mix well.
even in a medium containing 1.5% ar-
(6) Distribute in tubes or flasks.
senious acid.
Sterilization: Sterilize (2) and (3) at 100
Reference: Thoinot (1898 p. 126).
for 30 minutes. Sterilize (4) by itself
817. Omeliansky's Dilute Bouillon (method not given).
Constituents Use: Enrichment medium for typhoid
1. Distilled water 50.0 cc. group. B. coll and other forms are
2. Bouillon 50.0 cc. inhibited.
3. Gypsum 5.0 g. Reference: Muller and Malvoz (1923 p.
Preparation 435).
(1) Method of preparation of bouillon

not given. 819. Fremlin's Ammonium Sulphate
(2) Dilute 50.0 cc. alkaline bouillon with Bouillon
an equal volume of distilled water. Constituents
(3) Add 5.0 g.gypsum to (2). Water
1. 1000.0 cc.
Sterilization: Not specified. 2. (NH4)2S04 1.0 g.
Use: To study nitrification. The author
3. Potassium phos-
reported that using a mixture of Bac.
phate 1.0 g.
ramosus, Nitrosomonas and Nitrobacter,
4. MgCOs 10.0 g.
ammonia, nitrites and nitrates were
5. Peptone beef broth. 0.1 to 100.0 cc.
formed. Using Bac. ramosus and Nilro- Preparation
somonas, ammonia and nitrites were
formed. Using Bac. ramosus and Nitro-
(2) Dissolve 4 in the remainder of 1.
bacter only ammonia was formed. Using
(3) Mix and sterile (2) and add
sterile (1)
Nitrosomonas and Nitrobacter there was
various amounts of sterile 5.
only organic nitrogen present.
Variant: Omeliansky omitted the gypsum
Use: To study nitrogen oxidation by
and added 10.0 cc. alkaline bouillon to
nitroso bacteria.
818. Muller and Malvoz's Iodine Bouillon 820. Stitt's Carbonate Bouillon
1. Bouillon 1000.0 cc. 1. Bouillon.
2. CaC03 50.0 g. 2. CaC03.

3. Sodium hyposulphite. Preparation:(1) Add small fragments of
4. KI. marble (CaCOs) to bouillon.
5. Iodine. Sterilization: Not specified.
Preparation Use: General culture medium.
(1) Add 5 parts pure CaCOa to each 100 Reference: Stitt (1921 p. 35).
821. Dalimier and Lancereau's Opsine (4) Strain broth thru cheese cloth.
Bouillon (5) Measure broth and add 3, 4 and 5.
(6) Boil until and 5 are in solution.

3, 4
Constituents
(7) Correct to +1, phenolphthalein as
1. Bouillon.
indicator, with N/10 NaOH.
2. Opsine.
(8) Filter thru 2 layers absorbent cotton
Preparation
directly into 2 liter Erlenmeyer flask,
(1) Method of preparation of bouillon
leaving only enough space to allow
not specified. Reaction alkaline.
for expansion of broth in Arnold.
(2) Add opsine. (Opsine is a commercial
Sterilizatiori Sterilize in Arnold 90 minutes
:
mixture of amino acids.)

on first day and 1 hour on second day.
Use: Study toxin production by B. tetani
Use: General culture medium. Authors
and preliminary cultures for inoculation
reported the growth of staphylococci,
in toxin broth.
typhoid and paratyphoid bacilli, Micro-
Variants
coccus melitensis, B. prodigiosus, Vibrio
(a) Walbum studied diphtheria toxin pro-
cholerae, pneumococci and others.
duction on a veal infusion medium
Variants The authors suggested the addi-
:
containing 1.5% Witte peptone, 0.5%

dion of glycerol.
NaCl and 0.2% glucose. The me-
Reference: Dalimier and Lancereau (1913
dium was sterilized unadjusted, and
p. 419).
the sterile medium adjusted to pH
822. Frieber's Trypsinized Bouillon
= 7.0 by adding sterile calcinated
(Committee S. A. B.) soda. He reported that the addition
of small amounts of manganese chlo-
Constituents
ride (final concentration 0.01 molar)
in many cases markedly increased the
2. Physiological salt Solution. . 3000.0 cc.
toxin production.
3. Trypsin 0.2 g.
(b) Park, Williams and Krumwiede pre-
Preparation: pared the medium as above, but did
(1) Add 0.2 g. trypsin to aliter of peptone
not give the adjustment of reaction
bouillon.
and heated for 90 minutes at 90C.
(2) Add chloroform and toluol.
instead of 90 minutes in Arnold.
(3) Incubate for 24 to 48 hours at 37C. Park, Williams and Krumwiede used
(c)
(4) Filter. a similar medium for the production
(5) Dilute with 3 parts physiological salt This medium
of diphtheria toxin.
solution.
was prepared as follows:
Mix 20 pounds of lean chopped veal
(1)
Use: To determine the production of indol. with 20 liters of tap water.
Reference: Committee Society American Place in the ice box over night.
(2)
Bacteriologists (1923 p. 31). Strain the next morning.
(3)
(4) When chilled add 10.0 cc. of a
823. Wilcox's Glucose Veal Infusion Broth
24 hour culture of B. coli for each
Constituents liter and incubate at 37C. over
1. Water 1000.0 cc. night.
2. Veal (Market) 1.0 lb. (5) In the morning add 1 egg for each
3. Peptone (Witte) 10.0 g. 2 liters.
4. Glucose (anhydrous) 10.0 g. (6) Boil 20 minutes and strain.
5. NaCl 5.0 g. (7) Adjust the reaction to 0.5 acid to
Preparation phenolphthalein.
(1) Add water to chopped veal and let (8) Add 1.0% Park Davis & Co. pep-
stand in ice chest over night. tone, and 0.5% NaCl and heat to
(2) Place infusion over free flame and boiling to dissolve.
heat to 45C. for 1 hour, (9) Readjust the reaction.
(3) Boil briskly for ^ hour. (10) Filter thru paper and cotton.
(11) Distribute 800.0 cc. in 2 liter flasks. Reference: Park, Williams and Krum-
(12) Autoclave for 30 minutes at 15 wiede (1924 p. 133).
pounds pressure.
825. Reeser's Potato Veal Infusion Broth
(13) Add 8.0 cc. of a 10.0% glucose solu-
tion, sterilized in the Arnold on Constituents
each of 3 successive days, for 1. Distilled water 3000.0 cc.
30 minutes to each flask. 2. Potato
References: Wilcox (1916 p. 334), Walbum 3. Peptone 30.0 g.
(1922 p. 25) taken from (1922 p. 109), 4. NaCl 15.0 g.
Park, Williams and Krumwiede (1924 5. Glycerin 150.0 g.
pp. 132, 133). 6. Veal 1500.0 g.
Preparation
824. Park, Williams and Krumwiede's (1) Peel potatoes and carefully remove
Glycerol Veal Infusion Broth all eyes.
Constituents: (2) Cut into thin slices.

1. Water 1000.0 cc. (3) Allow to stand in water for a day,
2. Veal 1.0 lb. renewing the water occasionally.
3. Peptone (Fairchild's (1.0%). 10.0 g. (4) Soak in distilled water over night.
4. NaCl (0.5%) 5.0 g. (5) Pour off the water and heat the
5. Glycerin (5.0%) 50.0 g. pieces of potato for f hour in auto-
Preparation clave at 1.5 atmospheres pressure.
(1) Mince 1.0 pound of lean veal free (6) Add 3.0 liters of distilled water.
from fascia with 1 liter of water. (7) After sterilization allow the po-
(2) Macerate over night at room tem- tatoes to stand in water for one day.
perature. (8) Filter the well extracted potato
(3) Heat to 45 to 48 for one hour. slices and make the filtrate up to
(4) Boil up strongly. 3 liters.
(5) Strain thru cheese cloth. (9) Grind 1500.0 g. of lean veal in a meat
(6) Add 1.0% Fairchild's peptone and chopping machine.
0.5% NaCl. (10) Add (8) to (9) and place in the ice
(7) Boil to dissolve the peptone. box over night.
(8) Titrate and adjust the reaction to (11) Filter thru a linen towel.
+2.5 to phenolphthalein (room (12) Add water to make 3 liters.
temperature) (about pH = 6.0 (13) Boil 10 minutes.
to 6.4). (14) Filter thru a flannel cloth.
(9) Autoclave for 15 minutes at 15 (15) Add peptone and 0.5% NaCl
1%,
pounds pressure, to clear. which one has prepared a paste of
(10) Filter thru cotton and paper. with a little of the hot liquid.
(11,' Measure the filtrate and add 5.0% (16) Add 5.0% glycerin.
glycerol. (17) Neutralize with N/1 Na2C03 using
(12) Distribute in 250.0 cc. quantities in litmus as an indicator.
each quart Blake bottle or liter flask. (18) Add 3.0 cc. N/1 NajCOa per liter to
Sterilization: Sterilize in the autoclave for give a slight alkaline reaction or
30 minutes at 15 pounds pressure. adjust the reaction so that 5.0 cc. of
Use: Cultivation of B. mallei and malein the bouillon to which phenolphtha-
production for diagnosis of glanders. lein has been added, will be colored
Cultivation of tubercle bacilli and tuber- weakly red when 0.4 cc. N/1 NaOH
culin production. or NaoCOs be added.
Variants: The authors used the medium (19) Sterilize for 2 hours, method not
with a different reaction to cultivate given.
tubercle bacilli and for the production of (20) If not transparent after sterilization
tuberculin. This medium was adjusted thru paper or flannel.
filter
to +1.3 to phenolphthalein (pH about (21) Distribute in sterile Roux flasks.

7.1) and the final reaction +1.5 to phenol- Sterilization: Sterilize (6) by autoclaving
phthalein or pH about 6.9. for 45 minutes at 1.5 atmospheres of
pressure. Sterilize (19) for two hours, Preparation

method not given. (1) Prepare veal broth but use 0.2%
Use: Cultivation of tubercle bacilli and NaaHPOi instead of NaCl.
preparation of tuberculin. (2) Adjust to pH = 7.8.
Reference: Reeser (1908 p. 152). (3) Add 10.0 cc. of blood to 100.0 cc. of
sterile (2).
826. Mellon's Serum Veal Infusion Broth
Incubate at 37C.
(4)
Constituents: Sterilization: Sterilize (2) at 115 for
1. Veal Infusion Broth 1000.0 cc. 30 minutes.
2. Glucose (1.0%) 10.0 g. Use: Isolation of a streptothrix from the
3. Serum (human or rabbit). blood.
Preparation Reference: Thjotta and Gundersen (1925
(1) Prepare a 1.0% glucose veal infusion p. 1).
broth.
829. Robinson and Header's Liver Veal
(2) Adjust (1) to +1.6 to phenol-
Infusion Broth
phthalein.
(3) Add a few drops of sterile human or Constituents
rabbit serum to each tube of (2). 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Lean veal 500.0 g.
Use: Cultivation of diphtheroids. 3. Peptone 20.0 g.
Reference: Mellon (1916 p. 84). 4. NaCl 5.0 g.
5. Liver (Fresh sterile guinea pig).
827. Albert and Kelman's Blood Veal Preparation
Infusion Broth
(1) Mince lean veal fine, and soak in
Constituents water in ice box for 18 to 24 hours.
1. Veal Infusion Broth 1000.0 cc. (2) Filter thru wire gauze and heat to
2. Blood, rabbit defibrinated. . 100.0 cc. 80 for 5 minutes.
Preparation (3) Cool and remove fat from the sur-
(1) Prepare 1 liter of veal infusion broth. face.
(2) Adjust to neutral to phenolphthalein. (4) Add 3 and 4 to (3).

(3) Distribute in 50.0 cc. lots in Erlen- (5) Adjust to 0.3% acid to phenol-
meyer flasks. phthalein.
(4) Add 5.0 cc. of sterile defibrinated rab- (6) Boil 5 minutes and adjust again if
each flask.
bit blood to necessary.
(5) Heat the mixture over a water bath (7) Filter thru wire gauze and sediment
at 75C. until the blood coagulates allowed to settle.
and settles on standing (3 to 5 minutes (8) Distribute in 125.0 cc. lots in 300.0 cc.
after reaching the temperature). Florence flasks.
Sterilization: Method not given. (9) Kill a large guinea pig by a blow on
Use Cultivation of B. infliienzae and toxin
: the head and plunge the guinea pig
production. After incubation at 37C. into a 0.5% lysol solution for a few
for 24 hours, centrifuge to remove the minutes.
coarse particles and then filter thru a (10) Open the body cavity aseptically and
mandler candle filter. remove the liver.
Reference: Albert and Kelman (1919 p. (11) Introduce a piece of liver slightly
434). larger than a 25 cent piece into each
flask containing 125.0 cc. sterile
828. Thjotta and Gundersen's Blood Veal medium.
Infusion Solution by heating at 15
Sterilization: Sterilize (8)
Constituents: pounds pressure for 15 minutes.
1. Veal broth. Use: Cultivation of diphtheria bacilli, and
2. Sodium phosphate (secondary) production of toxin by the Park Williams
(NajHPOO. strain ^8. Contamination from liver
3. Blood. may be detected in smear preparations
and also
at the time of testing the toxin 2. Blood, defibrinated, rabbit
by the odor of the broth. or dog 100.0 cc.
Reference: Robinson and Meader (1920 3. Egg yolk.
p. 107). Preparation: (1) Add 50.0 cc. of egg yolk
and 100.0 cc. of dog or rabbit defibrinated
830. Starln and Dack's Casein Digest Veal
blood to 1000.0 cc. of veal infusion broth.
Infusion Broth
Constituents Use: Cultivation of tubercle bacilli.
1. Veal infusion 1000.0 cc. Reference: Klimmer (1923 p. 224).
2. Peptone 10.0 g.
3. NaCl 5.0 g. 833. Jackson and Muer's Glucose Liver
4. Casein digest (Wolf's) 50.0 g. Infusion
Preparation
Constituents:
(1) Method of preparation or composi-
1. Water 1000.0 cc.
tion of veal infusion not given.
2. Liver, beef 500.0 g.
(2) Dissolve 2, 3 and 4 in (1). The prepa-
ration of Wolf's casein digest was not
4. Glucose 10.0 g.
given by the authors. It was pre-
pared according to Kahn's method.
5. K2HPO4 1.0 g.
Preparation
See 648.
(1) Chop 2 into small pieces and add 1.
(3) Filter to remove any precipitated
Weigh the infusion and container.
material.
(2) Boil slowly for 2 hours in a double
(4) Adjust reaction to pH = 7.8 to 8.0
boiler, starting cold and stirring it
occasionally.
Use: Cultivation of Clostridium botulinum.
(3) Make up the loss in weight by evapo-
Reference: Starin and Dack (1923 p. 173).
ration and strain thru a wire strainer.
831. Bacto Veal Infusion Medium (4) To the filtrate add 3, 4 and 5. Weigh
(Dehydrated) the infusion and container.
Constituents (5) After warming this mixture in a

1. Distilled water. double boiler and stirring it for a few
2. Veal infusion 8.0 g.
minutes to dissolve ingredients, ti-
trate with N/20 sodium hydrate, using
3. Peptone, Bacto 10.0 g.
4. Agar, Bacto 10 g. phenolphthalein as an indicator and
Preparation neutralize with normal sodium
Dissolve 19.0 of Bacto Veal Infu- hydrate.
(1) g.
sion Medium (Dehydrated) in (6) Boil vigorously for 30 minutes in a
1000.0 cc. of distilled water by boil- double boiler and 5 minutes over a
ing. (Low agar content according free flame with constant stirring to
to Hitchens. prevent the caramelization of the

dextrose.
(2) Restore the loss in weight if neces-
sary. (7) Make up any loss in weight by
(3) If sterilized at 15 pounds for 20 evaporation and filter thru cotton
minutes pH = 7.5. flannel and filter paper.
Sterilization: Sterilize at 15 pounds for Sterilization : Tube and sterilize in an auto-
20 minutes. clave for 15 minutes.
Use: General culture medium. Use: Presumptive test for gas formers in
Reference: Digestive Ferments Co. (1925 water. Author reported that this me-
p. 11). dium gave a larger amount of attenuated
forms, had greater rejuvinating power,
832. Bruschettinis' Blood Egg Broth gave fewer abnormalities and greater and
(Klimmer)
more rapid gas production than did the
Constituents usual dextrose broth. B. coli produced
1. Veal Infusion Broth 1000.0 cc. gas.
Variants References: Jackson and Muer (1911
(a) Harvey prepared a similar medium p. 290), (1911 p. 727), Levine (1921 p. 110),
as follows: Harvey (1921-22 p. 110), Klimmer (1923
(1) Mince finely ox liver. p. 201).
(2) Add 500.0 g. to 1000.0 cc. distilled

water. 834. Haslam's Brain Liver Infusion Broth
(3) Heat the mixture 20 minutes at a
Constituents:
temperature not exceeding 50C.
1. Water 1000.0 cc.
(4) Skim off fat floating on surface.
2. Beef liver 500.0 g.
(5) Raise the temperature to boiling
3. Brain 500.0 g.
point.
4. Peptone (1.0%) 10.0 g.
(6) Boil 10 minutes.
5. NaCl (0.5%) 5.0 g.
(7) Pour the mixture on to a wet,
Preparation
thick, clean cloth.
(1) Grind liver and to each liter of water
add 500.0 g. liver.
the cloth together with that ob-
(2) Bring to boil slowly and cook for
tained by squeezing the meat in
about 30 minutes or until the liquid
the cloth.
is clear.
(9) Filter the fluid collected thru well-
(3) Pour off the clear broth, and add 1.0%
wetted, thick filter paper.
peptone and 0.5% NaCl.
(10) Add to the filtrate, peptone 10.0 g.
(11) Bring the volume up to 1000.0 cc.
(5) Grind brain, using the coarse grinder
by the addition of water.
and cook at 3 pounds pressure for
(12) Adjust the reaction.
about one and one half hours.
(6) Fill flasks f full with mixture of brain
(14) Filter, while hot, thru well-wetted,
tissue and liver broth (amounts of
thick filter paper.
each not given).
(15) Dissolve 10.0 g. glucose and 1.0 g.
K.HPO^ in the filtrate.
sure for three hours.
(16) Harvey further modified the me-
Use: Cultivation of B. chauvoei and
dium in that he added 1.0 cc. of
streptococci.
defibrinated rabbit blood to each
Variants
10.0 cc. of the medium.
(a) Gross, Barabin and Haines prepared
the medium as follows:
(b) Klimmer prepared a medium as
(1) Grind beef liver and brain sepa-
follows:
rately.
(1) Boil finely chopped liver with a
(2) To ground liver add 1000.0 cc.
double amount of water.
water.
(2) Add 1.0% peptone and 0.5% NaCl Cook ground brain and
(3) (2) in flow-
to the juice of (1).
ing steam for 1 hour.
(3) Neutralize to phenolphthalein by
(4) Strain liver broth thru cheese cloth
the addition of NaOH.
and cotton.
(4) Boil.
(5) Add peptone (1.0%) and NaCl
(5) Filter.
(0.5%).
(6) Add 2.0% glucose.
Cut the boiled liver in pieces (6) Titrate to pH = 8.2.
(7)
1-2 ccm. (7) Tube, two parts (6) to one part
(Method not given). brain.

(8) Sterilize (7)
When ready for use, add about 4 (8) Autoclave for one hour at 15 pounds
(9)
pieces of (8) to a series of tubes and pressure.
add (6) until the pieces of liver are (b) Long and Cornwell attempted to cul-
covered to a depth of 3 cm. tivate streptococci from a measles
(10) Sterilize (method not given).

patient on a medium prepared as
(11) Add a 2 cm. layer of sterile paraffin follows:
to each tube after inoculation. (1) Heat 500.0 g. of ground fat free beef
liver in 1000.0 cc. of water in flowing (8) HgS and

Filter off the precipitated
steam two hours.
for free the heavily
pigmented filtrate
(2) Add 10.0 g. peptone and 5.0
g. NaCl. from Ba with H2SO4. Dilute to
(3) Adjust to pH = 8.2. 100.0 cc.
(4) Strain through gauze. (9) Dissolve 4, 5, 6, 7, 8 and 9 in 1000.0 cc.
(5) Free beef brain from blood and of water.
membrane and make a mixture of (10) Make the following mixture:
medium consistency of 500.0 g. Decolorized infusion
brain and 200 to 300.0 cc. water. (2) 25.0 cc.
(6) Heat (5) in flowing steam for two Glucose salt solution
hours. (9) 25.0 cc.
(7) Mix 120.0 cc. of (4) with 60.0 cc. HgS04 precipitate frac-
of (6). tion (8) 0.25 or 2.5 cc.
(8) Final reaction, following sterilizing (11) To the remainder of the precipitate
pH = 7.6. fraction add 10.0 cc. of a 10.0% solu-
Autoclave at 15 pounds pressure for
(9) tion of HgS04 and allow to stand
one hour. over night.
References: Haslam (1920 p. 540), Goss, (12) Filter on Buchner funnel.
Barabin and Haines (1921 p. 615), Long (13) Free precipitate and filtrate from
andCornwell (1927 p. 409). H2SO4 and Hg by making slightly
alkaline with Ba(0H)2 and bubbling
835. Mueller's Heart Infusion Aminoid in H2S.
Solution (14) Free from Ba with H2SO4 and dilute
to 100.0 cc.
Constituents :
1. Water 600.0 cc.
(15) Make the following mixtures:
Decolorized infu-
2. Heart, beef 1.0 lb.
sion (2) 25.0 cc.
3. Aminoids 10.0 g.
Glucose salt solu-
4. NaCl 10.0 g. (a)
tion (9) 25.0 cc.
5. MgS04 0.4 g.
]
6. CaClz 0.2 g.
HgS04 filtrate frac-
[tion (14) 0.5 or 1.0 cc.
7. K2HPO4 2.0 g.
8. Glucose 2.0 g. Decolorized infu-
9. Phenol red (0.02% soln.) .... 80.0 cc. sion (2) 25.0 cc.
Preparation Glucose salt solu-
(1) Prepare a beef heart infusion by tion (9) 25.0 cc.
(b)
heating 1 pound of chopped beef HgS04 filtrate frac-
heart with 500.0 cc. of water to the tion (14) 1.0 cc.
boiling point. Strain and filter. HgS04 precipitate

(2) Decolorize by boiling for 25 minutes Infraction (8) 1.0 cc.
with 10.0% "Norit" a commercial Decolorized infu-

grade of wood charcoal. sion (2) 25.0 cc.
(3) Filter thru paper. Glucose salt solu-
(4) Dissolve 10.0 g. aminoids in 100.0 cc. tion (9) 25.0 cc.
(c)
of water. HgS04 filtrate frac-
(5) Precipitate aminoids from (4) with tion (14) 0.5 or 1.0 cc,
10.0% solution of HgSOi
100.0 cc. of HgS04 precipitate
in 5.0% H2SO4. fraction (8) 0.5 or 1.0 cc.
(6) Allow to stand over night and filter (16) Bring (10) and (15) (a), (b), (c) to
on a Buchner funnel and wash with pH of 7.4 to 7.8. Filter if necessary.
water. Sterilization: Tube and sterilize at 10
(7) Suspend filtrate in water, make pounds pressure for 10 minutes.
slightly alkaline with Ba(0H)2 and Use: To study food requirements of strep-
decomposed with H2S with warming. tococci and pneumococci. Aminoids are
an enzyme digest of milk to the amino 837. Huntoon's Hormone Heart Infusion
acid stage. Author reported no growth Broth
in medium (15b).
Constituents
Reference: Mueller (1922 p. 331).
Water
1. 1000.0 cc.
2. Heart, beef 500.0 g.
836. Mueller's Heart Infusion Peptone 3. Peptone (Bacto) 10.0 g.
Solutions 4. Gelatin 10.0 g.
5. Salt 5.0 g.
Constituents
6. Whole egg 1
Water (tap) 500.0 cc
7. Glucose 1.5 g.
Heart, beef 1.0 lb
8. Laked blood
Peptone (Difco) 10.0
Preparation :
NaCl 10.0
(1) Mix Bacto peptone, 10.0 g.
10.0 g.
MgS04 0.4
gelatin, 5.0 g. NaCl, one whole egg
6. CaCl, 0.2
and 500.0 g. of finely chopped beef
7. K0HPO4 2.0 g.
heart in a liter of water. Place in an
8. Glucose 2.0 g.
enamel ware vessel or a large coffee
9. Phenol red (0.02% soln.) .... 80.0 cc.
pot.
Preparation
(2) Heat over a free flame with constant
(1) Prepare beef heart infusion by heat-
stirring until the red color of the
ing 1 pound of chopped beef heart
meat infusion changes to brown at a
with 500.0 cc. of water to the boiling
temperature of about 68C. Do not
point. Strain and filter.
go beyond this temperature.
(2) Decolorize by boiling for 25 minutes
(3) Adjust to slightly alkaline to litmus
with 10.0% "Norit" a commercial
with N/1 NaOH and then add 1.0 cc.
grade of wood charcoal.
per liter of medium.
(4) Cover the vessel and place in an
(4) Make up one of the following media:
Arnold sterilizer or in a water bath
Decolorized infu-
at 100 for one hour.
sion 25.0 cc.
Glucose salt solu-
(5) Remove the vessel from the sterilizer
(a)
and separate with a glass rod the firm
tion 25.0 cc.
clot which has formed from the side
Peptone (Difco) .... 0.0 or 0.5 g.
of the vessel.
Infusion (not de-
(6) Return to the Arnold sterilizer at
colorized) 25.0 cc.
(b) Glucose salt solu-
100 for U hours.
(7) Remove the vessel and allow to stand
tion 25.0 cc.
at room temperature for about 10
Peptone 0.0 or 0.5 g.
minutes in a slightly inclined posi-
'Water 25.0 cc. tion.
Glucose salt solu-
(8) Pipette off the fluid portion or
(c)
jtion 25.0 cc. decant. If it is poured thru a fine
[Peptone 0.5 g. wire sieve, many of the fine pieces
(5) Bring each lot to pH = 7.4 to 7.8. of meat clot may be caught. (Avoid
(6) Filter if necessary. filtering thru cheese cloth, cotton
(7) Tube. or other adsorptive materials.)
Sterilization: Sterilize at 10 pounds pres- (9) Allow (8) to stand in tall cylinders
sure for ten minutes. for 15 to 20 minutes until the fat
Use: To study food requirements of strep- present has risen to the surface and
tococci and pneumococci. Author re- removed.
ported that no growth in (3) (a) occurred (10) The medium may be further cleared
without peptone, no growth in (3) (c). by filtering thru glass wool, asbestos
All others gave growth. wool, sedimentation or centrifu-
Reference: Mueller (1922 p. 325). gation.
(11) Add 0.15% dextrose and enough (5) Allow to stand at room temperature
laked blood to give a slight pink over night.
tint. Sterilization: Not specified.
(12) Tube in 10.0 cc. lots. Use: Isolation of gonococci.
by the intermittent
Sterilization: Sterilize Reference: Macnoughton (1923 p. 297).
method.
839. Ficker and Hoffmann's Caffeine
Use: To cultivate highly pathogenic organ-
Infusion Broth
isms. Author reported that if spinal
fluid be taken from a meningitis case in Constituents:
which no organism can be found in 1. Distilled water 4000.0 cc.
smears, a distinct growth appeared in 2. Beef 1000.0 g.
this medium after 9 hours rendering a 3. Peptone (Witte) (6.2%) .... 120.0 g.
positive diagnosis possible. Organisms 4. NaCl (0.5%) 10.0 g.
multiplied in the leukocytes transferred. 5. Caffein (0.6%) 24.0 g.
Variants 6. Crystal violet (0.1%)
(a) Author used fresh beef steak instead (solution) 28.0 cc.
of heart. Preparation
(b) Bailey prepared a similar medium as (1) Pour 2 liters of distilled water over
follows: 1000.0 g. finely chopped lean beef
(1) Dissolve 10.0 g. gelatin in 1000.0 cc. in an enamel kettle.
distilled water and heat to 50 to (2) Weigh the kettle and contents.
60C. (3) Heat at 50 to 55C. for 30 minutes.
(2) Add 500.0 g. of moderately fine (4) Stir with a glass rod and heat to
chopped beef to (1). boiling.
(3) Bring to a boil and cook slowly for (5) Weigh and add distilled water to
15 to 20 minutes. make up the loss in weight.
(4) Filter thru a 16 mesh (cullender (6) Filter thru filter gauze.
type) until clear. (7) Measure and add 6.0% (not given as
(5) Add 10.0 g. peptone and 5.0 g. 0.6%) Witte peptone and 0.5% NaCl.
NaCl. (8) Heat until the peptone is dissolved.
(6) Boil 5 minutes. (9) Filter.
(7) Adjust to the desired reaction (pH (10) Distribute in Erlenmeyer or beer
= 7.5). flasks.
(8) Allow to stand several minutes (11) Provide each flask with an absorbent
and decant the supernatant fluid. paper cap.
(9) Tube. (12) Prepare a 1.2% caffeine solution in
(10) Sterilize fractionally or at 5 sterile distilled water. Shake, but
pounds pressure for 5 minutes. heating is not necessary to obtain
Reference: Huntoon (1918 p. 172). Bailey complete solution. Weigh the caf-
(1925 p. 341). feine on a chemical balance.
(13) Measure 100.0 cc. of sterile (11) into
838. Macnoughton's Blood Infusion Broth
a sterile Erlenmeyer flask and adjust
Constituents to 2.7 to phenolphthalein.
1. Heart infusion broth. (14) Measure 105 cc. of (12) in a sterile
2. Blood. measuring flask and add to each
Preparation 100.0 cc. of cool (13) under aseptic
(1 Prepare infusion broth using ox heart, conditions. Do not heat after the
and adjust to pH between 7.4 and 7.5. addition of caffeine.
(2) Tube in 10.0 cc. lots. (15) Prepare a 0.1% crystal violet solu-
(3) The evening before the medium is re- tion (use chemical balance) in sterile
quired, add 1.0 cc. of sterile human distilled cold water.
blood to each 10.0 cc. of (2). (16) Add 1.4 cc. of (15) by means of a
(4) Thoroughly mix by rolling the tubes sterile pipette to each flask (14).
between the hands. Sterilization: Sterilize (1) in streaming
steam for 2 hours. Sterilize (13) in (2) Dissolve 2, 3, 4 and 5 in (1).

streaming steam for 10 minutes. (3) Adjust the reaction to pH = 7.2.
Use: Diagnosis of typhoid. Enrichment Sterilization: Method not given.
medium for typhoid. Emulsions were Use: To show hydrolysis of sodium hippu-
made from the patients' feces and rate by haemolytic streptococci. When
smeared, following 13 hours' incubation, more glucose was added, and phosphate
on this medium. omitted, allowing the hydrogen ion con-
Variants centration to increase, the streptococci
(a) Lubenau used 0.3% caffeine instead continued to hydrolyse the sodium hip-
of 0.6% and plated on solid medium. purate. The development of acidity had
(See medium 1749.) little effect.
(b) Abel gave the following method of Variants: The authors used 2.0 to 5.0 g.
preparation: glucose with or without 10.0 g. of K2HPO4.
(1) Chop 500.0 g. of fat free meat and Reference: Ayers and Rupp (1922 p. 391).
add to a liter of water at 50C.
(2) Keep at 50C. for 30 minutes and 841. MacConkey's Bile Salt Infusion Broth
boil for 30 to 45 minutes. (Heinemann)
(3) Filter or strain the fluid from the
Constituents:
meat.
1. Infusion broth 100.0 cc.
(4) Make up the fluid to one liter.
2. Sodium taurocholate (0.5%). 0.5 g.
(5) Add sufficient NaOH so that the
3. Peptone (2.0%) 2.0 g.
reaction is alkaline to phenol-
4. Glucose (0.5%) 0.5 g.
phthalein.
5. Litmus.
(6) Sterilize for 10 minutes in the
Preparation
steamer.
(1) Prepare infusion broth.
(7) Add 1050.0 cc. of a 1.2% caffeine
(2) Dissolve 2, 3 and 4 in (1) by heating,
solution and 14.0 cc. of a 0.1%
(3) Filter.
crystal violet solution (both the
caffeine and dye are dissolved in (4) Add sufficient litmus solution to
color.
sterile cold water).
Add the stools directly to this
(8)
medium.
Use: Culture medium used in water
analysis.
(c) Harvey used infusion broth prepared
Reference: Heinemann (1905 p. 129).
according to variant (bb) 779, ad-
justed permanently alkaline to
to
841a. Smith's Glucose Infusion Broth
phenolphthalein and mixed with
equal parts of a 1.0% caffeine solu- Constituents
tion. No crystal violet was em- Infusion broth 1000.0 cc.
ployed. Peptone, Witte 20.0 g.
References: Ficker and Hoffmann (1904 NaCl 5.0 g.
p. 255), Lubenau (1907 p. 248), Abel (1912 Glucose 1 .0 g.
p. 131), Harvey (1921-22 p. 92), Klimmer Preparation

(1923 p. 216). (1) Prepare infusion from beef in the
usual manner.
840. Ayers and Rupp's Hippurate Infusion Add Na2C03, normal
(2) solution, to
Broth
adjust to 1.5 to 2.0% acid.
Constituents (3) Heat to 40C. and inoculate with
1. Meat infusion 1000.0 cc. 30-50.0 cc. of a 12 to 14 hour bouillon
2. Peptone (Park Davis) 10.0 g. culture of B. coli and incubate for
3. Sodium hippurate 10.0 g. 16 hours or over night.
4. Glucose 2.0 g. (4) Mix with the white of egg, one egg
5. K2HPO4 1.5 g. per liter.
Preparation (5) Boil in a water bath or in an Arnold

(1) Method of preparation of meat infu- sterilizer for 45 to 60 minutes.
sion not given. Cool and add 2.0% Witte peptone,
and 0.5% NaCl. Dissolve by gentle (b) Copeland and Boynton used a me-
heat. dium prepared as follows to study
(7) Add N/1 NaaCOa until 0.8% acidity the Voges-Proskauer reaction by the
is reached. colon group.
(8) Boil or steam 20 or 30 minutes. (1) Prepare an infusion from fresh
(9) Filter. beef steak.
(10) Distribute into Fernbach flasks in (2) Dissolve 10.0 g. Witte's peptone,
layers 2.5 ctm. deep. Each flask 5.0 g. NaCl and 10.0 g. of water-
should have two or three cotton- free glucose in (1).
plugged openings. (3) Adjust to 1.0% acid to phenol-
(11) Add 0.1% glucose to each sterile flask phthalein.
of (10) from a sterile 20.0% glucose (4) Sterilization not specified.
solution. (c) Avery and Cullen prepared a medium
Sterilization: Sterilize (10) by heating in as follows:
the autoclave at 110 to 115 for 30 (1) Prepare a beef infusion.
minutes. Method of sterilization of glu- (2) Dissolve 10.0 g. peptone, 10.0 g.
cose solution not specified. glucose and 5.0 g. NaCl in (1).
Use: Cultivation of B. diphtheriae and (3) Adjust to pH = 7.6 to 7.8.
toxin production. Author reported that (4)Tube and sterilize.

the medium may be regarded as having They reported that in this medium
the maximum toxicity when reaction is the bovine type of Streptococcus
distinctly alkaline to phenolphthalein. haemolyticus gave a final pH of 4.3
Other investigators used a similar me- to 4.5 while a human type gave a final
dium for different purposes. pH of 5.0 to 5.3.
Variants References: Smith (1899 p. 375), Migula
(a) Migula prepared a medium as follows: (1901 p. 19), Copeland and Boynton (1905
(1) Mix 500.0 g. of finely chopped lean p. 242), Avery and Cullen (1919 p. 218).
beef with one liter of water and
842. Mueller's Meat Infusion Broth
allow to stand in the ice box for
12 to 24 hours. Constituents
(2) Press the liquid thru a towel and 1. Water 1000.0 cc.
make up the volume to 1 liter. 2. NaCl 10.0 g.
(3) Boil in the steam cooker for 30 3. MgS04 0.4 g.

minutes. 4. CaCh 0.2 g.
(4) The infusion may be boiled for an 5. K2HPO4 2.0 g.
hour before removing the meat 6. Glucose 2.0 g.

and then filtered thru paper. If 7. Phenol red (0.02% soln.) ... 80.0 cc.
the liquid is still red, boil again 8. Meat infusion 1000.0 cc.
for 15 minutes. 9. Peptone 20.0 g.
(5) Filter whey cold to remove the Preparation

fat. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
(6) Add g. Witte peptone and

10.0 (2) Mix equal parts (1) and meat infu-
5.0 g.NaCl. sion, and add 1.0% peptone. (The
(7) Neutralize by the addition of a author added no peptone.)
concentrated solution of Na2C03 (3) Adjust to pH = 7.8. (No peptone
until litmus is colored violet. medium pH = 7.6.)
Add the desired amount of soda. Sterilization: Sterilize in autoclave at 10

(8)
Generally, 10 cc. of 15.0% soda pounds for 10 minutes.
solution is added per liter. Use: To study the food requirements for
(9) Boil and filter. the growth of streptococci and pneumo-
(10) Add 1.0 or 2.0% glucose. cocci. The author reported that in the
(11) Distribute in tubes or flask. above medium the peptone had little
(12) Boil for one hour to sterilize. influence on the growth. Using the media
given under variants, all media not con- Variants: Authors adjusted the reaction to
taining peptone gave poor growth. + 1.0 to phenolphthalein instead of +0.6,
Variants: The author gave the following M.L.D. = 0.005 cc.

method of preparing different media: Reference: Robinson and Rettger (1917
(1) Soak one pound of meat with 500.0 cc. p. 363)
of cold water, heat to 55 for a few
minutes and strain. Boil this extract 844. Bronstein and Griinblatt's Indicator
to remove coagulable materials and Glucose Infusion Broth
filter. This is Extract I.
Constituents
(2) Take meat from extract I, add
1. Meat infusion broth 1000.0 cc.
500.0 cc. H2O, boil 5 minutes, strain
2. Glucose (0.5%) 5.0 g.
and filter. This is Extract II.
3. Indigo carmine solution.
(3) Take meat from extract II, add
4. Fuchsin solution (acid).
500.0 cc. of H2O, boil 5 minutes,
Preparation
strain and filter. This is Extact III.
(1) Prepare a neutral 0.5% glucose meat
(4) Divide each e.xtract into two equal
infusion broth.
parts.
(5) Mix each of these two parts with an
(3) Prepare a 2.0% watery solution of
equal amount of glucose salt solution
indigo carmine.
(see step (1) in the medium).
(4) Dissolve 10.0 g. of acid fuchsin in
(6) Add 2.0% peptone to one of these
parts, and make no addition to the
100.0 cc. of 1.0% KOH solution.
(5) Mix 2 parts (3) with 1 part (4) and
other.
add to 22 parts distilled water.
(7) Adjust to pH = 7.8 when containing
peptone and pH = 8.0 when not con-
Use: Differentiation of diphtheria organ-
taining peptone.
isms. After 24 hours incubation add 3
Reference: Mueller (1922 pp. 316, 320, 321).
drops of (5) to each tube. The authors
843. Robinson and Rettger's Glucose reported that the control tube was blue;
Infusion Broth diphtheria cultures were ruby-red; pseudo
diphtheria cultures were green after
Constituents:
several minutes.
1. Water 1000.0 cc.
Reference: Kolle and Wasserman (1912
2. Lean beef 1.0 lb.
p. 414).
4. Glucose 1.0 g.
845. Ayers, Rupp and Johnson's Glucose
Preparation
Infusion Broth
(1) Extract 1.0 lb. of chopped lean beef
in one liter of water in the cold for Constituents
24 hours. 1. Meat infusion 1000.0 cc.
(2) Coagulate on a boiling water bath. 2. Peptone (Park Davis & Co.) 10.0 g.
(3) Filter and add 2.0% Witte's peptone 3. Glucose 5.0 g.

to the filtrate. 4. Sodium ricinolate (Castor oil soap).
(4) Heat on the water bath for about 20 Preparation
minutes. (1) Prepare 1000.0 cc. of meat infusion.
(5) Adjust the reaction to +0.6 to phenol- (2) Add 2 and 3 to (1).
phthalein. (3) Adjust reaction to pH = 7.5.
(6) Filter and distribute into 150.0 cc. (4) Add castor oil soap (sodium ricino-
Erlenmeyer flasks in 20.0 cc. lots. late) or other surface reducing ma-
(7) After sterilization add enough sterile terial in sufficient amounts to lower
glucose to each flask to make 0.1% the surface tension to about 50 and
glucose. 43 dynes.
Sterilization: Sterilize at 12 to 14 pounds Sterilization: Method not given.
pressure for 15 minutes. Use: To study effect of surface tension on
Use: Cultivation of B. diphtheriae and growth of streptococci. Authors re-
toxin production. ported that in general when the surface
tension was reduced to from 40 to 41 dynes Preparation

the growth of streptococci was prevented. (1) Prepare meat infusion broth.
Reference: Ayers, Rupp and Johnson (1923 (2) Add 10.0 g. glucose to 1.
p. 204). (3) Add acetic acid to (2) until the reac-

tion is 0.2, 0.1 or 0.05 normal acetic
846. Ayers, Rupp and Mudge's Glucose acid, using phenolphthalein as an
Infusion Broth indicator.
Constituents (4) Tube in 10.0 cc. lots.

(5) Plug with paraffined cotton.
1. Meat infusion 1000.0 cc.
2. Peptone (Bacto) 10.0 g.
Use: To study fecal flora.
3. K2HPO4 10.0 g.
Reference: Torrey (1915 p. 74).
4. Glucose 10.0 g.
Preparation 849. Sherman's Lactose Infusion Broth
(1) Prepare 1000.0 cc. meat infusion.
Constituents
(2) Add 2, 3 and 4 to (1).
1. Beef infusion 1000.0 cc.
2. Peptone 30.0 g.
(4) Tube in 15 cc. lots in Eldredge tubes.
3. Na2HP04 10.0 g.
Use: To study fermentation of glucose by

4. KH0PO4 5.0 g.
5. Lactose 20.0 g.
streptococci.
Preparation
Reference: Ayers, Rupp and Mudge (1921
(1) Prepare 1000.0 cc. beef infusion.
p. 258).
(2) Dissolve 2, 3, 4 and 5 in (1).
847. Torrey's Brilliant Green Infusion
Use: Show CO2 production in highly
Broth
buffered medium by Streptococcus kefir,
Constituents Author reported that in this buffered
1. Meat infusion broth 1000.0 cc. solution CO2 production approached that
2. Glucose 10.0 g. in digested milk.
3. Brilliant green (1.0% aq. Reference: Sherman (1921 p. 130).
soln.) 15.0 cc.
Preparation 850. Harvey's Starch Infusion Broth
(1) Prepare meat infusion broth in the
Constituents:
usual manner.
1. Water 1000.0 cc.
(2) Neutralize to phenolphthalein. The
2. Beef 500.0 g.
final reaction must be neutral.
3. NaCl 10.0 g.
(3) Dissolve 10.0 g. glucose in (2).
4. Starch (Corn flour) 10.0 g.
(4) Tube in e.xactly 10.0 cc. quantities.
Peptone
5. 10.0 g.
(5) Prepare a 1.0% solution of Griibler's
6. Litmus solution.
brilliant green in distilled water.
Preparation
(6) Add 0.15 cc. of (5) to each sterile tube Mince finely fat-free beef.
(1)
of (4).
Note: Veal, chicken, ox or horse
Sterilization: Sterilize in the Arnold
heart, horse flesh, rabbit flesh, fish,
steamer. Time not specified.
blood, placenta, liver, spleen, kid-
Use: Enrichment paratyphoid-
of the
neys, brain and vegetable materials,
enteritidis group. Author reported that
such as yeast, wheat, etc., may serve
other dominant fecal bacteria were in-
to furnish the extract used as basis
hibited by the dye.
for medium.
water or clear tap water.
848. Torrey's Acetic Acid Infusion Broth
(3) Heat the mixture 20 minutes over a
Constituents: free flame, at a temperature not ex-
1. Meat infusion broth 1000.0 cc. ceeding 50C.
2. Glucose 10.0 g. Note: Or simply keep in a cool
3. Acetic acid. place over night.
(4) Skim off fat floating on the surface, Use: Enrichment of the colon-typhoid
(5) Raise the temperature to boiling group.
point. Variants: Klimmer prepared the medium
(6) Boil 10 minutes. as follows:
(7) Pour the mixture on to a wet, thick, (1) Dissolve 2, 3, 4, 5, 6 and 7 in meat
clean cloth. infusion broth.
(8) Add sodium chloride 5.0 g. to the (2) Flask in 100.0 cc. quantities.
filtrate. (3) Sterilize on each of 3 successive days
(9) Steam 45 minutes. for 10 minutes in streaming steam.
(10) Bring the volume up to 1000.0 cc. (4) Add 3.0 cc. of a 2.0% solution of 120
by the addition of water. Hochst malachite green to each
. (11) Estimate and adjust the reaction, 100.0 cc. lot of sterile (2).
(12) Steam for 30 minutes. (5) Tube in 3.0 or 4.0 cc. lots in sterile
(13) Filter while hot thru well-wetted, tubes.
thick filter paper. References: Loeffler (1906 pp. 289, 295),
(14) Make a paste of 10.0 g. of corn flour Klimmer (1923 p. 213).
starch and 200.0 cc. of (13).
(15) Heat 20 minutes at 20 pounds pres-
852. Duval and Lewis' Inulin Bouillon
sure. Constituents
(16) Add 10.0 g. peptone, 5.0 g. NaCl 1. Meat infusion broth 1000.0 cc.
and 800.0 cc. of (13) to (15). 2. Inulin 10.0 g.
(17) Steam 45 minutes. Preparation
(18) Make the reaction 0.2% acid to (1) Prepare boiullon according to stand-
phenolphthalein. ard methods from beef. (Method or
(19) Steam 30 minutes. reference not given.)
(20) Filter while hot thru well-wetted, (2) Adjust from 0.2 to 0.4% normal to
thick filter paper. phenolphthalein. After sterilization
(21) Add
saturated litmus solution to reaction from 0.5 to 0.8%,
make a deep
blue color. (3) Distribute into clean tubes in 9.0 cc.
(22) Distribute into test tubes. lots.
Sterilization: Sterilize at 100C, for 20 Prepare a 10.0% inulin solution in
(4)
minutes on 3 days. distilled water.
Use: Cultivation of meningococci. (5) Add 1.0 cc. of sterile (4) to each 9.0 cc.
Reference: Harvey (1921-22 p. 112), of sterile (3).
Sterilization: Sterilize (3) method not
851. Loeffler's Malachite Green Infusion
given. Sterilize (4) in the autoclave at
Broth
15 pounds pressure for 15 minutes.
Constituents Use: Cultivation of pneumococci.
1. Beef infusion 1000.0 cc. Reference: Duval and Lewis (1905 p. 484).
2. Peptone (2.0%) 20.0 g,
Lactose (5.0%)
853. Bulir's Mannitol Infusion Broth
3. 50.0 g.
4. Dextrose (1.0%) 10.0 g. Constituents
5. Sodium sulfate (0.5%) 5.0 g. 1. Water 2000.0 cc.
6. KNO3 (2.0%) 20.0 g. 2. Beef 1000.0 g.
7. KNO2(1.0%) 10.0 g. 3. Peptone (Witte) 25.0 g.
8. Malachite green 2% solution 30.0 cc. 4. NaCl 15.0 g.
Preparation 5. Mannite 30.0 g.
(1) Prepare a beef infusion from one 6. Neutral red.
pound of beef. Preparation
(2) Dissolve 2, 3,t, 5, 6 and 7 in (1). (1) Macerate 1000.0 g. finely chopped
(3) Add 3.0% of 2.0% malachite green lean beef with 2000.0 cc. water for
solution to (2), 24 hours.
Sterilization: Sterilizeon each of 3 suc- (2) Filter thru linen, and press the meat
cessive days for 10 minutes in streaming free from water.
steam. (3) To the liter of meat infusion thus
obtained add 25.0 g. Witte's peptone, 855. Harvey's Lead Acetate Infusion
15.0 g. NaCl and 30.0 g. mannite. Broth
(4) Boil for 1.5 hours over a flame. Constituents
(5) Neutralize by the addition of soda. 1. Water 1000.0 cc.
(6) Filter. 2. Peptone 10.0 g.
(7) Dissolve 0.1 g. neutral red in 100.0 g. 3. Beef 500.0 g.
distilled water. 4. NaCl 5.0 g.
(8) When ready for use add 3.0 cc. of 5. Lead acetate 1.0 g.
sterile (7) to each 150.0 cc. of the Preparation
medium. (1) Prepare meat infusion broth accord-
(9) Distribute in 15 to 20.0 cc. lots in ing to Harvey's method, see variant
fermentation tubes. (bb) 665.
Sterilization: Sterilize streaming
(6) in (2) Add 1.0 g. of lead acetate to (1).
steam. Sterilize (7) method not given. Sterilization: Not specified.
Use: Detection of Bacterium coli in water. Use: General culture medium.
When desired for use, add 100.0 cc. of the Reference: Harvey (1921-22 p. 107).
water being investigated to 50.0 cc. of
sterile (6). Then add 3.0 cc. of neutral 856. Omeliansky's Formate Infusion Broth
red solution as indicated in step (8). Constituents
The author reported that after 12 to 24 1. Meat infusion broth 1000.0 cc.
hours incubation at 46C. that the pres- 2. Sodium formate 5.0 g.
ence of Bad. coli was indicated by a 3. Phenolphthalein drops
uniform turbidity in the fermentation Preparation
tube, the formation of gas, and a change (1) Prepare meat infusion broth from
in color from a red to a yellow-greenish meat and peptone.
fluorescence. (2) Add 0.5% sodium formate to (1).
Variants: Klimmer added 25.0 g. Witte's (3) Add several drops of phenolphthalein.
peptone, 15.0 g. NaCl and 30.0 g. man- Sterilization: Not specified.
nitol in a liter of meat infusion, boiled Use: Differentiation of colon typhoid
1.5 hours, neutralized, tubed and steril- group. Author reported that Bad. typhi
ized in streaming steam for 2 hours. The abdom. did not decompose the formate.
addition of neutral red or test water not Bad. coli commune decomposed the
specified. formate with the production of gas.
References: Bulir (1907 p. 11), Klimmer Bad. paratyphi A and B decomposed the
(1923 p. 216). formate with production gas. Bact.
dysenteriae and Bac. faecalis alcaligenes
854. Harvey's Ferric Tartrate Infusion did not decompose the formate. Some
Broth of the organisms not producing gas may
turn the phenolphthalein red due to the
Constituents: decomposition of some albuminous ma-
1. Water 1000.0 cc.
terial to amino acids or other compounds.
2. Beef 500.0 g.
Variants: The author omitted the phenol-
3. Peptone 10.0 g.
phthalein.
4. NaCl 5.0 g.
Reference: Omeliansky (1905 p. 674, 1906-07
5. Ferric tartrate 1.0 g.
p. 158).
Preparation
(1) Prepare infusion broth according to 857. Jordan's Phenol Infusion Broth
Harvey's method, see variant (bb) Constituents:
665. 1. Infusion broth 900.0 cc,
(2) Add 1.0 g. of ferric tartrate to a liter 2. Phenol (1.0% soln.) 100.0 cc.
of (1). Preparation
Sterilization: Not specified. (1) Prepare infusion broth according to
Use: General culture medium. standard methods (see committee
Reference: Harvey (1921-22 p. 107). A. P. H. A. 1899, 665).
(2) Neutralize to phenolphthalein and cated above, to 5 parts meat infusion

then add 5.5 cc. ofnormal acid per broth.
liter. Reference: Harvey (1921-22 p. 86).
(3) Distribute (1) in 9.0 cc. lots in test
tubes. 859. Olitsky and Kligler's Egg White
(4) Add 1.0 cc. of a 1.0% aqueous solution Infusion Broth
of carbolic acid.
Constituents
Sterilization: Sterilize in the Arnold steri-
lizer (time not given).
Use: Enrichment medium in water analysis.
3. Egg white 10.0 cc.
Variants
Preparation:
(a) Harvey added phenol
1.0 to 5.0 g. of
(1) Prepare plain meat infusion broth
to infusion broth prepared according
(method not given).
to Harvey (see variant (bb) 665).
(2) Add one volume of egg white to 9
(b) Harvey added 4.0 cc. of strong HCl
volumes of distilled water.
to 100.0 cc. of 5.0% phenol solution
(3) Mix 300.0 cc. of (1) and 100.0 cc. of
and added 0.1, 0.2 or 0.3 cc. of this
(ôf (2) by volume).
(2),
mixture to 10.0 cc. of sterile meat
(4) Adjust to pH" from 7.6 to 7.8.
infusion (see 665).
(5) Distribute in 500.0 cc. lots into 2 liter
Reference: Jordan (1901 p. 298), Harvey
flasks to permit sufficient aeration.
(1921-22 p. 90).
858. Harvey's Egg Infusion Broth 15 pounds pressure.
Use: Cultivation of Bacillus dysenteriae
Constituents
Shiga, and study of toxin production.
1. Water 2000.0 cc.
After 5 days incubation at 37 C. filter thru
2. Beef 500.0 g.
Berkefeld N candle. This bacteria free
3. Peptone 10.0 g.
filtrate constitutes the exotoxin.
4. NaCl 5.0 g.
Variants: Dopter and Sacquepee gave the
5. Whites of 4 eggs
following method of preparation:
6. Yolks of 2 eggs
(1) Place the whites of two eggs in a glass
7. NaOH (normal) '
12.0 cc.
graduate.
Preparation
(2) Shake constantly, adding little by
(1) Prepare meat infusion broth accord-
little three times their volume of dis-
ing to Harvey's method (see variant
tilled water.
(bb) 665).
Prepare an egg stock solution by (3) Add 2.0 cc. of 10.0% soda solution
(2)
(0.5 cc.per 100.0 cc). Mix well.
mixing the whites of 4 eggs, the yolks
of 2 eggs and 12.0 cc. normal NaOH.
(5) Prepare meat infusion peptone solu-
(3) Add 1000.0 cc. of water to (2).
tion (500.0 g. beef, 5.0 g. NaCl and
(4) Heat very slowly to 90 C.
20.0 g. peptone per liter).
(6) Mix one part (4) with 5 parts (5)
(6) Mix equal parts of (5) and sterile (1)
(7) Distribute.
when ready for use.
(8) Sterilize for 15 minutes at 112C.
Sterilization: Sterilization of (1) in the
autoclave or steamer. Sterilize (5) in the
860. Weiss and Wilkes-Weiss Egg
autoclave.
"Hormone" Broth
Variant: Harvey used a similar egg medium Constituents
by preparing the egg stock solution as 1. Hormone broth 400.0 cc.
indicated above, or by mixing the whites 2. Whole egg 100.0 cc.
of 4 eggs and 8.0 cc. of normal NaOH with Preparation: (1) Mix whole egg and "hor-
660.0 cc. of water and then diluting to a mone" broth in the ratio of 1:4.
liter. When ready for use add 1 part of Sterilization: Heat for 30 minutes in the
the egg stock solution, prepared as indi- Arnold sterilizer.
Spirochaeta pallida. 2. Saline solution.

Use: Cultivation of
The authors reported that the value of 3. Heart (beef).
the medium would be greatly improved Preparation
if a small quantity of agar be added (1) Chop beef heart into small pieces.
(2) Divide (1) into six equal portions.
(0.2%).
(3) Take up each portion of (2)
with
Reference: Weiss and Wilkes-Weiss (1924
9 volumes of saline solution.
p. 226).
(4) Treat each one of the suspensions
in
861. Kohman's Brain Infusion Medium one of the following ways:
(a) Keep in the ice box over night and
Constituents
1. Meat infusion broth steam in the Arnold sterilizer for
2. Brain. an hour.
Grind beef brain and add (b) Keep in the ice box over night and
Preparation: (1)
meat infusion broth to cover. filterthru a Berkefeld candle.
sufficient
Sterilization: Sterilize in the autoclave. (c) Keep at 55C. over night and heat
in the Arnold sterilizer for an hour.
Use: To cultivate meningococci and to
(d) Keep at 55C. over night and filter
determine the effect of oxygen tension
thru a Berkefeld candle.
on growth.
Reference: Kohman (1919 p. 577). (e) Extract by boiling one hour and
then steam for an hour in the
862. Kreidler's Glucose Brain Broth Arnold.
(f) Extract by boiling for one hour.
Constituents
(5) Take one of (4) and add
1.0 cc. of to
1. Water 1000.0 cc.
meat infusion broth.
sterile
2. Beef 500.0 g.
3. Peptone (Difco) 10.0 g.
Use: To study the effect of growth stimu-
4. NaCl 5.0 g.
lating materials on the growth of patho-
5. Glucose (1.0%) 10.0 g.
genic bacteria. Author reported that
6. Brain.
heat destroyed the growth stimulating
Preparation
materials.
(1) Infuse 500.0 g. of finely chopped lean
Reference: Kligler (1919 p. 43).
beef in 1000.0 cc. water in an ice box
over night.
(2) Boil 30 minutes over a free flame. 864. Havens and Taylor's Kidney and Blood
Filter through gauze and then Infusion Broth
(3)
through paper to remove the fat.
Constituents
(4) Make up to 1000.0 cc. volume. 1000.0 cc.
and 1. Meat infusion
(5) Add 10.0 g. peptone (Difco)
10.0 g.
2. Peptone (1.0%)
5.0 g. NaCl.
3. Na2HP04 (1.0%) 10.0 g.
(6) Boil to dissolve.
4. Glucose (0.5%) 5.0 g.
5. Kidney (rabbit).
(8) Filter through paper.
6. Blood, defibrinated, (sheep or rabbit).
(9) Add 10.0 g. glucose.
Preparation
(10) Fill long tubes and add several pieces
(1) 3 and 4 to 1000.0 cc. of ordinary
Add 2,
of calf brain, one cubic centimeter in
meat infusion made with distilled
size, washed in running water.
water.
(2) Adjust to pH 8.0 to 8.2.
Tube in 10.0 cc. quantities.
Use: To study the bacteriology of endo- (3)
carditis. (4) Add a fragment of sterile rabbit
Reference: Kreidler (1926 p. 190). kidney and 1.0 cc. of defibrinated

sheep or rabbit blood to each tube
863. Kligler's Heart Infusion Solutions of sterile (3).
Constituents Sterilization: Sterilize (3) in the autoclave

at 10 pounds pressure for 10 minutes.
1. Meat infusion broth.
Use: Cultivation of hemolytic strepto- (6) Reduce the filtrate to 1.0% acid.
cocci. (7) Mix equal parts of sterile (6) and
Reference: Havens and Taylor (1921 sterile (1).
p. 313). Sterilization: Method of sterilization of (1)
not given. Sterilize (6) by repeated
865. Robertson's Cooked Meat Medium
heating at 65C. or by filtering.
(Torrey)
Use: Cultivation of Bacillus influenzae,
Constituents The author reported that better growth
1. Water 1000.0 cc. was obtained using double strength
2. Beef heart 500.0 g. broth.
3. Peptone 10.0 g. Variants: Author mixed (6) with equal
Preparation parts of double strength broth.
(1) Mix 500.0 g. finely minced fresh beef Reference: Wade and Manalang (1920
heart with 10.0 g. peptone and a liter p. 98).
of water.
867. Harvey's Ascitic Fluid Infusion Broth
(2) Cook in a double boiler with just
enough heat to cause a slight simmer- Constituents:
ing for about 10 minutes. 1. Water 1000.0 cc.
(3) Adjust to pH = 7.2. 2. Peptone 10.0 g.
(4) Continue to cook for 1| hours. 3. Beef 500.0 g.
(5) Readjust the reaction if necessary. 4. NaCl 5.0 g.
(6) Decant the broth. 5. Ascitic fluid 500.0 cc.
(7) Place the meat in test tubes to a Preparation
height of 5 cm. (1) Prepare meat infusion broth accord-
(8) Add 5.0 cc. of sterile broth to each ing to Harvey's method, (see variant
sterile tube of (7). (bb) 665).
Sterilization: Sterilize flasks of (6) and (2) Mix 2 parts (1) with one part ascitic
tubes of (7) separately in the autoclave fluid.
at 15 pounds pressure for 15 minutes. (3) Test sterility before use by incuba-
Following mixing, place the tubes in the tion 48 hours.
Arnold sterilizer on each of two successive Sterilization: Sterilize in the water bath
days for 20 minutes. 30 minutes at 56C., on each of five suc-
Use: Analysis of fecal flora. cessive days.
Reference: Torrey (1926 p. 355). Use: Cultivation of parasitic and saphro-
phytic bacteria.
866. Wade and Manalang's Blood Infusion
Broth
868. Stryker's Serum Infusion Broth
Constituents
1. Distilled water. Constituents
2. Meat infusion broth. 1. Meat infusion broth 900.0 cc.
3. Blood. 2. Serum 100.0 cc.
(1) The beef infusion broth contains (1) Prepare meat infusion broth.
Witte's peptone and NaCl in various (2) Mix 1 part highly potent anti-pneu-
concentrations. Exact amounts not mococcus horse serum with 9 parts
given. normal horse serum.
(2) Thoroly lake sheep or horse blood by (3) Mix 100.0 cc. of (2) with 900.0 cc.
adding 20.0 cc. blood to 100.0 cc. dis- of (1).
tilled water. Sterilization: Method not specified.
(3) Heat to 80 to 85C. Use: To study variation in pneumococcus.
(4) Precipitate the proteins while hot by Other investigators used similar media
adding strong hydrochloric acid. for different purposes.
(5) Filter first thru gauze, then thru Variants
paper. (a) The author used plain serum instead
of a mixture of immune and plain Preparation

serum. (1) Prepare beef infusion broth.
(b) Lucke and Rea cultivated strepto- (2) Adjust (1) to pH = 6.3.
cocci on a similar medium. They (3) Prepare (2) 10.0% glucose solution.
reported that typical streptococci (4) Mix (1) and (2).
gave a heavy white sediment consist- (5) Add sterile 3 to sterile (4) in suflB-
ing of albumin. Their medium was cient quantity to give a 1.0% con-
prepared as follows: centration of glucose.
(1) Prepare a meat infusion broth (2.0% Sterilization: Sterilize (3) method not
peptone). given. Sterilize (4) at 15 pounds pressure
(2) Boil 1000.0 CO. of (1) to 80.0% of its for 15 minutes.
original volume and adjust to 0.4+. Use: To study protein and carbohydrate
(3) Tube and sterilize for 3 days in metabolism of Streptococcus hemolyticus
steam. General culture medium.
(4) Obtain horse serum aseptically and Variants: Harvey cultivated meningococci
heat to 60C. for 1 hour in a water on a medium containing 1 part unheated
bath to destroy antihemolysins. clear sterile horse serum to 20 parts in-
(5) Add 2.0 cc. of (4) to each 8.0 cc. of fusion broth (see variant (bb) 770) to
(3). which was added 1.0% glucose.
(c) Stevens, Brady and West studied the References: Foster (1921 p. 222), Harvey
and hemol-
relation between virulence (1921-22 p. 80).
ysin. They reported that a strain
of streptococcus whose virulence had 870. Beach and Easting's Serum Infusion
been did not produce
increased and Extract Broth
greater concentrations of hemolysin.
Constituents:
Their medium was prepared as
1. Water 1000.0 cc.
follows Lean beef 450.0 g.
2.
(1) Prepare a meat infusion broth with 3. Peptone (Difco) 15.0 g.
2.0% peptone. 4. K2HPO4 7.5 g.
(2) Titrate (1) so that it is pH = 7.6
5. Liebig's beef extract 4.0 g.
after sterilization (method not
Glycerol 55.0 cc.
6.
mentioned). Aminoid peptone from beef
7.
(3) Distribute into 250.0 cc. Pyrex (Arlco) 5.0 g.
flasks in 80.0 cc. lots.
8. Serum (sterile blood) 100.0 cc.
(4) Add 20.0 cc. of fresh horse serum to
Preparation
each flask. with
lean beef
(1) Extract 450.0 g.
(5) Inactivate the contents at 56C. on 1000.0 cc. water for 3 hours. Bring
3 successive days, and store on ice
the water to 45C. After an hour
until ready for use.
raise the temperature to 50C. and
(d) Harvey mixed 2 parts serum with
during the third hour to 55C. Stir
1 part infusion broth, (see variant frequently.
(bb) 665). The mixture was steri-
Remove the major portion of the
(2)
lized for 60 minutes at 57C. on each liquid from the meat.
(3) Heat meat and remaining liquid to
References: Stryker (1916 p. 50), Lucke
100C. This causes the meat par-
and Rea (1919 p. 535), Stevens (1921
ticles to shrink giving the maximum
p. 224), Harvey (1921-22 p. 82). amount of liquid.
(4) To the liquid (2) plus that of (3)
869. Foster's Serum Infusion Broth add 10.0 g. peptone, 5.0 g. K2HPO4,
Constituents: 2.0 g. Liebig's beef extract and
1. Beef infusion broth 1000.0 cc. 50.0 cc. of glycerol.
2. Glucose 10.0 g. (5) Adjust the reaction to pH of 7.5
3. Horse serum broth 5.0 g. to 7.8.
(6) Distribute in 100.0 cc. lots in 872. Park, Williams and Krumwiede's
500.0 cc. Erlenmeyer flasks. Potato Infusion Broth
(7) Seed half the sterilized flasks with Constituents:
the grass bacillus of Karlensky and 1. Water 1000.0 cc.
the remainder with the glass bacillus 2. Meat infusion 1000.0 cc.
of Moeller. 3. Potato 1.0 lb.
(8) Incubate at 37.5C. for two weeks. 4. Peptone 20.0 g.
(9) Then heat to 100C.
5. NaCl 10.0 g.
(10) Cultures are then filtered and fil-
Preparation
trate made up to 1000.0 cc.
(1) Grate 1.0 pound of white potatoes
(11) Add 5.0 g. peptone, 2.5 g. K2HPO4, or run thru a chopping machine.
2.0 g. Liebig's beef extract, 50.0 cc. Soak in a liter of water over night.
(2)
glycerol and 5.0 g. of aminoid pep- Heat to boiling.
(3)
tone from beef (Arlco). Press thru cheese cloth.
(4)
(12) The reaction is left unchanged. (5) Add one egg per liter.
(13) Filter and distribute 50.0 cc. lots
(6) Autoclave one hour to clarify.
in 500.0 cc. Erlenmeyer flasks. (7) Filter thru cotton (very tedious).
(14) Add 5.0 cc. of sterile horse blood (8) Flask.
serum (serum must be raw) just (9) Soak 1 pound of finely chopped lean
before inoculation. beef in a liter of tap water over night
Sterilization: Sterilize (6) and (13) sepa- in the ice box or at room tem-
rately in the autoclave, time not given. perature.
Use: Preparation of johnin. Johnin is a (10) Weigh (9) and heat at 45C. for 1
material used for detection of Johnin's hour and then boil for 30 minutes.
disease, much as tuberculin is used for (11) Make up the loss in weight by the
tuberculosis. The medium was inocu- addition of hot water.
lated heavily with the organism causing (12) Strain thru cheese cloth and squeeze
Johnin's disease. Cover the flasks with by twisting the cloth or use a meat
tin foil to prevent evaporation and incu- press.
bate 10 to 16 weeks. (13) Mix equal parts of (8) and the juice
Reference: Beach and Hastings (1922 from (12).
p. 72). (14) Dissolve 20.0 g. peptone and 10.0 g.

NaCl in (13).
871. Harvey's Blood Infusion Broth (15) Distribute as desired.

Constituents 15 pounds pressure.
1. Water 1000.0 cc. Use: General culture medium.
2. Beef 500.0 g. Reference: Park, Williams and Krum-
3. NaCl 14.0 g. wiede (1924 p. 122).
4. CaCla 0.25 g.
5. KCl 0.42 g. 873. Lubinski's Potato Infusion Broth
6. Glucose 25.0 g. Constituents
7. Blood, citrated plasma. . . 200.0 cc. 1. Water 1500.0 cc.
Preparation 2. Potato 1000.0 g.
(1) Prepare meat infusion solution as 3. Meat 500.0 g.
indicated in medium 729. 4. Peptone 10.0 g.
(2) Dissolve 3, 4, 5 and 6 in 1. 5. NaCl 5.0 g.
(3) Tube in 5.0 cc. quantities. 6. Glycerol 40.0 g.
(4) Add 1.0 cc. of citrated humas plasma Preparation
to each tube of (3). (1) Wash potatoes clean and cut them
Sterilization: Method not given. in small pieces.
Use: Cultivation of gonococci. (2) Boil for 3 or 4 hours under a free
Reference:' Harvey (1921-22 p. 82). flame or in a steamer.
(3) Filter. (10) Add 3.0% glycerin and add a satu-

(4) To one liter of (3) add 500.0 g. finely rated solution of sodium carbonate
chopped meat. until the reaction is decidedly
(5) Allow to stand for 24 hours. alkaline.
(6) Strain. (11) Boil in the autoclave for i to i hour
(7) Add and and 5 in (6).
dissolve 4 at 118-120C., cool and filter.
(8) Boil to precipitate albuminous ma- (12) Distribute as desired.

terials. Sterilization: Sterilize at 115C. for ^ to
(9) Filter. 1 hour.
(10) Add 4.0% glycerin. Use: Cultivation of tubercle bacilli. Me-
(11) Neutralize (may be used without dium should be dark brown in color.
neutralization). Variants
Sterilization: Not specified. (a) The author used 0.25% HK2PO4
Use: Cultivation of tubercle bacilli. instead of NaCl.
Reference: Lubinski (1895 p. 126). (b) Kolle and Wesserman gave the fol-
lowing method of preparation:
874. Jurewitsch's Potato Infusion Broth
(1) Prepare a pulp from clean peeled
Constituents: potatoes.
1. Water 1500.0 to 2000.0 cc. (2) Add 1000.0 cc. of water to 500.0 g.
2. Potato 500.0 g. of (1) and allow to stand for one
3. Meat 500.0 g. day in the cold.
4. Peptone (Witte or (3) Shake thoroughly and filter
Chapoteaut) 0.5% through linen.
5. NaCl 0.25% (4) Allow to settle for 15 to 30 minutes
6. Glycerol 3.0% and decant the clear water.
Preparation (5) Soak 500.0 g. of lean meat in
(1) Peeled potatoes are cut into large 1000.0 cc. of water for 24 hours.
pieces, washed off with water and (6) Press thru linen.
pulverized. (7) Mix equal parts of (4) and (6).
(2) Weigh the potato pulp and add an (8) Add 1% peptone and 0.5% NaCl
equal weight or double weight of and boil for one hour.
water depending on the type of (9) Filter.
potato. If the potato contains lots (10) Add 3% glycerol and make dis-
of juice do not add so much water. tinctly alkaline by the addition
(3) Allow (2) to infuse in the cold for of a saturated soda solution.
one day, shake and strain thru a (11) Autoclave for 15 to 30 minutes.
straining cloth. (12) Filter.
(4) After -\ to i hour pour the liquid once more.
(13) Sterilize
from the sediment. Reference: Jurewitsch (1908 p. 664), Kolle
(5) During this time prepare a meat and Wasserman (1912 p. 409).
infusion using 500.0 g. of finely
chopped meat to 1000.0 875. Olitsky and Gates' Bacteria Infusion
g. of water.
Broth
Press thru a straining cloth after
24 hours. Constituents
(6) Mix equal parts of (5) and (4). 1. Meat infusion broth 1000.0 cc.
(7) Add 0.5% Chapoteaut's or Witte'a 2. Glucose 10.0 g.
peptone and 0.25% NaCl. 3. B. coll communis
(8) Warm gently until peptone is com- Preparation
pletely dissolved. (1) Prepare meat infusion broth with
(9) Boil in a Koch steamer for 1 hour 1.0% peptone.
and filter warm thru a folded filter. (2) Add 1.0% dextrose to (1).
It maybe necessary to allow the (3) Distribute in 100.0 cc. lots.
mixture to settle and pour off the (4) Seed with a broth culture of B. coli
clear liquid and filter that. communis.
(5) Incubate until a faint haze is formed (4) Heat to 80C. to stop digestion.
(usually 1 to 2 hours). (5) Pass thru a -layer of absorbent
(6) Cover with a layer of sterile paraffin. cotton.
Sterilization: Steam (6) at 100 for 15 to (6) The digest may be stored without
30 minutes. sterilization.
Use: Cultivation of Bacleriun pneumo- (7) Heat the filtrate from (5) to 70 C.
sintes. The medium was inoculated (8) Neutralize to litmus at 70C.
while the layer of paraffin was still (9) Mi.x an equal volume of (5) and infu-
liquid. sion broth, prepared according to
Reference: Olitsky and Gates (1922 p. 818), Park, Williams and Krumwiede
(see variant (ii) medium 779).
876. Ogata's Porphyra Infusion Broth
(10) Adjust to the desired reaction at
Constituents room temperature.
1. Meat infusion broth 1000.0 cc. (11) Autoclave for 15 minutes to clear.
2. Glucose 25.0 g. (12) Readjust the reaction as necessary.
3. Porphyra vulgaria 50.0 g. (13) Filter thru paper and cotton to
Preparation clear.
(1) Prepare meat infusion, using 500.0 g. Sterilization: Sterilize in the autoclave at
of meat per liter. 15 pounds pressure for 30 minutes.
(2) Dissolve 2 and 3 in (1). Use: General culture medium.
(3) Boil. Reference Park, Williams and Krumwiede
:
(4) Neutralize. (1924 p. 118).

(5) Filter.
(6) Tube. 878. Kitchen's Yeast Autolysate Blood
Sterilization: Method not given. Solution
Use: Isolation and cultivation of protozoa Constituents:
(infusoria). For isolation a capillary 1. Beef infusion.
tube is filled with the medium, inoculated 2. Blood clot 200.0 cc.
by placing one end of the tube into the 3. Peptone (1.0%) 10.0 g.
culture and the tube sealed. 4. NaCl (0.5%) 5.0 g.
Reference: Ogata (1893 p. 168). 5. Agar (0.07%) 0.7 g.
6. Glucose (3.0%) 30.0 g.
877. Park, Williams and Krumwiede's
7. Maltose (3.0%) 30.0 g.
Stomach Digest Infusion Broth Yeast.
8.
1. Water 2000.0 cc. (1) Dissolve 200.0 cc. of strained horse
2. Meat 1.0 lb. blood clot, 10.0 g. peptone, 5.0 g.
3. Peptone (1.0%) 10.0 g. NaCl and 0.7 g. agar in sufficient
4. NaCl (0.5%) 5.0 g. beef infusion to make a liter.
5. Stomach (hog) 200.0 g. (2) Adjust the reaction to pH 5.4.
Preparation (3) Autoclave and filter while hot.
(1) Clean 5 pigs' stomachs, remove the (4) Distribute in 500.0 cc. lots in liter
fatand mince finely. (A number of flasks.
stomachs should be used to equalize (5) After sterilization, add sufficient
the peptone content. In this way sterile solutions of glucose and mal-
an almost average composition in tose to make a 3.0% solution of each
peptone is obtained.) of the sugars.
(2) Mix 200.0 g. of (1), 10.0 g. HCl, pure, (6) Inoculate with yeast and keep at
and 1000.0 cc. of water at 50C. room temperature until ready for
(3) Incubate at 50C. for 20 to 24 hours use. (One week is sufficient.)
in a glass or porcelain vessel, not (7) Place the yeast cultures at 55C. over
enamel. It is most important not night and then bring rapidly to the
to allow the digest to come in con- boiling point over a free flame.
tact with any metal until neutral- (8) Heat at 95C. for 15 minutes.
ized. (9) The autolysate may be filtered thru
a mandler or allow to sediment and 881. Kitchen's Glucose Agar Infusion

pour off the supernatant fluid. Solution (Mulsow)
(10) Add 75.0 cc. of (9) to flasks contain- Constituents:
ing 1500.0 cc. of beef infusion. 1. Water 1000.0 cc.
Sterilization: Final sterilization not Beef 1.0 1b.
2.
specified. 3. Peptone 20.0 g.
Use: Cultivation of influenza bacilli, pneu- 4. KNO3 2.0 g.
mococci and streptococci. 5. Agar 1.0 g.
Reference: Kitchens (1922 p. 35). 6. Glucose 2.0 g.
Preparation
879. Kitchens' Semisolid Glucose Agar
(1) Grind one pound of lean beef and
(Mulsow)
infuse in 500.0 cc. of water at 37C.
Constituents: for 48 hours.
1. Water 1000.0 cc. (2) Add 20.0 g. peptone, 2.0 g. KNO3 and
2. Beef 1.0 pound an equal amount of an 0.2% agar
3. Peptone 20.0 g. solution, heated and cooled to 60C.
4. KNO3 2.0 g. to the juice from (1).
5. Agar (0.2%) 1.0 g. (3) Adjust to +0.9 to phenolphthalein.
6. Glucose 2.0 g. (4) Heat in the autoclave at 15 pounds
Preparation pressure for 25 minutes.
(1) Add 500.0 cc. of water to 1 pound of (5) Filter and adjust the reaction if
ground lean beef, and allow to stand necessary.

at 37C. for 48 hours. (6) Add 2.0 g. glucose.
(2) Express the juice and add 20.0 g. pep- (7) Final reaction to be between 6.6 and
tone, 2.0 g. KNO3 and an equal 6.8 with an optimum at pH 6.8.
amount (500.0 cc.) of a 0.2% agar Sterilization: Final sterilization not
solution heated and cooled to 60C. specified.
(3) Adjust the reaction to +0.9 to phenol- Use: Isolation of gonococci. The author
phthalein. reported that the gonococci grew as well
(4) Heatin the autoclave at 15 pounds if not better than the contaminating
pressure for 25 minutes. organism the first 12 to 18 hours. The
(5) Filter and readjust the reaction if addition of one part ascitic fluid to four
necessary. parts agar gave only slightly better
(6) Add 2.0 g. glucose. results.
Sterilization: Final sterilization not spec- Reference: Mulsow (1925 p. 420).
ified.
882. Kreidler's Trypsinized Broth
Use: Cultivation of gonococcus. Mulsow
reported that the gonococcus grew as well Constituents
if not better than any contaminating 1. Water 1000.0 cc.
organism during the first 12-18 hours. 2. Beef 500.0 g.
Reference: Mulsow (1925 p. 423). 3. Peptone (Difco) 10.0 g.
4. NaCl 5.0 g.
880. Menten and Manning's Lactose Bile 10.0 cc.
5. Trypsin (10% soln.)
Infusion Solution Preparation
(1) Infuse 500.0 g. finely chopped
Constituents lean
1. Infusion broth 100.0 cc. beef in 1000.0 cc. water in an ice box
2. Bile, fresh ox 900.0 cc. over night.
3. Peptone 10.0 g. (2) Boil 30 minutes over a free flame.
4. Lactose 10.0 g. (3) Filter thru gauze and then thru paper
Preparation: (1) Dissolve 2, 3 and 4 in 1. to remove the fat.
Sterilization: Not specified. (4) Make up to 1000.0 cc. volume.

Use: Enrichment of enteritidis-paraty- (5) Add 10.0 g. peptone (Difco) and
phoid B organisms from animal tissue. 5.0 g. NaCl.
Reference: Menten and Manning (1925 (6) Boil to dissolve.
p. 401). (7) Adjust to pH = 7.8.
(8) Filter thru paper. Preparation

(9) To 1000.0 cc. of sterile (8) add 10.0 cc. (1) Prepare an infusion from 1 part
of a sterile 10% solution of trypsin horse meat and 4 parts water.
in normal NaCl solution. (2) Dissolves, 4 and 5 in (1).
Sterilization: Sterilize (8) by the frac- (3) Adjust so that there is 10.0 cc. of N/1
tional method in the Arnold. Sterilize alkali per liter using litmus as an
the trypsin solution by passing through indicator.
a porcelain filter. (4) Distribute in 40.0 cc. lots in Erlen-
Use: To study the bacteriology of endo- meyer flasks.
carditis. Sterilization: Method not given.
Reference: Kreidler (1926 p. 190). Use: Cultivation of tubercle bacilli.
Reference: Siebert (1909 p. 305).
883. Richardson's Carcinoma Infusion
Broth
885. Peklo's Potato Horse Meat Infusion
Broth
Constituents
Constituents
1. Water 1000.0 cc.
1. Potato juice 500.0 cc.
2. Tumor mass 500.0 g.
S. Peptone 10.0 g.
2. Meat (horse infusion) 500.0 cc.
NaCl 3. Peptone (Witte's) 5.0 g.

4. 5.0 g.
5. Glucose 20.0 g.
4. KH2PO4 2.5 g.
5. Glycerol 30.0 g.
Preparation
Preparation
(1) Prepare a pulp from raw potatoes.
(1) Remove the tumor mass from the
(2) Add a little distilled water.
breast and strip off as much fat as
(3) Allow to stand in the cold for 24
possible. A few pieces of muscle
hours.
clear of all fat are also included.
(4) Press thru a towel.
(2) Weigh this material, chop finely and
make up in the same proportions as in (5) Heat until no more coagulum is
formed.
ordinary bouillon. (Exact propor-
tions not given.) (6) Filter.
(7) Prepare a meat infusion from horse
(3) Boil thoroly, filter and boil again.
While in boiling condition add pep- meat in the same manner as in steps
(4)
tone and salt as in ordinary bouillon, (1) thru (6).
(Exact amounts not given.) (8) Mix 500.0 cc. of (6) and (7).
Neutralize carefully (method not (9) Dissolve 3, 4 and 5 in (8).

(5)
given), boil again for a short while (10) Add NasCOa so that the reaction will
and while hot add 2.0% glucose and be alkaline after sterilization.
Sterilization Method not given.
:
1.0% tartaric acid.
(6)
Cool in a flask boil again and filter
Variants: The author gave the following
and tube hot.
variants:
Sterilization : Sterilize in the Arnold steril-
izer 3 times, one-half hour each time.
(a) Omitted the K2HPO4 and glycerol.
When a tube
(b) Omitted the glycerol, used 5.0 g.
is to be inoculated, sterilize
for I hour just before inoculation.
K2HPO4 instead of 2.5 g. and added
1.0 g. K2CO3, and 1.5 g. MgS04.
Reference: Richardson (1900-01 p. 72).
The medium was filtered and steril-
ized.
884. Siebert's Horse Meat Infusion Broth
Constituents:
1. Water 1000.0 cc.
886. Hida's Horse Meat Infusion Broth
2. Horse meat 250.0 g. Constituents
3. Peptone (Witte's) 10.0 g. 1. Water 1000.0 cc.
4. NaCl 5.0 g. 2. Meat, horse 500.0 g.
5. Glycerol 37.0 g. 3. Burdock root 40.0 g.
4. Peptone (Witte) 20.0 g. Use: Isolation and enrichment of B. cellu-
NaCl 5.0 g. lose dissolrens.

5.
Preparation Reference: Khouvine (1923 p. 715).
(1) Boil 500.0 g. finely chopped horse

888. Cutler's Blood Clot Infusion Broth
meat and 40.0 g. of finely chopped
(Stitt)
burdock root in 1 liter of water for
two hours. Constituents:
(2) Filter.
1. Water 1000.0 cc.
Add 20.0 g. Witte's peptone and 5.0 g. 2. Blood clot (human) 500.0 cc.
(3)
NaCl to the bluish filtrate. 3. NaCl (0.5%) 5.0 g.
(4) Neutralize by the addition of alkali, 4. Peptone (1.0%) 10.0 g.
using phenolphthalein, or an indi- 5. Blood

cator. Preparation
Add 6.0 cc. of a normal alkali solution (1 Take 500 .0 cc of . human blood clot and
(5)
boil for one hour in 1 liter of water.
to (4).
Boil for another hour. (2) Filter.
(6)
(7) Cool and filter. (3) Add 1.0% peptone and 0.5% NaCl.
(8) Distribute in flasks. (4) Tube.
Sterilization: Sterilize in the autoclave. (5) a few drops of blood to each
Add
Use: Cultivation of diphtheria bacilli and tube just before use.
sterile
toxin production. Author reported that Sterilization: Sterilize (4) for 20 minutes
0.001 cc. of a 10 day culture at 33C. on each of 3 successive days.

killed a 250.0 g. guinea pig in 4 days. Use: Cultivation of Entamobea histolytica
The roots of the compositae generally and other intestinal protozoa or amoeba.
contain from 2 to 3% inulin. References: Cutler (1918 p. 22), Stitt (1923
Variants: The author substituted Arctium p. 51).
lappa compositae for burdock roots. Urea Extract Broth

889. Percival's
Reference: Hida (1910 p. 412).
Constituents
Water 1000.0 cc.
887. Khouvine's Fecal Infusion Broth 1.
2. Beef extract (Lemco) 50.0 g.

Constituents 3. Peptone (Witte) 10.0 g.
1. Water 750.0 cc, Urea 100.0 g.
4.
2. Fecal infusion 250.0 cc. Preparation
3. Cellulose 250.0 cc. Boil 3 and 4 in 1.
(1) 2,
4. CaCOs 2.0 g.
(2) Cool.
5. Peptone (pancreatic) 1-0 g. Medium should be slightly alkaline.
(3)
6. NaCl 1-0 g. If not, add a drop or two of
7. K2HPO4 1-0 g- (NH4)2C03 solution.
Preparation (4) Place in a 250.0 cc. flask.
(1) Mix one part feces with 8
to 10 parts Sterilization: Not specified.
distilled water. Use: Cultivation of soil forms, Urobacillus
(2) Filteron a Laurent filter until clear. Pasteurii. Medium inoculated with gar-
(3) Allow to settle and decant the clear den Author reported that at first
soil.
liquid. a stained mount showed Urococci or

(4) Mix 250.0 cc. of sterile (4) with 750.0 g. Planosarcins. After a few days, however,
of water and 250.0 cc. of precipitated a pure culture of Urobacillus Pasteurii
cellulose that has been dissolved in developed.
Schweitzer's reagent. Reference: Percival (1920 p. 223).
(5) Dissolve 4, 5, 6 and 7 in (4).

890. Stutzer's Dinitro Benzol Extract Broth
Sterilization: Sterilize the filtrate from (2)
in the autoclave at 110C. for 15 minutes. Constituents

Water 1000.0 cc.
Sterilization of other material or final 1.
2. Potassium citrate 10.0 g.

sterilization not specified.
3. Dinitro benzol 2.0 g. Variants: The author omitted the pan-

4. K2HPO4 1.0 g. creatin.
5. Peptone Reference: Sawin (1909 p. 388).
6. Meat extract (Schulke and Meyer)
893. Olszewski and Kohler's Trypsinized
Preparation :
Bile Salt Extract Broth

(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Reaction to be slightly alkaline. Constituents :
(3) Distribute in 10.0 cc. lots. 1. Water 1000.0 cc.

Sterilization: Method not given. 2. Peptone 10.0 g.
Use: To study denitrification. Author re- 3. Liebig's meat extract 5.0 g.
ported no growth using B. agilis, B. nitro- 4. NaCl 5.0 g.
vorus, B. Stutzeri, B. Hartlebi. 5. Sodium taurocholate (0.1%). 1.0 g.
Reference: Stutzer (1901 p. 86). Preparation
(1) Prepare ordinary nutrient bouillon
891. Harrison and Vanderleck's Aesculin using 10.0 g. peptone, 5.0 g. Liebig's
Extract Broth
meat extract and 5.0 g. NaCl.
Constituents : (2) Make alkaline by the addition of
1. Distilled water 1000.0 cc. 7.0 cc. N/1 soda solution after neu-
2. Peptone 10.0 g tralizing to litmus.
3. Liebig's Meat Extract 2.0 g (3) Boil.
4. Iron citrate 2.5 g (4) Cool to 40C. and add 0.2 g. trypsin,
5. Aesculin 1.0g 10.0 cc. chloroform and 5.0 cc. toluol.
6. Bile salt (Commercial) 5.0 g (5) Place in a glass stoppered flask and
Preparation : place in the incubator for 24 to 48
(1) Dissolve 2, 3, 4, 5 and 6 in 1. hours, shaking occasionally.
(2) Adjust reaction to +1.0. (6) Filter thru a moistened folded filter
Sterilization: Not specified. paper.
Use: Detection and B. typhosus.
of B. coli (7) Add 0.1% sodium taurocholate to (6).
B. coli caused a blackening of the medium. (Not specified if the salt or gall is to
Reference: Harrison and Vanderleck (1909 be added before the dilution with
p. 622). physiological salt solution or not.)
892. Sawin's Glycocholate Extract Broth Sterilization: Not specified.
Constituents :
Use: Indol production. Dilute one part of
1. Water 1000.0 cc. (6) with 5 parts physiological salt solution
2. Peptone 10.0 g. when desired for use.

3. Lactose 10.0 g. Variants Author used 2.0% sterile beef bile
:
4. Beef extract 7.5 g. instead of sodium taurocholate.

5. Gelatin 5.0 g. Reference: Olszewski and Kohler (1923
6. Sodium glycocholate 2.0 g. p. 6).
7. Pancreatin 1.0 g.
894. Frieber's Tryptophane Extract Broth
Preparation
(1) Heat 2, 3, 4 and 5 in 1 over boiling Constituents :
water or in the steamer for 30 minutes. 1. Water 1000.0 cc.

(2) Neutralize in the usual way (method 2. Peptone (1.0%) 10.0 g.
not given). 3. Meat extract (Liebig's 0.5%). 5.0 g.
(3) Add 2.0 g. of sodium glycocholate dis- 4. NaCl (0.5%) 5.0 g.
solved in a little hot water and 1.0 g. 5. Tryptophane (0.03%) 0.3 g.
pancreatin to (2). Preparation :
Sterilization: Not specified. (1) Dissolve 2, 3 and 4 in 1.
Use: Presumptive test for B. coli. En- (2) Neutralize and add 6.0 cc. of normal
richment medium in water analysis. soda solution per liter.
Author reported that best results were ob- (3) Dissolve 0.03% tryptophane in (2).
tained when pancreatin was present. Sterilization: Not specified.
B. coli produced gas. Use: Indol production. Author reported
that Salkowski's test (H2SO4 nitrites) + Sterilization: Method not given.

gave a positive test with materials other Use: To study fermentation and buffer
than indol. Indol was not formed in the effect.
presence of glucose. References: Rogers, Clark and Evans
Variants: The author added 3.0% glucose (1914 p. 103), Burton and Rettger (1917
and about 10.0% sterile CaCOa. p. 165).
897. Waksman and Joffe's Glucose Extract
895. Bachmann's Indicator Extract Broth Broth
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. Dextrose (0.5%) 5.0 g. 2. Peptone 10.0 g.
3. Peptone (0.5%) 5.0 g. 3. NaCl 5.0 g.
4. Meat e.xtract (0.5%) 5.0 g. 4. Liebig's meat extract 5.0 g.
5. Indigo carmine (0.04%) 0.4 g. 5. Glucose 10.0 g.
(1) Dissolve 2, 3, 4 and 5 in 1, (1) Dissolve 2, 3, 4 and 5 in 1.
(2) Adjust the reaction to a slightly alka- (2) Adjust reaction to pH = 8.0.
line reaction by the addition of (3) Tube in 10-12 cc. lots.
Na^COs. Sterilization: Sterilize at 15 pounds pres-
Sterilization: Not specified. sure for 15 minutes.
Use: Cultivation of obligate anaerobes. Use To study change in reaction by actino-
:
Other investigators used the medium for mycetes metabolism.

various purposes. Reference: Waksman and Joffe (1920 p. 41).
Variants: Rogers, Clark and Davis tested
the reduction ability of lactic acid organ- 898. Whipple's Glucose Extract Broth
isms using the following solution: Constituents:
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. Beef extract 4.0 g. 2. Beef extract (Liebig's) 10.0 g.
3. Peptone 10.0 g. 3. Peptone 10.0 g.
4. Glucose 5.0 g. 4. Dextrose 10.0 g.
5. Neutral red (Griibler's, Preparation
0.5%soln.) 10.0 cc. (1) Dissolve 2, 3 and 4 in 1.
References: Bachmann (1912-13 p. 7), (2) Adjust the reaction to 0.
Rogers, Clark and Davis (1914 p. 415). (3) Tube in Smith fermentation tubes.
Sterilization: Sterilize in the autoclave.
896. Rogers, Clark and Evans' Glucose
Use: Presumptive test for B.coli. Author
Extract Broth
reported that B. coli produced 25 to 70%
Constituents: gas in the closed arm. Different investi-
1. Distilled water 1000.0 cc. gators used similar media for various
2. Beef extract (Liebig) 4.0 g. purposes.
3. Peptone (Witte) 10.0 g. Variants
4. Dextrose (Kahlbaum) 10.0 g. (a) Henneberg cultivated lactic acid bac-
5. K2HPO4 5.0 g. teria in the same medium using 5.0%
Preparation glucose instead of 1.0%.
(1) Add 2 and 3 to 1, heat on steam bath (b) Heinze studied the nitrogen assimi-
for 20 minutes. lation of green algae, Chlorella,
(2) Filter and make up loss of water. Chlorothecium and Stichococcus, and
(3) Neutralize with N/1 NaOH using reported that nitrogen was not as-
phenolphthalein. similated. He used a medium con-
(4) Heat 5 minutes, filter. taining 0.5% meat extract, 0.5% pep-
(5) Test reaction and neutralize again tone and 0.5% glucose per liter of
and filter if necessary. water.
(6) Dissolve 4 and 5 in (5). (c) Buchan studied the ability of organ-
(7) Make up (6) to 1000.0 cc. isms found in ice cream to give the
Voges-Proskauer reaction and used Use: Cultivation of cholera and typhoid

the following medium: group. Other investigators used similar
1. Distilled water 980.0 cc. media for different purposes.
2. Glucose 5.0 g. Variants
3. Peptone 10.0 g. (a) Author used 5.0%, 10.0% or 20.0%
4. Lemco 5.0 g. sucrose instead of 1.0% and added
(d) Zikes cultivated, cladothrix, Clado- 1.0% normal NaOH solution.
thrix dichotoma and sphaerotilus in (b) Author used 20.0% sucrose and
the following medium: added 2.0% normal NaOH solution.
1. Distilled water 1000.0 cc. (c) Author used 10.0% sucrose, 1.0%
2. Peptone 2.5 g. peptone, 0.5% meat extract, omitted
3. Glucose 2.5 g. the litmus and added 2.0% normal
4. Meat extract 0.5 g. NaOH solution.
References: Whipple (1902 p. 424), Henne- Reference: Weisser (1886 pp. 335, 339).
berg (1903 p. 7), Heinze (1906 p. 647),
901. Koegel's Lactose Extract Broth
Buchan (1910 p. 108), Zikes (1915 p. 533).
Constituents
899. Johnson's Glucose Phenol Extract
1. Water 1000.0 cc.
Broth
2. Peptone 10.0 g.
Constituents 3. Lactose 10.0 g.
1. Distilled water 1100.0 cc. 4. Meat extract (Liebig's) .... 5.0 g.
2. Beef extract (Liebig's) 10.0 g. Preparation: Dissolve 2, 3 and 4 in 1.
(1)
3. Peptone 10.0 g. Sterilization: Method not given.
4. Glucose 10.0 g. Use: Cultivation of L. bulgaricus. Other
5. Phenol 5.0 cc. investigators have used similar media for
6. HCl 4.0 cc. a variety of purposes.
Preparation Variants
(1) Dissolve 2, 3 and 4 in a liter of dis- (a) Obst used a medium containing 1.0%
tilled water. peptone, 1.0% lactose and 0.5%
(2) Adjust to neutral to phenolphthalein. Liebig's meat extract for the pre-
(3) Dissolve 5.0 g. of phenol and 4.0 cc. sumptive test for B. coli in water
of HCl in 100.0 cc. of water. (This analysis. She reported that generally
is Parietti's solution.) this gave a more delicate test for gas
(4) Add 20.0 cc. of (3) to (2). production than a medium containing
Sterilization: Method not given. bile.
Use: Isolation of Bacillus coli communis (b) Ritter used 3.0 g. Liebig's beef
from alimentary tract of fish. extract, 10.0% peptone and 10.0 g.
Reference: Johnson (1905 p. 351). lactose per liter for the presumptive
test for B. coli in water analysis.
900. Weisser's Sucrose Extract Broth
(c) Gompertz and Vorkaus used a me-
Constituents dium containing 4.0 g. Liebig's meat
1. Water 1000.0 cc. extract, 10.0 g. Merck's peptone and
2. Sucrose (1.0%) 10.0 g. 5.0 g. NaCl, and 50.0 g. lactose per
3. Peptone (0.1%) 1.0 g. liter to cultivate B. acidophilus for
4. Meat extract (0.1%) 1.0 g. therapeutical purposes. The authors
5. Litmus to color reported that an incubation of 60
Preparation hours gave about one hundred mil-
(1) Dissolve 2, 3 and 4 in 1. lion bacilli to each cc. of clear cul-
(2) Make slightly alkaline by the addi- ture. The organisms were trans-
tion of soda. ferred to a sterile 8 ounce bottle and
(3) Add litmus until the solution is kept until desired for therapeutic
colored light blue. purposes.
(4) Distribute in Erlenmeyer flasks. (d) Winslow and Dolloff studied the
Sterilization: Not specified. effect of certain triphenyl-methane
dyes on the growth of the colon Preparation:

bacilli. He added 10.0% lactose to a (1) Dissolve 2, 3, 4 and 5 in 1.
solution of 8.0 g. of Difco dehydrated (2) Tinge with litmus.
nutrient broth and added the dyes Sterilization: Not specified.
from a 1.0% stock solution. He re- Use: Determine fermentation of starch by
ported that y. cholerae asiaticae. Author reported
Rosolic acid in dilution of 1 to 1000 that V. cholerae asiaticae produced acid
inhibited. within 24 hours. Vibrio Finkler and
Gentian violet in dilution of 1 to Prior gave only a feeble acid reaction
5000 to 50,000 inhibited. after 3 days. Staphylococci, strepto-
Brilliant green in dilution of 1 to cocci, B. diphtheriae, B. coli, B. enteri'
10,000 to 1,000,000 inhibited. tides (Gartner), B. typhosus, B. proteus,
References: Koegel (1914 p. 460), Obst all to ferment starch with the
failed
(1916 p. 76), Ritter (1919 p. 610), Gom- production of acid.
pertz and Vorhaus (1921 p. 498), Winslow Reference: Gordon (1906 p. 5).
and Dolloff (1922 p. 303).
904. La Garde's Dextrin Peptone Solution
902. Gottheil's Sucrose Extract Broth Constituents:
1. Water 1000.0 cc. 2. Peptone 5.0 g.
2. Meat extract (Liebig's) .... 10.0 g. 3. Dextrin 5.0 g.
3. Peptone 10.0 g. 4. Meat extract trace
4. Sucrose 10.0 g. Preparation: (1) Dissolve 2, 3 and 4 in 1.
Preparation: (1) Dissolve 2, 3 and 4 in 1. Sterilization: Not specified.
Sterilization: Method not given. Use: Cultivation of Phycomyces nitens,

Use: Cultivation of organisms found in the Mucor mucedo, Mucor Rouxii, Mucor
soil, on roots and on rhizomes. corymbifer, Mucor spinosus, Mucor
Variants: The author gave the following rasemosus, Mucor rhizopodiformis, Mucor
variants: stolonifer.
(a) Added 0.02% tartaric acid. Reference: La Garde (1911-12 p. 248).
(b) Used 10.0 g. peptone, 10.0 g. Liebig's

meat extract and 5.0 g. of sucrose to 905. Banning's Glycerol Extract Broth
1200.0 cc. of water and added 5.0 g. Constituents
glucose, 5.0 g. lactose and 1.0 g. of 1. Water 1000.0 cc.
Seignette salt (Rochelle salt). 2. Glycerin 10.0 g.
(c) von Wahl used the medium with only 3. Peptone 10.0 g.
1.0 g. sucrose instead of 10.0 g. and 4. Meat extract 10.0 g.
variant (b) to cultivate the carrot Preparation
bacillus (Bacillus daucarum) and Dissolve 2, 3 and 4 in 1.
(1)
other organisms causing vegetable
(2) Do not adjust the reaction.
spoilage. Sterilization: Method not given.
References: Gottheil (1901 p. 432), von Use: To study oxalic acid formation by
Wahl (1906 p. 495). Bact. Pasteurii, Bact. aceti, Bad. xyli-
num, Bact. Dortmund, Bact. ascendens.
903. Gordon's Starch Extract Broth
La Garde used a similar medium for the
Constituents: cultivation of a number of moulds.
1. Distilled water 1000.0 cc. Variant: La Garde used a solution con-
2. Lemco 10.0 g. taining 10.0 g. peptone, 5.0 g. glycerol
3. Peptone 10.0 . and a trace of meat extract per liter for
4. Starch 5.0 g. the cultivation of Phycomyces nitens,
5. Litmus to color Mucor mucedo, Mucor Rouxii, Mucor
6. NaHCOa 1.0 g. corymbifer, Mucor spinosus, Mucor race-
mosus, Mucor rhizopodiformis, Mucor 909. Albus and Holm's Beef Extract
stolonijer. Medium (Medium X)
References Banning (1902 pp. 395 and 425),
:
Constituents
La Garde (1911-12 p. 248).
:
906. Jacobson's Mannitol Extract Broth

1. Water 1000.0 cc.
2. Yeast (1.0%) 10.0 g.
Constituents :
3. Peptone (1.0%) 10.0 g.
1. Water 1000.0 cc. 4. Lactose (1.0%) 10.0 g.
2. Meat extract (Liebig's) .... 10.0 g. 5. Beef extract (1.0%) 10.0 g.
3. Peptone (Witte's) 10.0 g. 6. Brom cresol purple (5.0%
4. NaCl 5.0 g. alcoholic soln.) 0.5 cc.
5. Mannitol 10.0 g. 7. Sodium recinolate.
6. Litmus solution 50.0 cc. Preparation :
Preparation and 6 in 1.
(1) Dissolve
:
2, 3, 4, 5
(1) Prepare a sugar free bouillon con- (2) Divide in four and keep one as
lots,
taining 1% Liebig's meat extract, 1% a control. To the other three add
peptone and 0.5% NaCl. varying quantities of sodium recino-
(2) Add about 20.0 cc. N/1 NaOH to late to lower the surface tension.
neutralize. Surface tension determined by weight
(3) Boil for about one hour. drop method following sterilization.
(4) Filter. Sterilization: Method not given.
(5) Add 1% mannitol and 5.0% litmus Use: To determine the eflfect of surface
solution. tension on growth of Lactobacillus bul-
(6) After sterilization, distribute in ster- garicus and Lactobacillus acidophilus.
ile tubes. The author reported that L. bulgaricus
steam bath.
Sterilization: Sterilize in a
was inhibited at a surface tension lower
Use: To determine fermentation of man- than 40 dynes while L. acidophilus ex-
nitol by the typhoid bacilli. hibited good growth in the same media
Reference: Jacobson (1910 p. 211). depressed to 36 dynes.
907. Harrison and van Der Leek's Citrate Variants: The authors used sodium tauro-
Aesculin Extract Broth cholate and sodium glycocholate instead
of sodium recinolate to lower the sur-
Preparation and composition the same face tension.
as for medium 891 except no bile salt Holm
Reference: Albus and (1926 p. 14).
is added.
908. De Gaetano's Potato Extract Broth 910. Flint's Serum Extract Broth
(Kamen) Constituents :
Constituents : 1. Water 750.0 cc.

1. Potato infusion 1000.0 cc. 2. Serum, blood 250.0 cc.
2. Meat extract (Liebig's) 20.0 g. 3. Peptone 15.0 g.
3. Peptone 10.0 g. 4. Glucose 15.0 g.
4. Dextrose 100.0 g. 5. Salt 3.0 g.
5. Tartaric acid 5.0 to 10.0 g. 6. Meat extract 3.0 g.
Preparation : Preparation :
(1) Preparation of potato infusion (Kar- (1) Mix 1, 2, 3, 4, 5 and 6.

tofi'elaufguss) not given. (2) Make alkaline by the addition of
(2) Dissolve 2, 3, 4 in 1. KOH.
(3) Filter. (3) Filter.
(4) Distribute in 6.0 cc. lots in test tubes. (4) Tube.

Sterilization: Method not given. Sterilization: Sterilize in the Arnold.
Use : Cultivation of Saccharomyces sep- Use: Cultivation of streptococci. Author
ticus. reported that streptococci grew abun-
Reference: De Gaetano (1897 p. 200). dantly in 24 hours and caused a flaky
NaCl with 500.0 cc. of filtered and

precipitate filling the lower portion of
sterile gall.
the tube.
(2) Neutralize with N/1 soda solution to
Reference: Flint (1896 p. 4).
litmus and then add 3.5 cc. of the
911. Grace and Highberger's Ascitic Fluid normal soda solution.
Extract Broth (3) Cool to 40C. and add 1.0 g. trypsin
(Grubler's), 5.0 cc. chloroform and
Constituents:
Water 1000.0 cc. 1.0 cc. toluol.
1.
2. Beef extract (Liebig's) (4) Place in a glass stoppered flask, and

3.0 g. place in an incubator at 37C. for 36
(0.3%)
3. Peptone (Difco) (1.0%) .... 10.0 g. hours, shaking occasionally.
NaCl (0.5%) 5.0 g. (5) Filter thru a double filter paper,

4.
5. Glucose (0.2%) 2.0 g. moistened with gall.
Ascitic fluid (5.0%) 50.0 cc. (6) Dissolve 75.0 and 25.0 g.
g. lactose
6.
Preparation NaCl Kubel and Tie-
in 600.0 cc. of
3 and 4 in 1. mann's litmus solution (from Kahl-

(1) Dissolve 2,
Add 0.2% glucose and 5.0%, sterile baum's litmus).

(2)
ascitic fluid that has been made (7) Boil 15 minutes, filter and neutralize
alkaline by the addition of 2.5% of a with normal soda solution.

40.0% NaOH solution. Sterilization: Sterilize the filtrate from (5)
(3) Adjust to pH = 6.4, 6.8, 7.2, 7.6 or 8.0. method not given. Sterilize (7), method
(4) Heat for 15 minutes at 15 pounds not given.
pressure. Use: Indol production in water analysis.
(5) Filter. Before use mix 5 parts of sterile (5) with
Tube and sterilize. 6 parts of sterile (7) and distribute in
(6)
Sterilization: Sterilize (1) in the autoclave 10.0, 5.0, 2.5, and 1.0 cc. lots in such sized
by heating at 20 pounds pressure for 15 containers so that 100.0, 25.0 and 10.0 cc.
minutes. Sterilize the ascitic fluid by of water under investigation may be
autoclaving at 15 pounds pressure for added to the flasks.
15 minutes. Final sterilization in the Reference: Olszewski and Kohler (1923
autoclave. p. 312).
Use: To study the effect of reaction on

the growth of Streptococcus viridans. 913. Rosenow's Brain Bacto Broth (Haden)
Author reported that best growth was ob-
Constituents
tained in the pH range 6.4 and 7.6.
Growth in pure culture nearly as good 1.
2. Nutrient broth (Bacto) 8.0 g.

in a 1.0% glucose broth omitting the
ascitic fluid.
3. NaCl 8.0 g.
and Highberger (1920 4. Glucose (c.p.) 2.0 g.

Reference: Grace
5. Andrade Indicator 10.0 cc.
p. 451).
6. Calf brain.
912. Olszewski and Kohler's Bile Extract 7. Marble.
Broth Preparation
(1) Dissolve 2 and 3 in 1 by heating.
Constituents:
1. Gall 500.0 cc. (2) Cool and add 4 and 5.
50.0 g. (3) Tube in 6 by 0.75 inch test tubes so

2. Peptone (Witte)
25.0 g. that the depth is at least 3.5 to
3. Meat extract (Liebig)
50.0 g. 4.0 inches.
4. NaCl
Trypsin 10 g. (4) Add 3 pieces of crushed calf brain
5.
6. Litmus solution 600.0 cc. about 1.0 cmm. square and two or
Lactose 75.0 g. three pieces crushed marbles to each

7.
Preparation: tube.
peptone, 25.0 Sterilization: Autoclave for 20 minutes at
(1) Boil 50.0 g. Witte's g.
Liebig's meat extract and 25.0 g. 15 pounds pressure.

Use: Isolation of bacteria from infected Use: Enrichment of typhoid from the
teeth. blood. Saponin is added to lake the
Reference: Haden (1923 p. 831). erythrocytes.
Reference Otaki and Akimoto (1922
: p. 101)
914. Hoffmann and Fischer's Nutrose taken from (1923 p. 331).
Extract Broth (Heinemann)
917. Dimitroff's Egg Sea Water Medium
Constituents:
1. Water, tap 1000.0 cc. Constituents
2. Peptone 10.0 g. 1. Sea water (75.0%) 750.0 cc.
3. Extract, meat 3.3 g. 2. Extract broth (25.0%) 250.0 cc.
4. Nutrose (1.0%) 10.0 g. 3. Egg white.
5. Caffein (0.5%) 5.0 g. Preparation:
6. Crystal violet 1.0% solution. (1) Place small cubes of hard boiled egg
Preparation white in 10.0 cc. of beef extract broth.
(1) Prepare e.xtract broth according to (2) Mix 25.0% of sterile (1) with 75.0%
Heinemann's method using 1, 2 and sterile sea water.
3 (see medium 695). Sterilization: Method not given.

(2) Dissolve 1.0% nutrose, 0.5% caffein Use: Cultivation of Spirillum virginianum.
and 1.0% solution of crystal violet This medium supported life of the
in (1). organism for a few days.
Use: Enrichment of colon typhoid group. (a) The following medium gave good
Reference: Heinemann (1905 p. 130). results, according to the author.
Physiological salt solution. .. 200.0 cc.
915. Keim's Honey Extract Broth Egg cube medium (same as
(1)) 50.0 CO.
Constituents:
Liquid 2.0% agar in distilled
1. Water 1000.0 cc.
water 30.0 cc.
2. Peptone 10.0 g.
(b) The author reported that the follow-
3. Meat extract 10.0 g.
ing semisolid medium gave a luxu-
4. Honey 25.0 g.
riant growth of the organism. The
5. NaCl 5.0 g.
grew luxuriantly in this me-
spirilla
Preparation
dium even when the egg cube medium
was omitted.
(2) Neutralize.
Liquid 2.0% agar in distilled
water 40.0 cc.
Use: Substitute for glucose broth.
Reference: Keim (1923 p. 604).
Egg cube medium (same
as (1)) 20.0 cc.
Reference: Dimitroff (1926 p. 21-22).
916. Otaki and Akimoto's Sodium Citrate
Blood Extract Solution 918. Lohnis' Urea Bouillon
1. Water 1000.0 cc. 1. Bouillon 1000.0 cc.
2. Meat extract 10.0 g. 2. Urea (2.0 or 10.0%) 20.0 or 100.0 g.
3. Peptone 10.0 g. Preparation: (1) Dissolve 2.0% or 10.0%
4. Saponin (Merck) 1.0 g. urea in bouillon.
5. Sodium citrate (neutral) . . . 20.0 g. Sterilization: Sterilize in the usual manner.
6. Blood. Use: Demonstrate the decomposition of
Preparation urea.
(1) Dissolve 2 and 3 in 1 by boiling. Variants
(2) Neutralize (1). (a) Geilinger sterilized the bouillon,
(3) Add 4 and 5 to (2). added a watery urea solution
sterile
(4) To 5.0 cc. of (3) add 2.0 cc. of blood. to the bouillon to obtain the 10.0%
Sterilization: Not specified in the abstract. strength of urea. The urea solution
283
by heating at one-half (4) Prepare a solution of 1 part "Extrait

was sterilized
atmosphere pressure for 30 minutes. de fer pomme" to 100.
Cunningham added 2.0, 5.0 or 10.0% (5) Prepare a solution of 1 part potas-
(b)
urea to bouillon and sterilized by the
sium ferro-cyanide to 100.
Mix 1000.0 cc. 30.0 cc. of sterile
intermittent method in flowing steam. (6) (3),
Geilinger (4) and 30.0 cc. of sterile (5) under

Reference: Lohnis (1913 p. 95),
Cunningham (1924 p. 143). aseptic conditions.
(1917 p. 246),
(7) Distribute as desired into sterile
containers.
919. Baruchello's Caffein Bouillon
Constituents: or (5) not given.
(2), lactose, (4)
Water 650 .0 cc.
1.
Use: Differentiation of colon-typhoid ba-
Bouillon 350.0 cc.
2. cilli. Author reported that colon bacilli
Caffein 10-0 g-
3. caused a blue precipitate to develop.
Preparation Typhoid bacilli caused no change in
Method of preparation of bouillon
(1) color, a brown precipitate formed.
not given, Reference: Muller (1922 p. 984).
(2) Mix 1, 2 and 3.
(3) Adjustment of reaction not specified. 922. Frost's Glucose Bouillon

Constituents
Use: Caffein hindered development of
B. coll.
10.0 g.
2. Glucose (1.0%)
Reference: Baruchello (1905 p. 570).
Preparation
(1) Prepare bouillon and add 1.0%
920. Picker's Crystal Violet Caffein
glucose.
Bouillon (Bezanfon)
(2) Tube in test tubes or fermentation
Constituents tubes.
1000.0 cc.
1. Meat infusion Sterilization: Sterilize in the steamer.
2. Peptone 30.0 g. Use: General culture medium.
3. NaCl 2.5 g. Variants
6.0
(a) Kendall, Day and Walker specified
4. Caffein g.
5. Crystal violet 0.0007 g. the use of a sugar free bouillon.

Preparation: (b) Stitt added small pieces of CaCOa
(1) Dissolve 2, 3, 4 and 5 in meat infusion. (marble) to tubes of glucose bouillon.
(2) Neutralize to phenolphthalein by (c) Klimmer used a 0.5% glucose bouil-
adding 21.0 cc. of 10 normal soda lon, tubed in 5.0 cc. lots. After
solution. inoculation and incubation, 3 drops
Sterilization: Not specified. of the following indicator solution
Use: Enrichment of the colon-typhoid was added to each tube:
group. 1. Distilled water 22.0 cc.
Reference: Bezangon (1920 p. 345). 2. 2.0% watery indigo 2.0 cc.
3. Carmine solution
921. Miiller's Lactose Ferro-cyanide 4. 10.0% acid fuchsin solution. . 1.0 cc.
Bouillon Klimmer differentiated pseudo diph-
theria and diphtheria bacilli in this
Constituents:
1000.0 cc. medium. He reported uninoculated
1. Bouillon
25.0 g. bouillon blue, diphtheria culture red
2. Lactose
3. Potassium ferro-cyanide ... 30.0 cc. and pseudo diphtheria culture green
after a few minutes. Pseudo diphthe-
4. Extrait de fer pomme 30.0 cc.
ria culture red also after 36 hours.
Preparation
References: Frost (1903 Heinemann
p. 64),
(1) Prepare bouillon.
(1905 p. 127), Gage Bornand
(1908 p. 282),
(2) Neutralize (2) and add 10.0 cc. of
Kendall, Day and Walker
normal NaOH per liter. (1913 p. 516),
(1914 p. 419), Stitt (1921 p. 35), Klimmer
(3) Add 25.0 g. sterile lactose to ster-
ile (2).
(1923 p. 223).
923. Rivas' Glucose Bouillon (15) Distribute in tubes, and cover with
Constituents: a layer of sterile olive oil. Plug
1. Bouillon 958.0 cc. with cotton and seal with gum.
2. Glucose 10.0 g. This process should be done quickly.
3. Peptone 15.0 g. (16) Incubate at 37C. for 48 hours dis-
4. Ammono-sulpho-hydrate carding any tubes showing turbidity
water 40.0 cc. or any sign of color.
5. Sodium indigo sulphuric Sterilization: Sterilize (6) at 120C. for
acid (10.0% soln.) 2.0 cc. 15 minutes.
Preparation Use: Cultivation of anaerobes.
(1) Prepare bouillon. Variants The author omitted the ammono-
:
(2) Add 1.0% dextrose and 1.5% pep- sulpho-hydrate (steps (5) thru (14)), but
tone. prepared a 1.0% NaiSOs solution and
(3) Prepare a 10.0% sodium indigo sul- added 50.0 cc. to 948.0 cc. of glucose
phuric acid solution in distilled bouillon containing 2.0 cc. of a 1.0%
water. solution of sodium indigo sulphuric.
(4) Heat (3) at 100C. for one hour. The medium was covered with olive oil
(5) Prepare a 1.0% ammonia solution in as specified above. This medium is in-
sterile distilled water. one described above.
ferior to the
(6) Place 150-200.0 water

g. of distilled Reference: Rivas (1902 p. 836).
in an Erlenmeyer flask and stopper
924. MacConkey's Iodide Glucose Bouillon
with cotton. Place a bent rod thru
the cotton reaching the bottom of the Constituents:
flask. Plug the other end of the 1. Bouillon 1000.0 cc.
rod with cotton. 2. Glucose 20.0 g.
(7) When cool pass a stream of washed 3. KI 5.0 g.
hydrogen sulphide from a Kipp Preparation
generator thru the water for 5 (1) Prepare bouillon.
minutes. (2) Dissolve 2 and 3 in (1).
(8) Prepare 10 test tubes, containing (3) Tube in fermentation tubes.

10.0 cc. of (7) each. Sterilization: Sterilize in a steamer for 10
(9) To a series of tubes add 1 drop, minutes on each of two successive days,
2 drops, 3 drops, etc., of (5) to each taking care not to over heat.
tube. Shake thoroughly. Use: Enrichment of colon-typhoid group.
(10) To each tube of (9) add 3 drops of a Variants: Author added 10.0 or 20.0 g. KI
10.0% methylene blue solution in instead of 5.0 g.
50.0% alcohol. Reference: MacConkey (1905 p. 337).

(11) Observe in which tube a complete 925. Kitasato's Formate Glucose Bouillon
decolorization takes place in about (Tanner)
1 minute (usually required 3 to 6
Constituents
drops)
(12) When the proper amount of am-
2. Glucose 20 g.
monia has been determined, pour
3. Sodium formate 4.0 g.
20.0 cc. of (7) into a sterile cylinder.
Preparation
(13) Add the proper amount of am-
(1) Dissolve 2 and 3 in bouillon.
monia (usually 10 to 20 drops) to the
(2) Tube.
20.0 cc. of hydrogen sulphite water.
This solution is to be kept cool or a
turbidity will develop.
(14) To 958.0 cc. of sterile (2) add 21.0
cc. of (13) (20.0 cc. of H2S water 926, Heymann's Acetic Acid Glucose
and 1.0 cc. of indigo sulphuric acid Bouillon (Finkelstein)
solution). The medium should be- Constituents
come clear after 1 minute. 1. Bouillon 1000.0 cc.
2. Glucose 20.0 g. 929, Plimmer's Tartaric Acid Infusion Broth

3. Acetic acid 5.0 g.
Constituents
Preparation: (1) Add 2 parts glucose, and
1. Infusion broth 1000.0 cc,
0.5 parts acetic acid to bouillon.
2. Glucose 20.0 g.
Use: Enrichment of aciduric bacilli from
Preparation
stools. Heinemann and Hefferan iso-
(1) Prepare infusion broth in the usual
lated B. vulgaricus
manner.
Variants: Heinemann and Hefferan spec-
(2) Neutralize carefully.
ified the use of glacial acetic acid.
References: Finkelstein (1900 p. 263),
Heinemann and Hefferan (1909 p. 308).
Use: Enrichment of Krebs-organisms.
Reference: Plimmer (1899 p. 806).
927. Savage's Neutral Red Glucose Bouillon
Constituents: 930. Gage's Lactose Bouillon

1. Bouillon 1000.0 cc. Constituents
2. Glucose (0.5%) 5.0 g. 1. Bouillon 1000.0 cc.
3. Neutral red (Grubler's 0.5% 2. Lactose 10.0 g.
soln.) 10.0 cc. 3. Neutral red 0.5% soln 20.0 cc.
Preparation Preparation:
(1) Add 0.5% glucose to bouillon. (1) Exact method of preparation of bouil-
(2) Prepare a 0.5% watery solution of lon not given.
Grubler's neutral red. (2) Adjust (1) to +0.8.
(3) Add 0.1 cc. of (3) to every 10.0 cc.
(3) Add 1.0% lactose and 2.0% neutral
of (1). red (0.5% aqueous solution).
Sterilization: Method not given. (4) Filter.
"Use: Detection of Bacillus coli in water (5) Tube.
analysis. (Neutral red reaction.) Ba- Sterilization: Method not given.
cillus coli gave a yellow coloration to the Use: Primarily for the presumptive test for
medium. Bacillus coli in water analysis. Author
References: Savage (1901 p. 437), Gage and reported that Bacillus coli produced acid
Phelps (1902 p. 407), Irons (1902 p. 315). and a yellowish green fluorescence.
Variants
928. DeKorti's Oleic Acid Glucose Bouillon
(a) Koegel omitted the neutral red and
Constituents used 6.0% lactose instead of 1.0%.
1. Bouillon 1000.0 cc. The medium was used for the culti-
2. Oleic acid (1.0%) 10.0 g. vation of bulgaricus.
3. Glucose (1.0%) 10.0 g. (b) Cunningham used 1.0% of Andrades
Preparation indicator and 0.5% lactose instead of
(1) Prepare ordinary bouillon. neutral red and 1.0% lactose.
(2) Add 1.0% oleic acid and 1.0% glu- References: Gage (1908 p. 282), Koegel
cose to (1). (1914 p. 460), Cunningham (1924 p. 102).
(4) Pour a layer of vaseline on the sur- 931. Hall and Ellef son's Gentian Violet
face of the broth in each tube. Lactose Broth
Sterilization: Method not given. Constituents:
Use: Enrichment of acne bacilli. Medium 1. Water 1000.0 cc.
inoculated by adding a suspension of a 2. Bouillon 1000.0 cc.
comedo crushed in a little normal salt 3. Lactose (1.0%) 10.0 g.
solution. Seal the hole made by the 4. Gentian violet, 1:100,000 or 1:20,000
pipette by means of heat. Author re- Preparation
ported that after incubation at 37 for (1) Prepare 2.0% lactose bouillon.
4 days, large numbers of acne bacilli were (2) Add an equal volume of water to (1)
found present. giving a 1.0% concentration of
Reference: De Korti (1921 p. 250). lactose.
(3) Add 1 to 100,000 or 1 to 20,000 of Sterilization: Sterilize in the autoclave in

gentian violet. the usual manner.
Sterilization: Not specified. Use Cultivation of anthrax bacilli.
:
Use: Presumptive test for B. coli. Author Reference: Holzel (1913 p. 149).
reported that dye inhibited other organ-
isms. 935. Heinemann's Glycerol Bouillon
References Hall and Ellefson (1918
: p. 337),
Constituents
Levine (1921 p. 110).
932. Jouan's Citrate Lactose Bouillon 2. Glycerol (6.0%) 60.0 g.
(Besson) Preparation: (1) Add 6.0% glycerol to
bouillon.
Constituents:
1. Water 850.0 cc.
Variants
3. Lactose 10.0 g.
(a) Kendall, Day and Walker specified
4. Magnesium citrate 1-0 g.
the use of sugar free bouillon and
5. Litmus.
used 30.0 g. of glycerol instead of
Preparation
60.0 g. per liter.
(1) Dissolve 150.0 cc. of bouillon, 10.0 g.
(b) Owen also used a 3.0% glycerol bouil-
lactose and 1.0 g. of magnesium ci-
lon to determine the amount of
trate in 850.0 cc. of water.
alcohol formed in the fermentation
(2) Add sufficient litmus solution to give
of glycerol by Bacillus saccharalis.
a violet color.
References: Heinemann (1905 p. 127),
(3) Filter.
Kendall, Day and Walker (1914 p. 419),
(4) Tube.
Owen (1916 p. 241), Dopter and Sac-
Sterilization: Sterilize at 110 to 112C.
quepee (1921 p. 119), Stitt (1923 p. 35),
Use: Synthetic whey.
Cunningham (1924 p. 165).
Reference: Besson (1920 p. 208).
933. Wollman's Starch Bouillon 936. Kendall, Day and Walker's Mannitol
Bouillon
Constituents:
2. Bouillon 400.0 cc, 1. Bouillon 1000.0 cc.
3. Starch 30.0 g. 2. Mannitol 10.0 g.
4. Peptone (Chapoteaut) 2.0 g. Preparation

Preparation (1) Prepare nutrient sugar free bouillon,
(1) Prepare bouillon. (2) Dissolve 2 in 1.
(2) Add 400.0 cc. of (1) to 600.0 cc. of (3) Distribute in 100.0 cc. lots.
water. Sterilization: Not specified.
(3) Dissolve 3 and 4 in (2). Use: To study the metabolism of tubercle

Sterilization: Not specified. bacilli. Authors reported that they used
Use: Cultivation of Glycobacter proteo- alizarin, neutral red and phenolphthalein
lytic u:^. to study changes in reaction; Ziehl-

Reference: Wollman (1912 p. 616). Neelsen stain for staining.
Reference Kendall, Day and Walker (1914
:
934. Holzel's Glycogen Bouillon

p. 419).
Constituents
937. Giltner's Adonitol Bouillon
1. Bouillon 1000.0 to 1500.0 cc.
2. Glycogen 20.0 to 100.0 cc. Constituents
Preparation 1. Bouillon 1000.0 cc.
(1) Prepare sugar free bouillon. 2. Adonitol (0.5%) 5.0 g.
(2) Add 10.0 to 15.0 cc. of (1) to 0.2 to Preparation: (1) Dissolve 0.5% adonitol in
1.0 g. of Merck's glycogen. bouillon.
(3) Cool quickly after sterilization. Sterilization: Not specified.
Differentiation of colon-aerogenes Sterilization: Sterilization of yeast extract

Use:
Author reported that B. aero- effected in step (4) in the preparation.
group.
Sterilization of bouillon not specified.
genes and B. cloacae fermented adonitol.
Reference: Giltner (1921 p. 383). Use : To study bacterial nutrition of Bacil-
lus influenzae. Authors reported that the
Phenol Bouillon medium supported the growth of B.
938. Vincent's
influenzae. Growth accessory materials
Constituents resisted boiling for 10 minutes, were
1. Bouillon. autoclaving, would pass
destroyed by
2. Phenol. thru a Berkefeld filter but were absorbed
Preparation by charcoal. Broth containing extract
(1) Preparation of bouillon not
given.
alone, however, did not give continued
Distribute in 10.0 cc. lots in tubes.
(2) growth of the influenza bacilli. Primary
(3) To each tube add 5 drops of a 5.0% inoculations were made from blood broth.
phenol solution (1 drop of 5.0% phenol Reference: Thjotta and Avery (1921
to each 2.0 cc. bouillon).
p. 100).
Use: Enrichment of typhoid bacilli. 940. Thjotta and Avery's Tomato Bouillon
Water analysis. Add 5 to 10 drops of the Constituents:
water under investigation to each tube. 1. Bouillon.
Author reported that contaminated water 2. Tomatoes.
clouded the medium. Other organisms Preparation:
were inhibited. To obtain a pure cul-
(1) Preparation of bouillon not given.
ture of typhoid bacilli 3 or 4 sub-cultures
(2) Readjust the reaction to pH = 7.8.
were made in this medium.
(3) Sear the skin off a ripe tomato with
Variants: Kamer added 5.0 g. of phenol a red hot knife.
and pure HCl to 100.0 cc. dis-
4.0 g. of
Plunge a sterile fork thru this area.
(4)
tilledwater and then added 4, 6 or 9 drops Dip the tomato into alcohol and
(5)
(30 drops = 60 cc.) of this mixture to flame off, then plunge into boiling
10.0 cc. of sterile bouillon contained in water.
tubes. Remove the peeling with sterile
(6)
References: Vincent (1890 p. 63), (1900
forceps.
p. 45), Kamer (1892 p. 35).
Place the tomatoes in a sterile
(7)
enamel dish and crush with a sterile
939. Thjotta and Avery's Yeast Bouillon pestle. (Reaction of juice pH =
4.2.)
Constituents
Distilled water 400.0 cc. (8) Boil the juice 10 minutes.
1.
100.0 g. (9) Filter the juice thru a Berkefeld
2. Brewer's yeast
Plain broth candle, or clear by centrifugation.
3.
Preparation (10) Test sterility and store in its original
Exact method of preparation or com- acidity.

(1)
position of plain broth not given. (11) Just before use readjust the reaction
(2) Adjust the reaction of (1) to pH = 7.8. from pH = 7.3 to 7.5.
(3) Emulsify 100.0 g. of brewers yeast in (12) Make

dilutions of the extract in (2)
400.0 cc. of distilled water with the in the proportion of 1:10 and 1:100.
reaction of pH = 4.6. Sterilization: As indicated in steps (8)

and (9) of the preparation. Sterilization
(4) Boil over a free flame for 10 minutes.
room tem- of the bouillon not specified.
(5) Allow to sediment at
Use: To study bacterial nutrition of
perature.
supernatant Bacillus Authors reported
influenzae.
(6) Pipette off the clear
extract and test sterility. that the medium supported growth of
Store in ice chest until ready for use. B. influenzae. Growth accessory ma-
(7)
terials resisted boiling for 10 minutes,
Readjust the reaction of the extract
from pH = 7.3 to 7.5 just before use. were destroyed by autoclaving, would
pass thru a Berkefeld filter but were 2. Brewers yeast 100.0 g.

absorbed by charcoal. Broth containing 3. Plain broth
extract alone, however, did not give con- 4. Potato
tinued growth of the influenza bacilli. Preparation
Primary inoculations were naade from (1) Preparation of bouillon not given.
blood broth. (2) Adjust the reaction of (1) to pH
Reference: Thjotta and Avery (1921 = 7.8.
p. 100). (3) Emulsify 100.0 g. of brewers yeast
in 400.0 cc. of distilled water with
941. Thjotta Pea or Bean Bouillon the reaction of pH = 4.6.
Constituents (4) Boil over a free flame for 10 minutes.

1. Distilled water 100.0 cc. (5) Allow to sediment at room tem-
2. Green peas or beans 100.0 g. perature.
3. Plain broth (6) Pipette off the clear supernatant
Preparation extract and test sterility.
(1) Preparation of bouillon not given. (7) Store in ice chest until ready for use.
(2) Adjust the reaction to pH = 7.8. Readjust the reaction of the extract
(3) Flame the surface of green pea or from pH = 7.3 to 7.5 just before use.
string bean pods. (8) Procure sterile pieces of potato from
(4) Open the pods with sterile forceps the center of an old potato without
and crush out the separate seeds into abrasions or imperfections. Take
different sterile dishes. all possible precautions to remove
(5) Add an equal weight of distilled the pieces in as asterile a condition
water to the emulsion and adjust the as possible.
reaction of pH = 4.6. (9) Distribute
the sterile pieces of
(6) Boil 10 minutes. potato into (2).
(7) Strain thru glass wool and filter thru (10) Add yeast extract (7) amount not
a Berkefeld filter (N). given.

(8) Test sterility, and store on ice until Sterilization: Sterilization of yeast extract
ready for use. Readjust the reaction effected in step (4) in the preparation.
from pH = 7.3 to 7.5. Sterilization of bouillon not given.
(9) Dilute each of the extracts in (2) Use: To study bacterial nutrition of Bacil-
in the proportions 1:10 and 1:100. lus influenzae. Author reported that
Sterilization: Sterilization of pea or bean growth in unheated potato occurred
extract effected in steps (6) and (7) in equally well without the addition of yeast
preparation. Sterilization of bouillon extract. When the potato was heated at
not given. 120 for 45 minutes no growth occurred
Use: To study bacterial nutrition of unless the yeast extract was added.
Bacillus influenzae. Author reported Medium did not give continued growth.
that the medium supported growth of Initial inoculation made from blood
B. influenzae. Growth accessory ma- broth.
terials resisted boiling for 10 minutes, Variants: The authors omitted the yeast
were destroyed by autoclaving, would extract.
pass thru a Berkefeld filter, but were Reference: Thjotta and Avery (1921
absorbed by charcoal. Broth containing p. 109).
extracts alone, however, did not give
continued growth of the influenza bacilli. 943. Thjotta's Bacterial Emulsion Bouillon
Primary inoculations were made from Constituents
blood broth. 1. Bouillon.
Reference: Thjotta and Avery (1921 2. Bacterial emulsion.
p. 100). Preparation
(1) Prepare bouillon with a reaction of
942. Thjotta and Avery's Potato Bouillon
pH = 7.8.
Constituents: (2) Grow bacteria (mucoid bacillus
1. Distilled water 400.0 cc. (Friedlander's), Bacillus ozoenoe and
B. proteus were organisms used) on Sterilization: Method not given.

plain agar slants. Use: Cultivation of anaerobes. The tubes
(3) Suspend the growth of the organism are heated until a control tube containing
in a few drops or 1.0 cc. of sterile 1.0 cc. of 1.0% methylene blue shows con-
plain bouillon or sterile normal saline siderable reduction, then cooled to 45C.
solution. until inoculated.
(4) Treat the emulsion thus obtained in Added nutrients: 1.0% of any desired car-
one of the following ways: bohydrate, alcohol, etc. may be used.
(a) Dilute 0.5 cc. of the emulsion with Variants Andrade and phenol red may be
:
5.0 cc. of bouillon, and heat for used as indicators.

1 hour at 60C. in each of the cases Reference: Kitchens (1922 p. 36).
listed below.
(b) Dilute as above, but heat at 70C. 945. Hegner and Becker's Blood Agar
for 1 hour. Solution
(c) Boil the emulsion for 5 minutes
Constituents:
and then centrifuge to separate the
1. Water 270.0 cc.
clear fluid extract from the bac-
2. NaCl (0.85%) 2.3 g.
terial bodies.
3. Nutrient agar (2.0%) 30.0 cc.
(d) Add 0.5 cc. of the emulsion which
4. Blood, rabbit
had been heated to 60C. for 1 hour
Preparation
to two tubes of bouillon. Place in
(1) Add 30.0 cc. of ordinary 2.0% nu-
the ice box for one week. Centri-
trient agar (pH 7.6) to 270.0 cc. of
fuge one tube and use the clear
0.85% saline solution (pH 7.6).
liquid and also the centrifuged
suspension.
(3) Add 20 drops of blood directly from
(5) Add from 0.1 to 0.5 cc. of one of the
a rabbit's ear to each tube of sterile,
emulsions or extracts to a tube of
melted and cooled tube of (2).
bouillon.
(4) Incubate 24 hours to test sterility.
Sterilization: Sterilization of bacterial ex-
Sterilization: Sterilize (2) at 120C. in the
tracts are given in step (4). Sterilization
autoclave.
of bouillon not specified.
Use: Cultivation of Emhodomonas in-
Use: To study nutrition of Bacillus influ-
testinalis. medium
This is a modified
enzae. Author reported that growth
Noguchi's serum medium described by
appeared earlier and was heavier in bac-
Wenyon.
terial extract bouillon than in blood
Reference: Hegner and Becker (1922 p. 17).
bouillon. Using Bacillus proteus extract
growth of Bacillus influenzae occurred
946. Thjotta and Avery's Blood Cell Bouillon
only in 2 or 3 generations.
Reference: Thjotta (1921 p. 765). Constituents
1. Bouillon.
944. Kitchens' Basal Sugar Free Agar
2. Red blood cells.
Solution
Preparation
Constituents (1) Prepare plain bouillon.
1. Sugar free bouillon 1000.0 cc. (2) Adjust the reaction of (1) to pH
2. Agar (0.1%) 1.0 g. = 7.8.
Preparation: (3) Wash the red cells from 20.0 cc,
(1) Dissolve 0.1% washed or purified agar sterile defibrinated blood three times,
in sugar free bouillon. each time with 50.0 cc. sterile salt
(2) Tube. solution. The cells are removed by
(3) Add 1.0% of the desired added nu- centrifugation.
trients. These may be added before (4) Make up the cells in sterile distilled
sterilization or made up in concen- water to the original volume of blood.
trated solutions, sterilized and then (5) Dilute (4) with (2) in varying propor-
added to sterile (2). tions from 1:10 to 1:100,000.
Sterilization: Sterilization of bouillon not (d) Bull and Pritchett added fragments
given. of sterile skeletal muscle of the pigeon
Use To study bacterial nutrition of Bacillus
: or rabbit to 100.0 cc. quantities of
influenzae. Author reported no growth bouillon. They used the medium for
in dilution of 1 to 10,000 of blood cells. the production of toxin by Bacillus
Reference: Thjotta and Averj' (1921 welchii. Medium
incubated in a
p. 109). vacuum from which the oxygen
jar
has been removed. They also used
947. Smith, Brown and Walker's Tissue the above medium with the addition
Bouillon
of 0.0 to 1.0% glucose, 0.2 or 0.3%
Constituents giving the best results.
1. Bouillon. References: Smith, Brown and Walker
2. Tissue, Rabbit or Guinea Pig. (1905-06 p. 196), Wrzosek (1905 pp. 1268-
Preparation 1270), Harrass (1906 p. 2237), Bull and
(1) Prepare ordinary bouillon. Pritchett (1917 pp. 129, 871, 873, 875),
(2) Tube in fermentation tubes. G6zony (1920 p. 566).
(3) Kill a rabbit or guinea pig by chloro-
948. Orr's Glucose Heart Bouillon
form.
(4) Moisten the animal thoroly with Constituents:
water. 1. Bouillon 1000.0 cc.
(5) Reflect the skin and open the abdo- 2. Glucose (1.0%) 10.0 g.
men with sterile instruments. 3. Heart, beef 100.0 g.
(6) Remove the liver, spleen, kidneys and 4. Marble
bits of tissue with sterile forceps. Preparation
(7) Place these bits of tissue, just large (1) Prepare 1.0% glucose bouillon.
enough to pass into the narrow part (2) Add (1) to 100.0 g. of finely minced
of the fermentation tubes. The tis- beef heart.
sue is pushed into this narrow por- (3) Adda quantity of marble chips.
tion, but not necessarily beyond. (4) Adjust to pH = 8.0.
(8) Incubate two or more days to test (5) Cover with a layer of paraffin oil.
sterility. Sterilization: Method not given.
Sterilization: Sterilization of bouillon not Use: Cultivation of B. botulinus and toxin
given. production. Author reported that
Use: Cultivation of anaerobes and flag- M.L.D. varied from 0.0001 cc. to 0.05 cc.
ellates. depending on the strain used.
Variants : Reference: Orr (1920-21 p. 128).
(a) Wrzosek reported that the addition of
949. Park, Williams and Krumwiede's Meat
sterile tissue to a medium permitted
Medium
the growth of obligate anaerobes.
(b) Harrass sterilized at 100 C. for 90 Constituents
to 120 minutes the medium after the 1. Bouillon.
addition of animal tissue. He also 2. Meat.

suggested the use of water instead of Preparation: (1) Drop pieces of meat into
bouillon. tubes of bouillon.
(c) Grozony diluted equal parts of bouil- Sterilization: Method not given.
lon (containing 2.9% peptone) and Use: General culture medium.
water, and made slightly alkaline by Reference Park, Williams and Krumwiede
:
the addition of sodium carbonate. (1924 p. 125).

After sterilization in the autoclave,
950. Kligler's Tissue Infusion Bouillon
cooled to 45C. and a small piece of
dog kidney was added to each
sterile Constituents
tube under aseptic conditions. The 1. Bouillon.
medium was used for the cultivation 2. Physiological salt solution.
of flagellates. 3. Tissue.
Preparation: 952. Oberstadt's Egg Albumin Bouillon

(1) Prepare bouillon. Constituents
(2) Obtain tissue (beef heart, rabbit or 1. Bouillon.
cat tissue or organ or mucosa of 2. Egg albumin.
various organs were used) as free Preparation
from blood as possible under aseptic
(1) Boil eggs 10 minutes.
conditions.
(2) Shell the egg, separate the yolk and
(3) Wash (2) with saline solution to re- cut the white into small pieces.
move all visible traces of blood. (3) To 100.0 cc. of clear ordinary nutrient
(4) Weigh and chop into small bits and bouillon that is slightly alkaline to
suspend in 9 times the weight of litmus add the finely chopped albumin
saline solution. from one egg.
(5) Shake thoroly and place in the ice Heat at 100C. for 30 minutes and
(4)
box over night. allow to stand until the following day.
(6) Centrifuge and filter thru a Berkefeld Filter off the egg albumin.
(5)
candle. Distribute the filtrate as desired.
(6)
(7) Tube (1) in 5.0 cc. lots. Sterilization: Autoclave at 0.5 atmospheres
(8) Add from 0.01 cc. to 1.0 cc. of sterile pressure for 45 minutes. If the medium
(6) to each tube of sterile (7).
is not to be used within 8 days, sterilize
Sterilization: Sterilize (6) by filtering thru the medium at 0.5 atmospheres pressure
a Berkefeld filter. Sterilization of (7) not filtering the egg albumin. Then
without
specified. filter just before ready to use.
Use: To show influence of growth accessory Use: General culture medium. Author re-
substances on growth of pathogenic ported that growth of pathogenic and
bacteria. saphrophytic organisms was much better
Reference: Kligler (1919 p. 32). on this medium than on ordinary bouillon.
References: Oberstadt (1914-15 p. 137),
951. Capaldi's Egg Yolk Bouillon Klimmer (1923 p. 202).
Constituents 953. Lipschutz's Egg Albumin Bouillon

1. Bouillon.
Constituents
2. Egg yolk.
1. Water 100.0 cc.
Preparation 2. Egg albumin 2.0 g.
(1) Break an egg. Separate the white 3. Bouillon 200.0 to 300.0 cc.
from the yolk and place the yolk in a Preparation
sterile Petri dish.
(1) Prepare a 2.0% egg albumin solution
(2) Exact method of preparation of bouil- in water.
lon not given. (2) Add 20.0 cc. of 0.1 normal lye solution
(3) Place a glowing glass rod on a spot on foreach 100.0 cc. of (1).
the covering of the egg yolk. (This (3) Allow to stand for 30 minutes shaking
sterilizes the covering of the naturally occasionally.
sterile yolk.) (4) Filter and distribute into Erlen-
(4) Remove 3-4 loops ("oesen"), (exact meyer flasks in 30 to 50.0 cc. lots.
amount not given) of egg yolk thru (5) Mix 1 part sterile (4) with 2 or 3
the portion of covering of the yolk parts sterile bouillon.
that had been sterilized and add to Sterilization: Sterilize (4) by placing on an
each tube of (3). asbestos plate and bringing to a boil.
Sterilization: Sterilization of bouillon not This may be done on the same or suc-
given. cessive days, or the solution may be
Use: Cultivation of tubercle bacilli and sterilized in the steamer. Method of
diphtheria bacilli to study toxin pro- sterilization of bouillon not given.
duction by diphtheria bacilli. Use: Cultivation of gonococci.
Reference: Capaldi (1896 p. 801). Reference: Lipschlitz (1904 p. 744).
954. Kahn's Casein Digest Egg Albumin Variants

Bouillon (a) Stitt prepared the medium as follows:
Constituents: (1) Prepare a 10.0% egg white solution
1. Water 1000.0 cc,
by constantly heating the egg
2. Bouillon. white while distilled water is
3. Casein 200.0 g.
added, little by little.
(2) Filter (1) thru absorbent cotton.
Preparation
(3) Heat to 100C.
(1) Boil a few eggs for 15 minutes.
(4) Filter thru filter paper.
(2) Carefully separate the white from
the yolk and wash in distilled water. (5) Tube or flask.
(6) Sterilize at 115 for 20 minutes.
(3) Cut the washed coagulated albumin in
cubes of about 2 mm. (7) Add 1.0 cc. of normal NaOH, to
(4) Place a cube into each of several test 100.0 cc. of a 10.0% emulsion of
tubes. egg yolk in water. Add sufficient
(5) Prepare casein digest according to the NaOH to slightly clarify, but still
method given by Kahn (see 648). be opaque when in rather thick

layers.
(6) Add 10.0 cc. of (5) to each tube.
(7) Seal the tube with a vaseline cap (8) HeattolOOC.
Sterilization: Autoclave at 15 pounds pres- (9) Filter.
sure for 20 minutes. (10) Sterilize at 115C. for 20 minutes.

Use: To study proteolysis by spore forming (11) Mix 100.0 cc. sterile bouillon (con-
anaerobes. taining no NaCl) with 80.0 cc. of

Variants (6) and 20.0 cc. of (10) under
(a) Torrey and Kahn added 1.0 cc. of aseptic conditions.
casein digest containing 1.0% glu- (12) Distribute in sterile tubes or
cose or lactose to each tube, or they
added 20.0% lactose, or whey broth Stitt reported that tubercle bacilli
was added to the casein digest bouil- grew better if the peptone be omitted
lon. The authors reported that in a from the bouillon,
proper medium Bacterium acid- (b) Klimmer prepared the medium as
ophilus inhibited proteolysis by sev- follows
eral spore bearing proteolytic anaer- (1) Thoroly mix 1 part egg white with
obes including B. sporogenes, B. 10 parts distilled water.
histolyticus and B. botulinus for a (2) Boil.
period of 10 days or more. (3) Filter.
Reference: Kahn (1922 p. 174), Torrey and (4) Sterilize (method not given).
Kahn (1923 p. 483). (5) Thoroly mix 1 part egg yolk with
10 parts distilled water.
955. Besredka and Jupille's Egg Bouillon (6) Add 1.0 cc. normal soda solution
(Besson) to 100.0 cc. (5).
Constituents (7) Boil.
1. Bouillon 500.0 cc. (8) Filter.
2.Egg white (10.0% soln.) 400.0 cc.
3. Egg yolk (10.0% soln.) 100.0 cc.
(10) Mix 4 parts (4) with 1 part (9)
Preparation: (1) Mix 5 parts of bouillon and 5 parts nutrient bouillon.
with 4 parts of a 10.0% egg white solution
References: Besson (1920 p. 55), Stitt (1923
and 1 part of 10.0% egg yolk emulsion in Klimmer (1923 p. 202).
p. 34),
sterile water.
Sterilization: If the egg whiteand yolk are 956. Robertson's Alkaline Egg Bouillon
not obtained under aseptic conditions,
sterilize by heating at 55C. Constituents
Use: Cultivation of pneumococci, gono- 1. Water 1000.0 cc.
cocci, meningococci, various other highly 2. Eggs 4
parasitic forms as well as saprophytic 3. NaOH (normal) 12.0 cc.
organisms. 4. Bouillon
Preparation : fresh rabbit blood with two thirds

(1) Mix the yolks of 2 eggs, the whites of volume of bouillon to cultivate
4 eggs and 12.0 cc. normal NaOH Ducrey's bacillus (chancroid ba-
in 1000.0 cc. of water. cillus),
(2) Add 1 part (1) to 5 parts bouillon. (b) Wollstein cultivated Pfeiffer bacillus
Sterilization: Method not specified. on a medium prepared as follows:
Use: Cultivation of cholera organism. (1) Boil fresh rabbit blood for 2 minutes
Reference: Park, Williams and Krumwiede in a water bath.
(1924 p. 126). (2) Centrifuge.
(3) Add 0.5 cc. of the pale pink or yel-
957. Piorkowski's Alkaline Egg Albumin low fluid resulting from (2) to
Bouillon
20.0 cc. of bouillon adjusted to
Constituents pH = 7.8.
1. Bouillon 1000.0 cc. (c) Tanner and Besson added 0.5, 0.3 or
2. Glucose (1.0%) 10.0 g. 0.25 the volume of blood (or serum)
3. Peptone (2.0%) 20.0 g. to bouillon.
4. Egg albumin (2.0% dry) (d) Besson prepared a medium for the
5. NaOH cultivation of pneumococci, meningo-
Preparation cocci and gonococci as follows:
(1) Prepare a 1.0% glucose bouillon con- (1) Collect 400.0 cc. of beef blood in a
taining 2.0% peptone. sterile flask containing 30.0 cc. of a
(2) Distribute in 5.0 cc. lots. 1.0% ammonium citrate.
(3) Prepare a 2.0% solution of dry egg (2) Shake thoroly.
albumin in tap water. (3) Store for several hours at room
(4) Add 1.0 cc. (3) and 20.0% of a N/10 temperature.
NaOH solution. (4) Dilute (3) with 3 volumes of sterile
Sterilization: Method not given. physiological salt solution.
Use: Enrichment of streptococci from the (5) Add 1 part (4) to 15 parts sterile
blood. bouillon.
Reference: Piorkowski (1922 p. 69). (e) Thjotta and Avery boiled whole blood
for 10 minutes, centrifuged and
958. Hibler's Blood Bouillon
diluted the supernatant fluid 1 to 10,
Constituents to 1:10,000 with bouillon adjusted to
1. Bouillon. pH = 7.8. He reported no growth
2. Blood, dog. with dilutions of 1:1000 or greater.
Preparation References: Hibler (1899 p. 604), Davis
(1) Collect dog blood in sterile test tubes (1903 p. 405), Wollstein (1919 p. 556),
in about 5 cc. lots. (Blood may be Tanner (1919 p. 47), Besson (1920 p. 31),
collected in beaker and then the finely Thjotta and Avery (1921 p. 109).
divided clot with a corresponding
959. Dieudonne's Alkaline Blood Bouillon
amount of serum, be distributed in
tubes. Constituents:
(2) Add a few drops of bouillon (or 1. Bouillon 500.0 cc.
water). 2. Blood (defibrinated beef).... 250.0 cc.
Sterilization: Sterilize by heating at 58 or 3. KOH(normal) 250.0 cc.
97C. Before use heat the tubes for 15 Preparation
minutes in flowing steam. (1) Add 250.0 cc. of normal KOH to
Use: Cultivation of anaerobes and various 250.0 cc. defibrinated beef blood.
parasitic forms. Author reported that (2) Adjust nutrient bouillon (or peptone
the anaerobes grew better in a medium solution) to neutral point to litmus.
sterilized at 58C. Fresh blood gave Sterilization: Sterilization not specified.
best results. Author mentioned that (1) might be
Variants sterilized in the autoclave.
(a) Davis mixed one-third volume of Use: Enrichment of cholera vibrio.
Variants: Kraus, Zia and Zubrzciky incu- 961. Fildes' Pepsinized Blood Bouillon
bated the flasks for 3 hours at 50C. for Constituents
24 hours at 37C. before use. 1. Bouillon
References Dieudonnc (1909 p. 108), Kraus,
:
2. Saline solution 150.0 cc.
Zia and Zubrzciky (1911 pp. 1084-1085). 3. Blood, defibrinated, sheep... 50.0 cc.
Preparation
960. Orcutt and Howe's Fat Blood Bouillon
(1) Place 150.0 cc. of saline solution, not
distilled water, 6.0 cc. pure HCl,
Constituents
1. Bouillon.
50.0 cc. defibrinated sheep blood and
1.0 g. pepsin (B.P. granulated) in a
2. Blood, whole defibrinated.
250.0 cc. bottle fitted with a well
3. Cream.
Preparation :
ground glass stopper. Add ingre-
dients in order specified.
(1) Distribute sterile bouillon in 1.0 cc.
lots in sterile test tubes. (2) Shake thoroughly.

Add 1.0 cc. of defibrinated blood to (3) Place in a water bath at 55 C. for 2
(2)
each tube. to 24 hours, shaking occasionally at
first.
(3) Allow milk to stand in sterile test
tubes. (4) Add 12.0 cc. of 20.0% NaOH.
Remove the cream for (3). (5) Adjust the reaction to pH 7.6 using
(4)
Dilute sterile (3) 1:10 with sterile cresol red as an indicator (per-
(5)
bouillon. manganate color).
(6) Add 0.02 cc. of (5) to each tube of (3). (6) Add pure HCl, drop by drop until
Sterilization: Sterilization of bouillon not
pH 7.0 to 7.2 is reached, using phenol
Sterilize by steaming at red as an indicator (red).

specified. (4)
100C. for 20 minutes on each of 3 suc- (7) Add 0.25% chloroform and dissolve
cessive days.
by shaking.
Use: To study the influence of fat on (8) When ready for use, shake the flask
hemolysis by staphylococci. Authors and remove the pepsinized blood with
a sterile pipette. Add from 2.0 to
reported that with the organism studied
hemolysis took place in the presence of 5.0% to bouillon.
whole milk, cream, butter, olive oil and
triolein, but not in the presence of fat

Use: Cultivation of B. influenza.
milk, nut butter, triacetin, tri- Reference: Fildes (1920 p. 129).
free
butyrin or pork fat.
962. Dunham's Glucose Serum Bouillon
Variants: The authors used one of the
following instead of cream: Constituents :
(a) Remove the cream from milk by 1. Water 300.0 cc.

centrifugation. Add 0.01 cc. of the 2. Bouillon.
it has been sterilized by
milk, after 3. Glucose 10.0 g.
the fractional method in steam, to 4. Serum (sheep) 100.0 cc.
each tube of (3) above. 5. Litmus (1.0% solution of Kahlbaum)
(b) Make a suspension of one of the fats Preparation
(olive oil, butter, nut butter, tri- (1) Prepare bouillon.
acetin, triolein, tributyrin and pork (2) Dissolve 10.0 g. of glucose in (1).
fat were used) by shaking 1.5 cc. of (3) Dilute 1 part sheep serum with 3
the fat in 10.0 cc. bouillon for one to times its volume of water.
2 hours using a mechanical shaker. (4) Mix 1 part of sterile (3) with 3 parts
Add sufficient of one of these emul- sterile (2).
sions to bouillon so that each cubic (5) Add a sufficient quantity of sterile
cm. of bouillon shall contain 0.002 1.0% Kahlbaum's purified litmus
cc. of fat. solution to impart a distinct color to
Reference: Orcutt and Howe (1922 p. 412). the whole.
(6) Incubate 2 or 3 days to detect any- (1) Mix one part rabbit serum with 2
accidental contamination. parts physiological salt solution.
Sterilization: Sterilize (2), (3) and litmus (2) Prepare a bouillon containing 10.0%
solution separately in the Arnold on each peptone.
of 3 successive days. (3) Add 1.0% of (2) to (1).
Use: To study fermentation by diplococci. (4) Adjust (3) to pH = 7.2.
Reference: Dunham (1906 p. 19). (5) Distribute into 3 to 4.0 cc. lots into
test tubes approximately 1.0 cm. in
963. Shmamine's Liver Serum Bouillon
diameter.
Constituents (6) Inoculate with one drop of infected
1. Bouillon 100.0 cc. blood, or if subcultures are being
2. Sodium nucleate 1.0 to 2.0 g. made, add a drop of fresh rabbit
3. Physiological salt soln. 20.0 cc. blood and rotate the tube gently.
4. Serum, horse 100.0 cc. (This furnishes the necessary fibrin.
5. Liver, rabbit Instead of fibrin, 0.05 to 0.1% agar
Preparation may be used.)
(1) Dissolve 1 to 2.0 g. of the sodium salt (7) Cover with a layer of oil about
of nucleic acid in 20.0 cc. of physio- 1.5 cm. high.
logical salt solution. (8) Incubate at 28-30C.
(2) Boil in the water bath for 15 minutes. (b) Klimmer added one part serum to 3
(3) Mix with 100.0 cc. of sterile clear
(2) to 20 parts bouillon, under aseptic
transparent horse serum. conditions.
(4) Add a small piece of rabbit liver that (c) Stitt cultivated Borrelia recurrentis
has been burned off in the flame. on a medium prepared as follows:
(5) Mix equal parts sterile bouillon (1) Mix three parts of a 1.0% peptone
and (4). bouillon with one part rabbit or
(6) Following final sterilization pass C0> horse serum (ascitic fluid may be
thru the tubes for 2 minutes and then used).
seal with rubber stoppers and para- (2) Tube in tall tubes.
ffin. (The fact that the medium was (3) Cover with a layer of oil not to e.x-
to be tubed and a small piece of rab- ceed 1.5 cm. in height.
bit liver added to each tube was not References: Besson (1920 p. 31), Kligler
specified.) and Robertson (1922 p. 315), Klimmer
Sterilization: Method of sterilization of (1923 p. 200), Stitt (1923 p. 54).
serum or bouillon not specified. Sterilize
965. Veillon's Ascitic Fluid Bouillon
(5) for one hour on each of 3 successive
days at 60 C. Constituents:
Use: Cultivation of spirochetes. 1. Bouillon 1000.0 cc.
Reference: Shmamine (1912 p. 323). 2. Ascitic fluid 1000.0 cc.
Preparation
964. Besson's Serum Bouillon
(1) Adjust the reaction of bouillon to be
Constituents slightly alkaline.
1. Bouillon. (2) Add an equal amount of sterile ascitic
2. Serum. fluid to sterile (1).
Preparation: (1) Mix one-half, one-third or Sterilization: Method of sterilization of
one-fourth the amount of sterile serum bouillon or ascitic fluid not given.
with ordinary sterile bouillon under Use: Cultivation of gonococci and other
aseptic conditions. highly pathogenic organisms.
Use : General culture medium. (a) The author mixed bouillon with its
Variants volume of ascitic fluid.
(a) Kligler and Robertson cultivated (b) Elser and Huntoon isolated meningo-
Spirochaeta obermeieri on a medium cocci from blood or spinal fluid in a
prepared as follows: mixture of 1000.0 cc. of ascitic fluid
and 2000.0 cc. of bouillon with a re- inoculated thru the liquid vaseline and
action of +0.6 to phenolphthalein. cooled quickly, incubated for 24 hours
(c) Besson mixed ^, 3 or J the volume of and 0.1, 0.2, 0.4 and 0.5 cc. of the culture
sterile ascitic fluid with sterile transferred to 1.0 cc. of a 5.0% suspension
bouillon. of washed fresh sheep erythrocytes. Add
(d) Kligler and Robertson cultivated sufficient physiological salt solution to
Spirochaeta obermeieri in a medium each tube to bring the volume to 20.0 cc.
prepared as follows: and incubate at 37C. for one hour.
(1) Prepare a 10.0% peptone broth Reference: Kahn (1922 p. 198).
(exact method not given).
967. Lyall's Carbonate Ascitic Fluid
(2) Add 1.0% of (1) to undiluted ascitic
Bouillon
fluid.
(3) Adjust to pH = 7.2. Constituents

(4) Distribute in 3.0 to 4.0 cc. lots into 1. Bouillon 1000.0 cc.
test tubes approximately 1.0 cm. 2. Ascitic fluid 200.0 cc.
in diameter. 3. CaCOa
(5) Inoculate with 1 drop of infected Preparation
blood, or if subcultures are being (1) Preparation of bouillon not given.
made, add a drop of fresh rabbit (2) Mix one part ascitic fluid and 5 parts
blood and rotate the tube gently. of (1).
(This furnishes the necessary (3) Add CaCOs (amount not given).
fibrin.) Sterilization: Not specified.
(6) Cover with a layer of oil about Use: To determine hemolysis by strepto-
cm. high.
1.5 cocci. Grow the organism in the medium
They reported that the spirochetes for 18 hours. Add a definite amount of
grew well at 37C. but degeneration the 18 hour broth culture to 1.0 cc. of a
changes tended to set in early. 5.0% solution of washed red blood cells
After the culture was well started, of a sheep. Incubate in a water bath for
growth proceeded well at room 1 hour at 37.5C. Author reported that
temperature. three degrees of hemolysis are possible (1)
References: Veillon (1898 p. 24), Elser and complete solution of erythrocytes, (2)
Huntoon (1909 p. 382), Besson (1920 p. 31), change of undissolved cell from a bright
Kligler and Robertson (1922 p. 315). red to a dark brown due to a transforma-
tion of oxyhemoglobin to methemo-
966. Kahn's Casein Digest Ascitic Fluid globin, but cells are not dissolved, (3) no
Bouillon hemolysis or change in color of the cells.
Constituents Reference: Lyall (1914 p. 497).
968. Roddy's Bile Bouillon
2. Casein (2.0%) 20.0 cc.
3. Ascitic fluid 200.0 cc. Constituents:
4. CaCOs 1. Bouillon 1000.0 cc.
Preparation 2. Ox bile 1000.0 cc.
(1) Preparation of bouillon not given. Preparation: (1) Mix equal parts of ox bile
(2) Mix 1 part ascitic fluid and 5 parts and bouillon.
(1) adjusted to +0.3. Sterilization: Sterilize in the steam steril-
(3) Add CaCOs (amount not given). izer for 20 minutes on each of 3 successive
(This is Lyall's broth.) days.
(4) Add 2.0% casein digest as prepared Use: Culture medium for parasitic and
by Kahn (see 648). saprophytic organisms.
(5) Tube. Reference: Roddy (1917 p. 42).
(6) Seal the tubes with a vaseline cap by
969. Mayer's Mucin Bouillon
boiling the medium and vaseline.
Use: To determine hemolysis by spore 1. Bouillon.
forming anaerobes. The medium was 2. Mucin (Merck).

Preparation 972. Thjotta and Avery's Yeast Ascitic Fluid

(1) Dissolve Merck's mucin in bouillon. Bouillon
(Water or nutrient agar may be used Constituents
instead of bouillon.)
(2) Concentrate the solution by slow Brewer's yeast
2. 100.0 g.
evaporation to obtain the desired Bouillon
3.
strength of mucin.
4. Ascitic fluid
Preparation
Use: Cultivation of parasitic and sapro- Exact composition of bouillon not
(1)
phytic bacteria.
given.
Reference: Mayer (1899 p. 818).
Adjust the reaction to pH = 7.8.
(2)
970. Schloffer's Urine Bouillon (3) Emulsify 100.0 g. of brewer's yeast

in 400.0 cc. distilled water with the
Constituents pH =
reaction of 4.6.
(4) Boil over a free flame for 10 minutes.
2. Urine 1000.0 cc.
(5) Allow to sediment at room tempera-
Preparation
ture.
(1) Prepare bouillon. the supernatant
(6) Pipette off clear
(2) Mix 2 parts sterile bouillon and 1 part
extract and test sterility.
sterile urine.
(7) Store in ice chest until ready for use.
(3) Tube or flask.
Readjust the reaction of the extract
Sterilization: Obtain urine under sterile
from pH = 7.3 to 7.5 just before use.
conditions. The urine may be collected
Dilute hemoglobin free ascitic fluid
(8)
in sterile flasks and then heated to 70
with (2) in varying proportions from
to 80C. to sterilize.
1:2 to 1:100.
Use: Cultivation of diphtheria bacilli.
(9) Add varying amounts of the yeast
Authors reported that growth a little
extract (amount not given) to (8).
more luxuriant than on ordinary bouillon. Sterilization: Sterilization of bouillon not
Reference: Schloffer (1893 p. 659).
given. Yeast infusion is sterilized in the
971. Kligler's Nasal Secretion Bouillon process of preparation in step (4).

Use: To study bacterial nutrition of Bacil-
Constituents
lus influenzae. Author reported no
1. Bouillon.
growth of B. influenzae without yeast
2. Nasal secretions.
extract.Growth in dilutions of ascitic
Preparation
and 1:5 only.
fluid in 1:2
Reference Thjotta and Avery (1921 p. 109)
:
(2) Obtain nasal washings by using 50.0

cc. warm saline solution for each
973. Thjotta and Avery's Yeast Serum
washing of apparently normal in-
Bouillon
dividuals.
(3) Collect the washings in sterile bottles. Constituents
(4) Test sterility and tube in 2.0 cc. lots 1. Distilled water 400.0 cc.
in small test tubes. 2. Brewer's yeast 100.0 g.
(5) Mix 0.1 cc. of (4) with 5.0 cc. of (1). 3. Bouillon
Sterilization: Method of sterilization of 4. Serum
bouillon not given. Sterilize (3) by Preparation
filtering thru a Berkefeld filter. (1) Exact method of preparation of
Use: To show influence of nasal washing on bouillon not given.
growth of pathogenic bacteria. Author (2) Adjust the reaction of (1) to pH
reported that nasal secretions of some in- = 7.8.
dividuals stimulated the growth of some (3) Emulsify 100.0 g. of brewer's yeast
organisms. Saliva treated in a similar in 400.0 cc. distilled water with the
manner did not. reaction of pH = 4.6.
Reference: Kligler (1919 p. 40). (4) Boil over a free flame for 10 minutes.
(5) Allow to sediment at room tem- (10) Dilute the supernatant fluid from
perature. (9) invarying proportions from 1 10 :
(6) Pipette off the clear supernatant to 1:10,000 with bouillon.

extract and test sterility. (11) Add yeast extract (7) (amount
(7) Store in ice chest until ready for not given).
use. Readjust the reaction of the Sterilization: Sterilization of bouillon not
extract from pH = 7.3 to 7.5 just given. Sterilization of yeast infusion
before use. and blood is effected in steps (4) and (8)
(8) Separate serum from sterile defib- in the preparation.
rinated blood by repeated centrifu- Use: To study bacterial nutrition of Bacil-
gation. Serum is to be free from lus influenzae. Author reported nearly
blood pigment. as good growth without the yeast extract
(9) Dilute (8) in (2) in varying propor- as with it added. No growth after blood
tions from 1:10 to 1:10,000. dilution of 1:1000.
(10) Add yeast e.xtract (7) (amount not Reference: Thjotta and Avery (1921
given) to (9). p. 109).
Sterilization: Sterilization of bouillon not
given. Yeast infusion sterilized by boil- 975. Thjotta and Avery's Yeast Blood Cell
ing in step (4).
Bouillon
Use: To study bacterial nutrition of Bacil- Constituents
lus influenzae. Authors reported no 1. Distilled water 420.0 cc.
growth of B. influenzae without yeast 2. Brewer's yeast 100.0 g.
extract. No growth when serum diluted 3. Bouillon
1:10,000. 4. Red blood cells
Reference: Thjotta and Avery (1921 Preparation
p. 109). (1) Exact method of preparation of
bouillon not given.
974. Thjotta and Avery's YeastBlood
(2) Adjust the reaction of (1) to pH
Bouillon = 7.8.
Constituents (3) Emulsify 100.0 g. of brewer's yeast in
1. Distilled water 400.0 cc. 400.0 cc. distilled water with the
2. Brewer's yeast 100.0 g. reaction of pH = 4.6.
3. Bouillon (4) Boil over a free flame for 10 minutes.

4. Blood (5) Allow to sediment at room tem-
Preparation perature.
(1) Exact method of preparation of (6) Pipette off the clear supernatant
bouillon not given. extract and test sterility.
(2) Adjust the reaction of (1) to pH (7) Store in ice chest until ready for use.
= 7.8. Readjust reaction of the extract
(3) Emulsify 100.0 g. of brewer's yeast from pH = 7.3 to 7.5 just before use.
in 400.0 cc. of distilled water with (8) Wash the red blood cells from 20.0 cc.
the reaction of pH = 4.6. sterile defibrinated blood three
(4) Boil over a free flame for 10 minutes. times, each time with 50.0 cc. sterile
(5) Allow to sediment at room tem- salt solution. The cells are removed
perature. by centrifugation.
(6) Pipette off the clear supernatant (9) Take up the cells in sterile distilled
extract and test sterility. water to the original volume of
(7) Store in the ice chest until ready for blood.
use. Readjust the reaction of the (10) Dilute (9) with (2) in varying pro-
extract from pH = 7.3 to 7.5 just portions from 1:10 to 1:100,000.
before use. (11) Add yeast extract (7) (amount not
(8) Boil whole blood 10 minutes. given).
(9) Centrifuge repeatedly until all the Sterilization: Sterilization of bouillon not
coagulated proteins have been given. Sterilization of yeast infusion
thrown down. effected by boiling in step (4),
Use : To study bacterial nutrition of Bacil- 977. Savini and Savini-Castano's Bacteria
lus influenzae. Authors reported growth Blood Bouillon
in a blood cell dilution of 1:100,000.
Constituents:
Reference: Thjotta and Avery (1921
1. Bouillon.
p. 109). 2. Glycerol.
976. Thjotta and Avery's Yeast Hemoglobin 3. Blood.
Bouillon 4. Staphylococcus aureus.
Preparation
Constituents
(1) Place 5.0 cc. of glycerol in a small
Erlenmeyer flask containing glass
2. Brewer's yeast 100.0 g.
beads.
3. Bouillon
(2) Wash the growth from 2 or 3 cultures
4. Hemoglobin (crystalline) .... 1.0 g.
of Staphylococcus aureus (24 to 48
Preparation
hours cultures at 37C.) and place in
(1) E.xact method of preparation of
sterile (Bouillon cultures may
(1).
bouillon not given.
be used.)
(2) Adjust the reaction of (1) to pH Place in a paraffin oven at 58 to GO'C.
(3)
= 7.8.
over night until the bacteria are
(3) Emulsify 100.0 g. of brewer's yeast
kUled.
in 400.0 cc. distilled water with the
(4) Add 3.0 to 4.0 cc. of blood freshly
reaction of pH= 4.6.
drawn under aseptic conditions to (3).
(4) Boil over a free flame for 10 minutes. Shake until the blood is coagulated
(5)
(5) Allow to sediment at room tem- completely.
perature.
(6) Heat at 58 to 60C. for about an hour.
(6) Pipette off the clear supernatant
(When heating here and in step (3)
extract and test sterility.
place the cotton stopper in the tube
(7) Store in the ice chest until ready for or flask lightly to allow evaporation
use. Readjust the reaction of the of water.)
extract from pH = 7.3 to 7.5 just
(7) Add sufficient of (6) to sterile bouillon
before use.
until the bouillon takes on a weak
(8) Prepare crystalline hemoglobin by red color.
the method of Walker and William-
son. J. Biol. Chem., 41: 75, 1920.
glycerol not specified. Method of steril-
(9) Prepare a 10.0% solution of crys- ization of bouillon not given.
talline hemoglobin in water by
Use: Cultivation of influenza bacilli.
weight.
Variants: The author prepared a similar
(10))Dilute sterile (9) with sterile bouil-
medium using a hemoglobin solution in-
lon in varying proportions from
stead of blood. The powdered hemo-
1:10 to 1:200,000.
globin was sterilized at 100C. in the hot
(11) Add yeast extract (7) amount not air sterilizer after drying in the desic-
given.
cator. The powdered hemoglobin was
crushed in a sterile mortar with solution
bouillon not given. Sterilization of yeast
I,
obtained in step (3) above. Filter thru
infusion effected by boiling in step (4).
sterile filter paper. This solution was
the hemoglobin solution thru a
Filter
used instead of blood as indicated in
Berkefeld filter to sterilize.
step (6) and (7).
Use: To study bacterial nutrition of Bacil- and Savini-Castano
Reference: Savini
lus influenzae. Authors reported no
(1911 p. 494).
growth of B. influenzae without yeast
extract. Growth when hemoglobin solu-
tion diluted 1:200,000 (with yeast extract
present). Liquid media or basal solutions contain-
Reference: Thjotta and Avery (1921 ing a digest or autolysate other than a com-
p. 109). mercial digest.
Ai. Containing digests of plant origin only. El. Stomach only, digested.
Bi. Yeast digest employed. Martin's Stomach Digest Solution
Ci. No salts added. (Martin's peptone) 998
Kammen's Yeast Digest (Klimmer). 978 E2. Liver digested.
Ayers and Rupp's Yeast Digest Martin's Liver Digest Solution 999
Solution 979 Seallards and Bigelow's Liver Digest
Robertson's Glucose Asparagin Blood Solution 1000
Solution 980 Dubovsky and Meyer's Liver Digest
Kister's Yeast Digest 981 Solution 1001
Cj. Salts added. E3. Spleen digested.
Kligler's Yeast Autolysate Solution. 982 Emery's Spleen Digest Solution 1002
Jotten's Yeast Autolysate Solution. . 983 E4. Blood clot digested.
Abt and Blanc's Yeast Autolysate Stickel and Meyer's Blood Clot
Solution 984 Digest Solution 1003
Robertson and Davis' Yeast Autoly- Bramigk's Peptic Digest Solution. 1004 .
sate Solution 985 Harvey's Blood Clot Digest Solution 1005

Bj. Plant digests other than yeast em- E5. Digests of a variety of other materials
ployed. used.
Aj.* Containing digests of animal origin Martin's Stomach Digest Infusion
only. Broth 1006
Bi. Digests prepared by acid hydrolysis. Besredka and Jupille's Egg Stomach
Ci. Gelatin hydrolyzed. Digest Solution 1007
Davis and Ferry's Hydrolyzed Gela- Stickel and Meyer's Meat Digest
tin Solution 986 Solution 1008
Capaldi and Proskauer's Hydrolyzed D2. Digested by pepsin.
Gelatin Solution 987 Frieber's Gelatin Digest Solution., 1009
Capaldi and Proskauer's Mannitol Frieber's Fibrin Digest Solution 1110
Hydrolyzed Gelatin Solution 988 Bramigk's Peptic Tryptic Digest
C2. Casein hydrolyzed. Solution 1111
Di. Infusions or e.xtracts not added. Jensen's Milk Digest Solution 1112
Robinson and Rettger's Hydrolyzed C2. Tryptic digest only, (no peptic digest)
Casein Solution 989 added.
Cannon's Hydrolyzed Casein Solu- Di. Blood clots or blood digest specified.
tion 990 Stickel and Meyer's Trypsinized
Berthelot's Hydrolyzed Casein Solu- Blood Clot Solution 1113
tion 991 Spray's Blood Clot Digest Solution. 1114
D2. Infusions or extracts added. LofH's Trypsinized Blood Solution. 1115
Mueller's Hydrolyzed Casein Heart D2. Heart digest specified.
Solution 992 Gordon et al. Trypsinized Heart
Robinson and Rettger's Extract Hy- Solution (Tanner) 1116
drolyzed Casein Solution 993 Harvey's Trypsinized Heart Solu-
C3. Other materials hydrolyzed. tion 1117
Groer and Srnka's Hydrolyzed Pla- D3. Serum digest specified.
centa Solution 994 Distaso's Trypsinized Serum Solu-
Boyd's Hydrolyzed Meat Solution. . 995 tion 1118
Harvey's Hydrolyzed Meat Solution 996 D4. Meat digest specified.
Frouin and Ledebt's Hydrolyzed Hottinger's Trypsinized Meat Solu-
Serum Solution 997 tion (Klimmer) 1119
B2. Digest prepared by enzyme hydrolysis. Peckham's Trypsinized Beef Solu-
Ci. Peptic digest only, (no tryptic digest) tion 1120
added. Harvey's Trypsinized Meat and
Di. Digested by means of minced stomach. Kidney Solution 1121
Celozzi's Placenta Digest Blood
* See next page for A3. Solution 1122
Douglas Trypsin Broth (Hartley).. 1123 (6) Neutralize the liquid by the addition
Ds. Casein or milk digest specified. of NaOH.
Harvey's Basal Trypsinized Casein (7) Heat at 50 until all the liquid has
Solution 1124 evaporated.
Teruuchi and Hida's Trypsinized (8) Heat at 100 to 105C. for one hour.
Casein Solution 1125 (9) Pound to a powder.
Zipfel's Trypsinized Casein Solu- Sterilization: Material remains sterile in
tion 1126 powdered form.
Bacto Tryptophane Broth (Dehy- Use: Substitute for meat peptone.
drated) 1127 Variants
Bacto Peptonized Milk (Dehy- La) Klimmer added the supernatant fluid
drated) 1128 of (2) to the liquid obtained in step
Mueller's Trypsinized Casein Solu- (6) and prepared a powder as indi-
tion 1129 cated above,
Cole and Onslow's Trypsinized Ca- (b) Klimmer dissolved 10.0 g. of the
sein Solution 1130 powder obtained above, 8.0 g. NaCl
Berman and Rettger's Trypsinized and 0.1 g. KNO3 in a liter of water.
Casein Solution 1131 He specified that this medium could
Norris' Trypsinized Caseinogen be used as ordinary bouillon or might
Solution 1132 be used in the preparation of special
Cannon's Trypsinized Casein Solu- media.
tion (Norton and Sawyer) 1133 Reference: Klimmer (1923 p. 170).
De. Digest of a variety of materials spec-
979. Ayers and Rupp's Yeast Digest
ified.
Solution
Duval and Harris' Tryptic Digest
Solution 1134 Constituents
Harvey's Tryptic Digest Solution. 1135 . 1. Water 1000.0 cc.
Stickel and Meyer's Tryptic Digest 2. Yeast 100.0 g.
Solution 1136 Preparation
C3. Both peptic and tryptic digests added. (1) Add 100.0 g. dry fresh yeast to
Stickel and Meyer's Digest Solution 1137 1000.0 cc. distilled water with 1.0 g.
Frieber's Digest Extract Solution. . 1138 pepsin. Add HCl to obtain pH =
A3. Containing digests of both plant and 4.4.
animal origin. (2) Incubate at 40 for 24 hours.
Davis and Ferry's Hydrolyzed Gli- (3) Steam for 30 minutes.
adin Solution 1139 (4) Filter.
Robinson and Rettger's Hydrolyzed (5) Dilute to 1000.0 cc. with distilled
Edestin Casein Solution 1140 water.
978. Kammen's Yeast Digest (Klimmer) Sterilization: Method not given.
Constituents Use: General culture medium. Author re-
1. Water 20,000.0 cc. ported that this medium gave very good
2. Yeast 10,000.0 g. growth of the delicately growing strepto-
Preparation cocci.
(1) Autoclave 10,000.0 g. yeast with 20 Reference: Ayers and Rupp (1920 p. 96).
liters of water for 2 hours at 1.5 to 1
980. Robertson's Glucose Asparagin
atmospheres pressure.
Solution
(2) Allow to settle and decant the liquid.
(3) Add pepsin and about 10 liters of Constituents:
0.5% HCl solution to the thick yeast 1. Distilled water (sterile). . . 1000.0 cc.
residue from (2). 2. Asparagin (Merck) 3.4 g.
(4) Incubate (3) for 5 days at 37C. 3. CaCl2 0.1 g.
(5) Allow to settle and decant the liquid 4. Glucose 20.0 g.
or filter. 5. MgS04 0.2 g.
Preparation 982. Kligler's Yeast Autolysate Solution

(1) Dissolve 2, 3, 4 and 5 in 1 by boiling
Constituents:
3 minutes. Make up the loss of
Water
1. 1000.0 cc.
water.
2. Yeast 200.0
NaHjPOi
3. 2,0 g.
(3) Tube in 5 cc. quantities.
Preparation
Sterilization: Heat in the autoclave at 20
(1) Suspend 200.0 g. of drained or centri-
pounds pressure for 30 minutes.
fuged brewers yeast in a liter of water.
Use: To study the relation of substances
(2) Add 2.0 g. NaH2P04.
formed by B. coli to the growth of yeast.
(3) Add N/1 NaOH to obtain pH = 6.1.
The author reported that this medium
(4) Add 5.0 cc. chloroform.
did not support the continued growth of
(3) Shake well incubate at 37 for 2
yeast. The addition of aqueous extracts
days, shaking occasionally.
of autolized yeast and grated carrot to
(6) After incubation, adjust to pH = 7.4
the medium in high dilution permitted the
and heat in water bath or Arnold for
growth of yeast, but caused no increased
30 minutes.
growth of B. coli. The filtrate of the
synthetic medium on which B. coli had
(8) Tube.
been grown for 68 generations, per-
mitted the continued growth of yeast.
Use: Substitute for meat or meat infusion
Reference: Robertson (1924 p. 396).
media for the cultivation of colon-typhoid
981. Kister's Yeast Digest group and others.
References: Kligler (1919 pp. 183-186),
Constituents:
Harvey (1921-22 p. 120).
1. Water.
2. Yeast.
983. Jotten's Yeast Autolysate Solution
Preparation
(1) Dissolve the proteins and extractives Constituents
from dry yeast by means of water. 1. Water.
(2) Evaporate or condense the extract a 2. Yeast (brewer's) 10,000.0 g,
thick syrupy mass. This composes 3. NaCl 0.8 %
the yeast extract. 4. KNO3 0.01%
(3) To obtain a yeast peptone subject the Preparation
yeast extract to super-heated steam (1) Wash 10,000.0 g. of brewers yeast
for some time (exact time not spec- twice with 20 liters of water. Use a
ified). large centrifuge if possible.
(4) Change the hydrolysed mass into a (2) Add N/1 NaOH or (NH 4)200 3 until
light yellow powder. This gives you the whole mass has turned brown.
a yeast peptone soluble in water. Shake thoroly when adding NaOH or
Sterilization: Material required no further (NH 4)200 3.
sterilization. (3) Allow to stand for 30 to 60 minutes.
Use: In the preparation of yeast media, This dissolves some of the im-
the yeast extract replaces the meat purities.
extract and the yeast peptone 'replaces (4) Add fresh cold water to (2). This
the Witte or commercial peptone. causes the yeast to separate in flakes.
Variants: Kister reported that Yeast pep- (Amount of water not given.)
tone may also be prepared by an acid (5) Allow the yeast to settle for several
hydrolysis under pressure or by a pepsin hours.
hydrolysis, in an incubator, of the protein (6) Remove the water from the yeast.
of the yeast after the separation of the (7) Add fresh water (amount not given).
extractive materials. Exact method not (8) Distribute in 5 liter flasks, and place
given. at 45 for 48 hours.
Reference: Kister (1921-22 p. 478). (9) Pour off the brown liquid.
(10) Add water to the sediment and results. It might also be used as a
centrifuge. basis for the preparation of special
(11) Remove the supernatant liquid of media. For the enrichment of
(10) and add to the liquid obtained cholera allow the autolysis to con-
in (8). This finally leaves about 35 tinue for 3 days instead of 2 as indi-
to 40 liters of extract. cated in step (6).
(12) Boil for 90 minutes in the steamer Reference: Jotten (1920 p. 359), Klimmer
and make slightly alkaline to litmus. (1923 p. 171).
(13) Filter thru paper or cotton.
(14) Add 8.0 g. NaCl, 0.1 g. KNO3 and 984. Abt and Blanc's Yeast Autolysate
0.2 g. NasCOg per liter. Solution
Sterilization: Sterilize in the steamer on
Constituents:
each of 3 successive days.
1. Water.
Use: Meat extract substitute in the prepa-
2. NaCl (0.9%).
ration of general culture media.
3. Yeast (Brewers).
Variants
Preparation
(a) The author evaporated (1) to a quick
mass, similar to meat extract, using a (1) Wash yeast in water and decant after
"Faust-Heim" evaporating oven. settling. Repeat this several times.
Klimmer prepared the medium as (2) Dry the yeast.

(b)
(3) Prepare a physiological salt solu-
follows:
tion containing 9.0 g. NaCl per
(1) Wash 10,000.0 g. of bottom beer
1000.0 g. water.
yeast with 20 liters of water, twice,
using the centrifuge if possible. (4) Add 8 to 10 parts of dry washed
yeast to each 100.0 cc. of (3).
(2) Add about 1.5 cc. normal NaOH
Incubate at 48 to 50 for 24 to 36
or (NH 4)200 3 to the residue in (5)
hours.
thin stream, stirring constantly.
The liquid becomes dark brown. (6) Add two volumes of water to (5).
Add cold tap water after 30 to 60 (7) Heat to boiling.

(3)
(8) Filter.
minutes.
Allow to settle and decant the (9) Add soda to neutralize to phenol-
(4)
yeast water.
phthalein, or make slightly alkaline.
Stir the yeast mass with water. (10) Heat for 15 minutes at 115C.
(5)
Distribute in 5 liter flasks and (11) Filter.
(6)
incubate at 45C. for two days. (12) Evaporate 10.0 cc. of the filtrate to
Pour off the brown colored liquid. dryness to determine the dry ma-
(7)
Thoroly wash the residue with terial. Calculate the dry material
(8)
in 100.0 cc.
water, and centrifuge out the
residue. (13) Dilute (11) so as to have 2.1 or even
0.5 parts of dry material per 100.0 cc.
(9) Mix the liquid from (7) and (8).
Total volume 30 to 40 liters.
Boil for one hour in the steamer. Sterilization: Sterilize at 110 for 15
(10)
Filter thru cotton or paper. minutes.
(11)
(12) Neutralize to litmus. Use: General culture medium for colon-
(13) Add 0.8% NaCl, 0.01% KNO3 and typhoid group, parasitic and saprophytic
0.02% Na2C03 to (12). forms and others.
(14) Sterilize on each of three succes- Variants: Robertson and Davis prepared
sive days. a similar medium as follows:
(15) The liquid from (9) may be evap- (1) Grow yeast on dextrose agar slants.
orated to a thick brown paste, (2) Suspend in sterile distilled water,
and using 15.0 g. per liter to pre- throw down in centrifuge and wash
pare the medium. in sterile distilled water 3 times.
Klimmer reported that the medium (3) Kill yeast by heating at 70 C. for
might be used instead of ordinary 10 minutes. (The suspension con-
nutrient bouillon and give very good tained about 500,000 per cc.)
(4) Allow to autolyze for one week at 4. Tyrosine 1 -5 g.
37.5C. 5. Cystin 0.4 g.

(5) Pass thru a Berkefeld filter. 6. NaCl 5.0 g.
(6) Adjustment of reaction not given. 7. K2HPO4 3.0 g.
(7) Add sterile (method of sterilization 8. MgS04 0.5 g.
not given) physiological salt solution Preparation :
to (5). Amount of each not given. (1) Hydrolyze gelatin with 25.0% H2SO4
They used the medium to study the for 24 hours on sand bath. (Tem-
influence of vitamines on bacterial perature not given.)
growth. (2) Add Ba(OH) 2 until alkaline and filter.
Reference: Abt and Blanc (1921 p. 452), (3) Neutralize exactly the Ba(0H)2 with
Robertson and Davis (1923 p. 154). 10.0% H2SO4. Filter.
(4) Test filtrate for presence of free Ba
985. Robertson and Davis' Yeast Autolysate and SO4 ions.
Solution Concentrate to a thick syrup in a
(5)
Constituents vacuum.
1. water to.
Sterile distilled . 1000.0 cc. (6) Dilute to 2.0% solid material with
2. Asparagin (Merck) 3-4 g distilled water
3. CaCU 0.1 g (7) To each liter of (6) add 3, 4, 5, 6, 7
4. Dextrose 20.0 g and 8.
5. MgS04 0.2 g (8) Adjust reaction to pH 8.0-8.2 with
6. K2HPO4 1.0 g N/10 NaOH. See med. 474.

7. NaCl 5.0 g Sterilization: Heat for 20 minutes at 115C.
8. Yeast. Use: Cultivation of Bad. diphtheriae for

Preparation :
toxin production. Authors reported that
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1 by this medium was superior to most syn-
boiling 3 minutes. theticamino acid media. Amino acids
(2) Restore original volume with sterile used were chemically pure.
distilled water. Reference: Davis and Ferry (1919 p. 231).
(3) Adjust reaction to pH = 7.4. 987. Capaldi and Proskauer's Hydrolyzed
(4) Tube. Gelatin Solution
(5) Grow yeast on dextrose agar slants. Constituents:
(6) Suspend in sterile distilled water,
1. Water 1000.0 cc.
throw down in centrifuge and wash in
2. NaCl (0.02%) 0.2 g.
sterile distilled water 3 times.
3. MgS04 (0.01%) 0.1 g.
(7) Kill yeast by heating 70C. for 10
4. K2HPO4 (0.2%) 2.0 g.
minutes. (This suspension contained
5. Gelatin (2.0%) 20.0 g.
about 500,000 per cc.)
Preparation
(8) Allow to autolyze for 1 week at
37.5C.
(2) Boil gelatin for 6 hours in 1.0% HCl,
Sterilization: Sterilize (4) ty autoclaving
H2SO4 or HNO3.
at 20 pounds pressure for 30 minutes. Add 2.0% of (2) to (1) (or water).
(3)
Filter (8) thru a Berkefeld to sterilize.
Use: To study influence of vitamins on
Use: General culture medium. Author re-
!_ bacterial growth. Authors reported a
ported that typhoid bacteria showed no
^ luxuriant growth of yeast.
growth in this medium.
Reference: Robertson and Davis (1923
p. 154).
p. 460).
986. Davis and Ferry's Hydrolyzed Gelatin 988. Capaldi and Proskauer's Mannitol
Solution Hydrolyzed Gelatin Solution
Constituents : Constituents
1. Distilled water 1000.0 cc. 1. Water 1000.0 cc.
2. Hydrolyzed gelatin (syrup). 20.0 g. 2. Mannitol (0.1%) 1.0 g.
3. Tryptophane 0.4 g. 3. Gelatin (2.0%) 20.0 g.

Preparation (7) Mix 100.0 cc. (6) with 1000.0 cc. water.
(1) Dissolve 2 in 1. (8) Method of sterilization not given.
(2) Boil gelatin in 1.0% HCl, H2SO4, or This medium is inferior to opsine as
HNO3 for 6 hours. a general culture medium.
(3) Add 2.0% of (2) to (1). Reference: Robinson and Rettger (1917
Sterilization: Not specified. p. 364), (1918 p. 219).
Use: Acid production by colon-typhoid
group. 990. Cannon's Hydrolyzed Casein Solution
Constituents
p. 464).
989. Robinson and Rettger's Hydrolyzed 2. Asparagin 5.0 g.
Casein Solution 3. Ammonium lactate 5.0 g.

4. KH2PO4 2.0 g.
Constituents:
5. MgS04 0.2 g.
1. Water 1000.0 cc.
Casein 10.0 g.
6.
2. Casein 50.0 g.
Preparation
Preparation with
(1) Hydrolyze 10.0 g. of casein
(1) Boil 50.0 g. of casein with 10.0% HCl
200.0 cc. of 10.0% H2SO4.
under a reflux condenser until the
(2) Keep on a water bath for 24 hours
solution no longer responds to the
(temperature not given).
Biuret test.
(3) Neutralize by the addition of a satu-
(2) Evaporate on the water bath until
rated barium hydroxide solution and
nearly all the HCl is removed.
filter.
(3) Neutralize the remaining acid with
(4) Evaporate the solution until the
NaOH. This solution is of dark amino acids crystallize.
brown color and is known as "Ca-
sein C."
distilled water.
(4) Dissolve 50.0 g. of (3) in 1000.0 cc. of
(6) Add the crystals of (4) to one half
water.
of (5).
(5) Heat on water bath for 20 minutes.
(6) Adjust the reaction neutral to litmus.
Use: Rapid detection of indol production.
Variant: Tanner employed all the amino
Erlenmeyer flasks.
acid crystals in step (6) and not only
(8) After sterilization add 0.1% sterile
one-half.
glucose to one half of the flasks.
References: Cannon (1916 p. 535), Tanner
Sterilization: Sterilize at 12 to 14 pounds
(1919 p. 63).
pressure for 15 minutes.
Use: Cultivation of B. diphtheriae and
991. Berthelot's Hydrolyzed Casein Solution
to.xin Authors reported that
production.
the cultures were nearly non toxic. Constituents
Variants: The authors prepared a similar 1. Water 1000.0 cc.
medium as follows: 2. Casein 15.0 g.
(1) Heat 100.0 g. casein with 400.0 cc. Preparation
concentrated HCl for a few hours on (1) Hydrolyze casein to amino acids by
water bath under a reflux condenser. acid (Method not given).
(2) If charring occurs, dilute with (2) Dissolve 15.0 g. of the amino acids
150.0 cc. water and heat over free obtained from the hydrolysis in
flame for 8 hours. 1000.0 cc. water.
(3) Filter off humus residue. (3) Exactly neutralize with soda (Indi-
(4) Filtrate is biuret free and is evapo- cator not specified).
rated on water bath with frequent Sterilization: Sterilize with ether (Method
renewal of water. not given).
(5) Decolorize (method not given). Use: To.xin production hy Proteus vulgaris.
(6) Neutralize with NaOH Reference: Berthelot (1914 p. 916).
992. Mueller's Hydrolyzed Casein Heart 993. Robinson and Rettger's Extract
Solution Hydrolyzed Casein Solution
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. NaCl (1.0%) 10.0 g. 2. Casein 50.0 g.
3. MgS04 (0.04%) 0.4 g. 3. Meat extract (Liebig's)
4. CaCl. (0.02%) 0.2 g. (0.5%) 5.0 g.
5. K2HPO4 (0.2%) 2.0 g. Preparation
6. Glucose (0.2%) 2.0 g. (1) Boil 50.0 g. of casein with 10.0% HCl
7. Phenol red (0.02% solution). 80.0 cc. under a condenser until the
reflex
8. Heart, beef 1.0 lb. solution no longer responds to the
9. Casein. Biuret test.
Preparation (2) Evaporate on the water bath until
(1) Prepare beef heart infusion by heat- nearly all the HCl is removed.
ing 1.0 pound of beef heart in (3) Neutralize the remaining acid with
500.0 cc. of tap water to boiling. NaOH. This solution is of dark
Strain and filter. brown color and is known as "Ca-
(2) Decolorize by boiling for 25
(1) sein C."
minutes with 10.0% "Norit," a com- (4) Dissolve 50.0 g. of (3) in 1000.0 cc.
mercial grade of wood charcoal. water.
(3) Filter thru paper. (5) Decolorize by filtering thru animal
(4) Prepare casein hydrolysate by boil- charcoal.
ing commercial casein 18 hours with a (6) Add 0.5%Liebig's meat extract.
mixture of 6 times its weight of water (7) Heat on a boiling water bath for
and 3 times its weight of concen- 20 minutes.
trated H2SO4. (8) Adjust the reaction to +0.6 to phenol-
(5) Free from H2SO4 by the addition of phthalein, and filter.
Ba(0H)2. Wash precipitate with (9) Distribute in 20.0 cc. lots in 150.0 cc.
water and concentrate the filtrate Erlenmeyer flasks.
and washings. Sterilization: Sterilize at 12 to 14 pounds
(6) The quantity of casein hydrolysate pressure for 15 minutes.
used in the following preparations is Use: Cultivation of B. diphthcriae and
equivalent to 0.5 g. of the original toxin production.
casein. Reference: Robinson and Rettger (1917
(7) Dissolve 2, 3, 4, 5, 6 and 7 in 1000.0 cc. p. 363).
of water.
994. Groer and Srnka's Hydrolyzed
(8) Prepare the following solutions:
Placenta Solution
[Decolorized infusion (1).. 25.0 cc.
(a) i Glucose salt solution (7) 25.0 . . cc. Constituents:
[Casein hydrolysate (6) 1. Water 4000.0 cc.
to 0.5 g. 2. Placenta 4000.0 g.
[Water 25.0 cc. Preparation
(b) {Glucose salt solution (7).. 25.0 cc. (1) Mix about 4 liters (3 placenta to
[Casein hydrolysate 0.5 g. 1 liter on an average) placenta with
(9) Adjust to pH = 7.8. the blood, cut into medium sized
(10) Filter if necessary and tube. pieces, with 4 liters of water.
Sterilization: Sterilize at 10 pounds pres- (2) Add about 10.0 cc. concentrated HCl
sure for 10 minutes. and mix well.
Use: To study food requirements of strep- (3) brown mix-
Boil for about 2 hours to a
tococci and pneumococci. Author re- ture, adding water.
ported growth with casein hydrolysate (4) Strain and filter thru paper while
present. No growth in other solutions. still warm.
Reference: Mueller (1922 p. 327). (5) Add 10.0% NaOH until phenol-
phthalein is turned red (hot titra- (7) Neutralize the acid completely and
tion) to the clear light greenish make the medium faintly alkaline to
yellow filtrate. phenolphthalein.
(6) Boil for one hour. (8) Boil (7) for 15 minutes.
the albumin.
(7) Filter off (9) Filter hot thru filter paper.
(8) Readjust the reaction (pH = 7.8). (10) Dilute to 1000.0 cc. by the addition
(9) Distribute the crystal clear fluid into of water.
flasks. (11) Dissolve 3 and 4 in (10).
Sterilization: Method not given. (12) Make the desired final adjustment of
Use : Cultivation of diphtheria bacillus and the reaction.
toxin production. Sterilization: Sterilize in the autoclave.
Reference: Groer and Srnka (1918-19 Use: General culture medium.
p. 334). Variants
(a) The author suggested the steriliza-
995. Boyd's Hydrolyzed Meat Solution tion of solution (9) above by auto-
Constituents: claving and storing until ready for
1. Water, tap 1000.0 cc, use. When desired continue as in
2. H,S04 (strong) 100.0 cc. steps (10), (11) and (12) as indicated.
3. NaCl 5.0 g. (b) Norris prepared the medium as

4. KCl 2.0 g. follows
5. Lean meat 1.0 lb. (1) Add 500.0 g. lean meat to 40.0 cc. of
Preparation strong H2SO4.
(1) To 300.0 cc. of tap water add 100.0 cc. (2) Pound together in a mortar.
strong H2SO4 gradually with con- (3) Mix thoroly and
digest in a water
stant stirring. bath for 4 days at 70C.

Cut up 1 pound lean meat into small (4) Test for proteose with nitric acid
(2)
pieces and add to (1) after the reac- and then the Biuret test.
tion has subsided somewhat. The remainder of the preparation is
Place in a suitable water bath identical with Boyd's medium, step
(3) (2)
and bring quickly to a boil. Boil (5) thru (12).
for 3 to 4 hours with frequent References: Boyd (1917-18 p. 410), Norris
stirring. (1918-19 p. 176).
(4) After 3 hours withdraw about 10.0 cc.

996. Harvey's Hydrolyzed Meat Solution
and test for absence of proteoses
with nitric acid. If negative with- Constituents:
draw another 5 or 10.0 cc. and carry 1. Water.
out Biuret reaction. Whether hy- 2. Meat, goat, sheep or ox.
drolysis is to be continued or not Preparation
depends upon the color obtained. (1) Mince finely fat-free goat, sheep or
(5) When the color is satisfactory with- ox flesh.
draw the flask from the boiling water (2) Add to it 80.0 cc. strong commercial
and add calcium hydrate (quicklime, hydrochloric acid of specific gravity
CaO, that has been e.xposed to the 1.16, per kilogramme.
free air for some time is best) until (3) Mix thoroly.
a large precipitate of CaC04 is (4) Keep in water bath 4 days at 70C.
formed. Continue adding lime until (5) Bring the volume up to 1000.0 cc.
the precipitate occupies about 1 of for each kilogramme of minced meat
the fluid in the flask. used.
(6) Filter thru lint and wash thru the (6) Mix 1 part (5) with 2 parts boiling
lint with a little tap water. Con- water.
tinue the neutralization of the fil- (7) Add 75.0 cc. 40.0% sodium hydroxide
trate obtained with more lime, best for each kilogramme of minced meat
carried out in 3 stages so as to avoid used.
an excess of precipitate at any one (8) Make the reaction faintly alkaline to
time. litmus.
(9) Steam 60 minutes. (6) Heat the filtrate and make alkaline
(10) Pour the mixture on to a wet, thick, when the temperature reaches about
clean cloth. 80C.
(11) Collect the fluid which drains thru (7) Filter the fluid thru paper to clarify.
the cloth together with that ob- (8) Heat at 120C.
tained by squeezing the cloth. (9) Filter.
(12) Estimate and adjust the reaction of (10) Distribute in flasks.
the medium to a definite pH value or Sterilization: Sterilize by heating at 115
to faintly alkaline to litmus or 1.0% for 15 minutes.
acid to phenolphthalein. Use: Cultivation of diphtheria bacilli and
(13) Filter thru well-wetted, thick, filter toxin production. The author reported
paper. that the addition of 2.0 g. acetic acid per
(14) Add part of the filtrate to 2 parts
1 liter favored toxin production. Other
boiling water. organisms grew well on this medium.
Variants
Use: General culture medium. (a) Nicolle, Debains and Jouan culti-
Reference: Harvey (1921-22 p. 69). vated meningococci on a medium
997. Frouin and Ledebt'sHydrolyzed Serum prepared as follows:
Solution (1) Heat one liter of water to about
55C.
Constituents: Add 10.0 cc. of pure HCl (22
(2)
1. Water 1000.0 cc.
Baum's) to (1).
2. NaCl 6.0 g Add 300.0 g. of finely divided hog's
(3)
3. KCl 0.3 g stomach.
4. Potassium phosphate 0.5 g Regulate the temperature to about
(4)
5. MgS04 0.3 g 50 C. and keep at this temperature
6. CaCla 0.15 g
for 7 to 8 hours.
7. Serum albuminoids 10.0 g
(5) Heat to 80 or 90 degrees to destroy
Preparation
the pepsin and stop digestion.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(6) The peptone thus obtained may
(2) Hydrolyze 10.0 g. of serum albumi-
be preserved for several weeks.
noids in (1). (Method not given.)
(7) To use the peptone, make the
(3) Neutralize (indicator not specified).
peptone slightly alkaline to litmus
and heat at 120 C.
Use: Cultivation of colon typhoid group.
(8) Filter on a wet filter paper.
Variants: The authors added 2.0% glucose
or 3 to4.0% glycerol to the medium. (9) Add 2.0 g. of glucose.
Reference: Frouin and Ledebt (1911 p. (10) Sterilize at 112 to 115C.

24).
(b) Besson used 250.0 g. hog's stomach
998. Martin's Stomach Digest Solution instead of 200.0 g. and digested for
(Martin's Peptone) 20 to 24 hours instead of 12 to 24
Constituents hours.
1. Water 1000.0 cc. (c) Harvey digested for 20 hours and
2. Stomach (hog) 200.0 g. steamed for 30 minutes instead of
Preparation heating at 120C. as indicated in step
(1) Chop the stomachs of five hogs into (8) above.
small pieces. (d) See medium 877, step (1) thru (8)
(2) Mix 200.0 g. of (1), 10.0 g. pure HCl for Park, Williams and Krumwiede's
and 1000.0 cc. water at 50C. method of preparation.
(3) Allow to stand at 50C. for 12 to References: Martin (1898 p. 32), Thoinot
24 hours. and Masselin (1902 p. 22), Nicolle,
(4) Heatatl0OC. (Time not specified.) Debains and Jouan (1918 p. 151), Besson
(5) Strain or filter thru a layer of ab- (1920 p. 28), Harvey (1921-22 p. 98), Park,
sorbent cotton. Williams and Krumwiede (1924 p. 118).
999. Martin's Liver Digest Solution Sterilization: Sterilize (4) in the Arnold.
Method of sterilization of blood not
Constituents given. Sterilize the mi.xture of blood and
1. Water 2000.0 cc.
digest for 15 to 20 minutes in the Arnold.
2. Stomach (hog) 200.0 g.
Use: Cultivation of measles virus from
3. Liver, beef or hog 200.0 g.
patients' blood.
Preparation Reference: Sellards and Bigelow (1920-21
(1) Mi.x chopped hog stomach
200.0 g. of
p. 242).
and 200.0 g. chopped hog or beef liver,
and 20.0 cc. of commercial HCl in 2 1001. Dubovsky and Meyer's Liver Digest
liters of water. Solution
(2) Digest (1) at 50C. for 12 to 24 hours.
Constituents
(3) Boil the liquid, at the end of 24 hours.
1. Water, tap 5000.0 cc.
(4) Allow to cool and settle for 24 hours
2. Stomach, hog 400.0 g.
or less.
3. Liver 400.0 g.
(5) Decant or siphon off the clear liquid.
4. K2HPO4 (0.2%).
(6) Neutralize and make slightly alkaline
5. Heart 1000.0 g.
by the addition of soda. Indicator
Preparation
not specified.
(1) Wash clean and mince finely 5 or
(7) Heat at 120 degrees.
more large hogs' stomachs.
(8) Filter and distribute as desired.
(2) Mince an equal amount of clean
hog or beef liver.
Use: Cultivation of typhoid bacilli.
(3) Mi.x 400.0 g. (1), 400.0 g. (2), 40.0 g.
Author reported that typhoid and para
HCl and 4000.0 cc. tap water at
typhoid bacilli developed rapidly in this
50C.
medium, producing gas, and acid and
(4) Keep in glass or porcelain receptacles
finally the reaction became alkaline.
for 18-24 hours.
References: Martin (1915 p. 261), Harvey
(5) Make Biuret and tryptophan test,
(1921-22 p. 99).
when both are +
the digest is green-
yellowish and contains little un-
1000. Sellards and Bigelow's Liver Digest
digested debris.
Blood Solution
(6) Transfer to large bottles and steam
Constituents: 10 minutes at 100.0C. to stop
1. Water 1000.0 cc. digestion.
2. Veal 100.0 g. (7) Strain the digest thru cotton or
3. Stomach (hog) 150.0 g. preferably store over night in the
4. Liver 200.0 g. ice chest and decant after 24 hours.
5. Blood, horse or rabbit (8) Warm the digest to 70C. and neu-
(10.0%) 100.0 cc. tralize with 2 N Na2C03 using
Preparation litmus.
(1) Prepare apeptone by digesting (9) Filter the desired amount, add 0.2%
hog stomach and
100.0 g. veal, 150.0 g. K2HPO4 and adjust to "pH = 7.4.
200.0 g. liver in a liter of water con- (10) Slowly heat to boiling finely ground
taining 1.0% concentrated HCl, for 1000.0 g. fat-free heart and tap water
19 hours at 53C. 1000.0 cc.
(2) Heat in an Arnold to destroy the pep- (11) Adjust to pH = 8.0 or 8.2.
sin. (12) Cool and carefully skim off the layer
(3) Decant the supernatant fluid and of fat which floats on medium.
neutralize at room temperature first (13) To each liter of (12) add 2 liters
to litmus and then to china blue and of (9).
rosalic acid. (14) Adjust to pH = 7.2 or 7.4.
(4) Again decant and dilute with an equal Sterilization: Sterilize at 18 pounds pres-
volume of water. sure for one hour. Incubate 5 days and
(5) Add 10.0%, horse or rabbit blood to repeat the same sterilization for one hour
sterile (4). at 18 pounds pressure.
Use: Cultivation and toxin production by (10) Transfer to large bottles and steam
B. botulinus. Authors reported toxin for 10 minutes to stop digestion.
production very good in this medium. (11) Strain thru cotton or preferably
Reference: Dubovsky and Meyer (1922 store over night in ice chest and
p. 505). decant after 24 hours.
(12) Warm (5) to 70C. and neutralize
1002. Emery's Spleen Digest Solution with 2 normal Na2C02 to litmus.
Constituents (13) Filter into a flask.
1. Water 1500.0 cc. (14) Add the 2.0 g. K0HPO4.
2. Spleen (beef) 1500.0 g. (15) Adjust to reaction using
desired
3. Stomach (hog) 250.0 g. litmus or preferably to a definite
Preparation H-ion concentration (pH = 7.0
(1) Digest 250.0 g. of beef spleen pulp, to 7.5).

250.0 g. hog stomach with 1.5 liters (16) Clear (15) by adding 5-10.0% of the
of water acidulated with 15.0 cc. HCl decanted beef serum. Steam 45-60
for 24 hours. minutes.
(2) Add beef spleen pulp (500.0 g. to 1.0 (17) Remove (16) from steamer and
liter ofwater) to (1). allow the clot to form as a compact
(3) Boil 10 minutes. mass. Decant, or better centrifuge,
(4) Filter thru wet absorbent cotton. the medium to remove it.
Sterilization: Not specified. Sterilization: Sterilize at 100 for 30
Use: Differentiation of coli and typhoid minutes on 2 successive days.
bacilli. Use: General inexpensive culture medium.
Reference: Emery (1901 p. 979). Reference: Stickel and Meyer (1918 p. 81).
1003. Stickel and Meyer's Blood Clot Digest 1004. Bramigk's Peptic Digest Solution
Solution
Constituents:
1. Tap water 1000.0 cc. 2. Blood clot 1000.0 g.
2. Blood clots 100.0 g. 3. Stomach (hog) 2
3. Pig's stomach (minced) 100.0 g. Preparation
4. K2HPO4 2.0 g. (1) Obtain a bucket of blood clots from
Preparation the slaughter house.
(1) Obtain 10 liters fresh beef blood from (2) Wash the clots free from blood with
the abattoir. running water.
(2) Decant and store the serum (which (3) Press the clots free from water.
has separated on standing) in a (4) To 1000.0 g. clots (contains about
refrigerator. 230.0 g. fibrin) add 3 liters of water
(3) Weigh the blood clots and mix and 15.0 cc. of H2SO4.
100.0 g. with 1 liter tap water. (5) Allow to stand over night.
(4) Place mixture in an enameled pot, (6) Filter thru a sieve and press the
bring slowly to a boil and boil slowly water from the clot or fibrin.
for 5 minutes, stirring constantly. (7) Add the clot to 3 liters water and
(5) Wash and mince pig's stomach. 18.0 cc. H2SO4 heated to about 50C.
(6) Cool (4) to 50C. and add 100.0 g. of (8) Remove the mucous membrane from
minced pig's stomach for each liter two fresh pigs' stomachs.
of (4). (9) Run the stomachs thru a meat chop-
(7) Transfer to glass or porcelain re- ping machine.
ceptable and finally add 1.0% HCl. (10) Mix (9) with 1 liter of water and
(8) Digest at 50C. for 18-24 hours. 8.0 cc. H2SO4.
(9) Make a Biuret and tryptophane, (11) Heat to 35C.
test; when both are +, the digest is (12) Mix (11) and (7) thoroly and place
yellowish green and contains very at 37C. for 48 hours. Stir fre-
little indigested debris. quently. Some fat will settle out.
(13) Filter and neutralize with ammonia. (14) Collect the fluid which drains thru
(14) Distribute in flasks and sterilize. the cloth, together with that ob-
Sterilization: Method not given. tained by squeezing the cloth.
Use: General culture medium and substi- (15) Filter the fluid collected thru well-
tute for commercial peptone. wetted, thick, filter paper.
Variants: The author prepared a dry ma- (16) Bring the volume up to 1000.0 cc.
terialby treating the medium obtained (17) Filter thru well wetted, thick filter
above as follows: paper.
(1) Neutralize the digest with Ba(0H)2 (18) Make the reaction neutral to litmus.
(about 40.0 g.) and boil until the (19) Add 75.0 cc. of the decanted serum
upper fluid is clear. (3). _
(2) The reaction should be slightly acid, (20) Steam sixty minutes.
showing a light excess of H2SO4. (21) Allow the clot to form a compact
(3) Allow the digest to settle.
(4) Pour off the clear solution and filter (22) Decant the clear fluid.
the remainder, washing the pre- Sterilization: Sterilize at 100C.
cipitate with water. Use: General culture medium.
(5) This gives a light yellow solution con- Reference: Harvey (1921-22 p. 100).
taining about 10.0% peptone.
This fluid may be evaporated over a 1006. Martin's Stomach Digest Infusion
(6)
Broth
water bath or in a vacuum to a thick
syrupy paste and then drawn into Constituents:
threads and dried in a desiccator. 1. Water 2000.0 cc.
(7) This peptone is equal to Witte's pep- 2. Veal 500.0 g.
tone in the preparation of media. 3. NaCl 5.0 g.
Reference: Bramigk (1921 p. 429). 4. Stomach (hog) 200.0 g.
Preparation
1005. Harvey's Blood Clot Digest
(1) Infuse 500.0 g. of finely ground veal
Solution
with 1000.0 g. water for 20 hours
Constituents: at35C.
1. Water 1000.0 cc. (2) Strain out the meat and add 5.0 g.
2. Blood. NaCl to the fluid.
3. Pig's stomach 100.0 g. (3) Chop the stomachs of 5 hogs into
4. HCl 10.0 g. small pieces.
Preparation (4) Mix 200.0 g. of (3), 10.0 g. HCl (pure)
(1) Procure ox or sheep blood from the and 1000.0 cc. water at 50C.
slaughter house. (5) Allow to stand at 50C. for 12 to 24
(2) Allow to clot. hours.
(3) Decant and store the serum in an (6) Heat at 100C. (Time not spec-
ice chest. ified).
(4) Mince the clot. (7) Strain or filter thru a layer of ab-
(5) Add 100.0 g. clot to 1000.0 cc. tap sorbent cotton.
water. (8) Heat the filtrate and make alkaline
(6) Raise slowly to boiling temperature. when the temperature reaches about
(7) Boil 10 minutes. 80C.
(8) Cool to 50C. (9) Filter the fluid thru paper to clarify.
(9) Add 100.0 g. minced pig's stomach (10) .Mix 1 liter of (9)with 1 liter of (2).
and
10.0 cc. strong hydrochloric acid. (11) Heatto70C.
(10) Digest 20 hours. (12) Filter thru paper.
(11) Make a biuret and a tryptophane (13) Make alkaline (method not given).
test. Sterilization: Method not given.
(12) Boil 10 minutes to stop the digestion. Use: Cultivation of diphtheria bacilli and
(13) Pour the mixture on to a wet, thick, to.xin production. Author reported that
clean cloth. heating the medium decreased the ability
of the medium to produce toxin. Heat- (g) Klimmer used the stomachs of 5 hogs,
ing only to 70C. and filtering thru a but did not specify the use of 200.0 g.
Chamberland filter gave good results. He carried on the digestion for 24
Other investigators utilized a similar me- hours and then sterilized. Equal
dium for the production of tetanus toxin. volumes of the sterile digest
Variants (1000.0 cc.) and the infusion from
(a) Thoinot and Masselin used the 500.0 g. of veal to 1000.0 cc. of water
stomachs of 5 hogs, but did not were mixed, boiled and neutralized.
specify the use of 200.0 g. They also The reaction was made slightly alka-
used 500.0 g. beef instead of veal. line, or adjusted to any desired pH
(b) BezanQon digested the stomach for value, filtered, tubed and sterilized
24 hours instead of 12 to 24, infused in the usual manner,
beef instead of veal, heated the mix- (h) Park, Williams and Krumwiede in-
ture at 115 for 15 minutes instead of fused the veal and 1000.0 cc. of water
heating at 70 C. in step (11) above for 18 to 24 hours at 35C., heated at
and sterilized in the autoclave at 45 to 48C. for one hour, then boiled
115 for 15 minutes. briskly for 30 minutes and strained
(c) Besson used 250.0 g. hogs stomach thru cheese cloth. This infusion at
instead of 200.0 and added 7.0 cc.
g. 70 C. was mixed with an equal
of normal soda solution to the me- volume of stomach digest at 70C.,
dium after it had been neutralized to prepared according to Martin's
litmus. method, and heated to boiling. The
(d) Wilcox studied the production of reaction was adjusted to -f-0.5 to
tetanus toxin using one of the follow- phenolphthalein at room tempera-
ing combinations, prepared according ture. The medium was sterilized at
to Martin's process: 15 pounds pressure for 30 minutes
(1) (a) 200 g. minced stomach in after the reaction was readjusted
1000.0 cc. water. and filtered thru cotton and paper.
(b) 400 g. minced stomach in They prepared a similar medium with
1000.0 cc. water. a reaction of +1.0 to phenolphthalein
(c) 200 g. minced stomach in and added 1.0% glucose. This me-
1500.0 cc. water. dium was used for the production of
(d) 300 g. minced stomach in tetanus toxin.
1000.0 cc. water. References: Martin (1898 p. 35), Thoinot
(2) Veal infusions prepared from one of and Masselin (1902 p. 23), BezanQon
the following: (1920 p. Ill), Besson (1920 p. 29), Wilcox
(a) 500 g. veal to 1000.0 cc. water. (1921 p. 414), Dopter and Sacquepee (1921
(b) 500 g. veal to 500.0 cc. water. p. 119), Harvey (1921-22 p. 99), Klimmer
(3) Add 1.0% glucose if desired. (1923 p. 200), Park, Williams and Krum-
Author reported that 1 part (1) (a) wiede (1924 p. 133).
to 1 part (2) (a) gave highest toxin
production with organism studied. 1007. Besredka and Jupille's Egg Stomach
(e) Dopter and Sacquepee used beef in- Digest Solution
stead of veal, added 7.0 cc. of normal
soda to the medium after it had been Constituents:
neutralized to litmus and autoclaved 1. Distilled water.
2. Beef or veal 500.0 to 750.0 g.
at 117C. for 15 minutes instead of
heating at 70C. as in step (11) above. 3. Egg.
(f) Harvey neutralized the medium to 4. Stomach (hog).
litmus added 7.0 cc. of normal NaOH Preparation

per liter, then filtered thru well- (1) Beat the white of eggs with 10
wetted thick filter paper, steamed 30 volumes of distilled water, adding
minutes, filtered again thru well- the water little by little.
wetted thick filter paper while hot, (2) To clarify run thru a sieve covered
distributed and sterilized. with a thin layer of absorbent cotton.
(3) Heat to 100C. and then filter on a 2. Stomachs (hog) 400.0 g.

Chardin paper. 3. Liver, beef placenta or blood
(4) Distribute the opalescent fluid in clots 400.0 g.
flasks or tubes in 20.0 cc. lots. 4. K2HPO, 8.0 g.
(5) Dilute egg yolks with 10 volumes of Preparation

distilled water. (1) Wash clean and mince finely 5 or
(6) Clarify the egg yolk by the addition more large pig's stomachs. Mince
of normal soda solution. Generally an equal amount of clean pig's or
1.0 cc. of normal soda solution to beef liver, cheap fat-free beef, pla-
100.0 cc. suspension of egg yolk will centa or blood clots.
give the proper solution. However, (2) Mix 2, one of 3 and 40.0 g. of Baker's
different yolks may require only Chemical Co. HCl in 1 at 50C. and
half as much. The suspension keep at 50C. for 18 to 24 hours.
should be slightly yellow in a thin (3) Make a Biuret and tryptophane
layer. test. When both are + the digest
(7) Heat at 100C. is yellowish green and contains very
(8) Filter on Chardin paper. undigested debris.

little
(9) Distribute in flasks or tubes in (4) Transfer to large bottles and steam
20.0 cc. lots. for 10 minutes at 100C. to stop
(10) Prepare a bouillon from 500 to digestion.
750.0 g. chopped beef or veal with (5) Strain thru cotton or preferably
1000.0 cc. water and Martin's pep- store over night in the ice chest and
tone, by heating to boiling under a decant after 24 hours.
low flame and then boiling for (6) Warm (5) to 70C. and neutralize
30 minutes. See med. (1006). with 2 N Na2C03, to litmus.
(11) Filter and make slightly alkaline. (7) Filter into a flask.
(12) Heat at 120C. to activate pre- (8) AddS.Og. K2HPO4.
cipitation. (9) Adjust to desired reaction using lit-
(13) Filterthru double paper. mus or preferably to a definite H-ion
(14) Distribute in flasks or Rou.x tubes. concentration (pH 7.0 to 7.5).
(15) Add to 500.0 cc. of (14), 400.0 cc. of (10) Heat in steamer at 100 for 15
(4) egg white solution), and
(sterile minutes.
100.0 cc. (9) (sterile egg yolk solu- (11) Correct reaction and filter thru
tion) under aseptic conditions. paper.
(16) Distribute in sterile tubes in 10.0 cc. (12) Distribute in receptacles used for
lots under aseptic conditions. cultures.
Sterilization: Sterilize (4) and (9) at 115C. Sterilization: Sterilize at 100 for 30 min-
for 20 minutes. Sterilize (14) at 115C. utes on 2 successive days or at 10 pounds
for 25 minutes. pressure for 15 minutes.
Use: General culture medium, for highly Use: General inexpensive culture medium.
parasitic and saprophytic forms. To cul- Variants
tivate tubercle bacilli, add cold, and (a) The authors prepared a medium as
under aseptic conditions, 20.0 cc. of indicated from step (1) thru (6).
(4) (egg white solution) and 5 to 20.0 cc. The remainder of the preparation was
of (9) (egg yolk solution) to 100.0 cc. of as follows:
the bouillon, prepared as before but con- (7) Sterilize at 10 pounds pressure for
taining no peptone. 15 minutes in the autoclave or for
Reference: Besredka and Jupille (1913 30 minutes at 100 C. on 2 suc-
p. 1009). cessive days.
(8) Inoculate (7) with 1.0% of a
1008. Stickel and Meyer's Meat Digest 24 hour old broth culture of B.
Solution
saccharolyte or B. coli and incu-
Constituents: bate for 12-18 hours at 37C.
1. Water, tap 4000.0 cc. (9) Steam for 20 minutes.
(10) Adjust to desired reaction. (5) Allow the flask to stand at room
(11) Add 0.2-0.4% K2HPO4 and 2.0% temperature or in the incubator.
of purified talcum. Shake thoroly several times a day.
(12) Filter thru paper. (6) Add 5.0% HCI from time to time.
(13) Distribute for use. Use Congo red as an indicator (blue
(14) Sterilize at 100C. for 30 minutes color).
on 2 successive days or at 10 (7) About 10 to 14 days are required for
pounds pressure for 15 minutes. complete digestion of the fibrin.
(b) Park, Williams and Krumwiede omit- The reaction may be allowed to reach
ted the K2HPO4. acid to litmus at this time.
References : Stickel and Meyer (1918 pp. 78, (8) Allow the digest to stand undis-
79),Harvey (1921-22 p. 99), Park, Wil- turbed for 2 or 3 days.
liams and Krumwiede (1924 p. 119). (9) In order to obtain a fat-free digest,
remove the bottom dark brown
1009. Frieber's Gelatin Digest Solution digest by means of a rubber tubing.
Constituents: The fat floats on the top surface.
1. Water 2000.0 cc. (10) In order to obtain about a 1.0%
2. Gelatin 20.0 g. peptone solution dilute the stock
3. NaCl 5.0 g. solution about 3 with water and add
4. KH2PO4 2.0 g. 5.0% NaOH solution to neutralize
5. MgS04 0.2 g. to litmus.
6. Tryptophane 0.3 g. (11) A heavy flaky precipitate is formed.
Preparation Do not add an excess of NaOH or
(1) Add 20.0 g. of gelatin, 10.0 cc. HCI the precipitate will dissolve.
and 2.0 g. Witte's peptone to 2 liters (12) Boil and filter or allow the pre-
of water and allow to digest for cipitate to settle. This gives a light
several days. yellowish 1.0% peptone solution.
(2) Neutralize to litmus by adding It is necessary to add 0.5% NaCl.

NaOH. (13) Distribute in flasks.
(3) Dilute (2) with an equal volume of Sterilization: Method not given.
water. Use: Substitute for commercial peptone.
(4) Dissolve 3, 4, 5 and 6 in (3). Reference: Frieber (1921 p. 425).
(5) Add 7.0 cc. of normal soda solution.
Sterilization: Not specified. 1111. Bramigk's Peptic Tryptic Digest
Use: Indol production. Solution
Constituents:
1110. Frieber's Fibrin Digest Solution 1. Water 4000.0 cc.
2. Blood clot 1000.0 g.
Constituents
3. Stomach (hog) 2
1. Water.
Preparation
2. Fibrin.
0.5% (1) Prepare Bramigk's Blood Clot Digest
3. NaCl
solution, see medium 1004.
Preparation
Wash running water until
fibrin in (2) Add Ba(0H)2 to 1000.0 cc. of (1) until
(1)
Press out the water. the reaction is only slightly acid.
it is white.
The fibrin containing about 25% (3) Boil to clarify.
dry material is placed in a flask so (4) Add without filtration 5.0 cc. water
that the flask is about ^ full. free soda (strength solution not
Add to each liter flask about 3 to given), 5.0 cc. toluol, 5.0 cc. chloro-
(2)
5 grams pepsin powder and fill the form, and 2.0 g. pancreaten (Rhe-
flask with water. nania).
(3) For each liter of material add 10.0 cc. (5) Mix well and incubate at 37 for 120
of concentrated (1.19) HCI. hours, shaking occasionally. (Test
(4) Shake thoroly. for tryptophane using bromine
water.) Add HCI to stop the di- (5) Strain fluid thru cheese cloth and
gestion. pass the residue thru a fruit press,
Sterilization: Method not given. cool to 37C.
Use: Indol production. Use this digest in (6) Make the thick brownish fluid
1.0 or 2.0% solution for indol reaction. slightly alkaline to litmus.
Author reported that the addition of (7) Add 1.0% pancreatic extract and
0.1% of this digest gave equally as good incubate at 37 for 5, 24, 48 hours.
growth when added to nutrient medium (8) When the process is sufficiently ad-
as did 1.0% Witte's peptone. vanced, render slightly acid with
Reference: Bramigk (1921 p. 431). glacial acetic acid and boil slowly
for 15 minutes.
1112. Jensen's Milk Digest Solution
(9) Either filter or decant the clear fluid
Constituents: which results on placing the digest
1. Milk 1000.0 cc. over night in a cool place.
Preparation (10) Adjust the reaction as desired.
(1) To sterilized milk add 10.0 cc. of pure (11) Dissolve 3 in (10).
concentrated HCI per liter and 2.0 g. (12) Heat
for 15-30 minutes in the
of a pure pepsin preparation. steamer at 100 C.
(2) Incubate at 35-37 for 36 to 48 hours (13) Filter again if necessary.
shaking often at first until the casein (14) Clear (13) by adding 5-10.0% of the
has precipitated and to dissolve the decanted beef serum. Steam 45-
pepsin, and then only occasionally. 60 minutes.
(3) Approximately neutralize the acid (15) Remove (14) from steamer and allow
(acid hydrolyzes the lactose when clot to form as a compact mass.
heated) and heat in the autoclave at Decant, or better, centrifuge the
115 to 120 for 10 minutes. medium to remove the clot. * .
(4) Filter thru paper. Sterilization: Sterilize at 100 for 30 min-

(5) Adjust the reaction (neutral to lit- utes on each of 2 successive days.
mus). Use: Author reported that this medium
(6) May be clarified with egg white if was excellent for primary isolation of
desired. highly parasitic organisms.
SterilizationSterilize in the usual manner.
: Variants: Knorr prepared a similar me-
Use: Cultivation of lactic acid bacteria. dium as follows:
Reference: Jensen (1898 p. 199). (1) Mix 1 kilo of blood clots with
1500.0 cc. water, and boil for 1 hour
1113. Stickel and Meyer's Tr)rpsinized
on an open flame. Stir often.
Blood Clot Solution
(2) Press the fluid thru a filtering cloth
Constituents: and run the blood clot thru a meat
1. Water, tap 1000.0 cc. grinding machine.
2. Blood clots 500.0 g. (3) Add the ground blood clot to the
3. K0HPO4 2.0 g fluid. Stir.
Preparation (4) Add 2.0 g. pancreatin and
1.0 to
(1) Obtain 10 liters fresh beef blood from chloroform and digest for 6 to 7
the abattoir. days at room temperature. Mix the
(2) Decant and store the serum (which chocolate brown liquid often.
has separated on standing) in a (5) Acidify to stop the digestion.
refrigerator. (6) Filter thru paper.
(3) Weigh the blood clots and mix (7) Add enough water to the residue to
500.0 g. with 1 liter tap water. make a 4000.0 cc. stock solution.
(4) Place the mixture in an enameled (8) Add 100.0 g. of rock salt (cattle salt)
pot, bring slowly to a boil, and boil and potassium phosphate.
15.0 g.
slowly for 5 minutes, stirring con- (9) Filter. This is the stock solution.
stantly. It gives a tryptophane reaction.
(10) When ready for use, use in 40.0% (5) Precipitate out the oxalate by the
strength. addition of calcium acetate.
(11) Sterilization of stock solution not (6) Add 40.0 g. trypsin to the plasma and
specified. allow it to act for 8 days.
References: Stickel and Meyer (1918 p. 81), (7) At the end of this time, boil for a few
Knorr (1921 p. 598), Harvey (1921-22 minutes, allow to stand and filter.
p. 117), Klimmer (1923 p. 176). Method not given.
Sterilization:
Use: Inexpensive medium. Author re-
1114. Spray's Blood Clot Digest Solution ported that this medium contains about
Constituents: 8.0% nutrient materials and should be
1. Water. diluted 1:3 before use.
2. Blood clot. Reference: Loffl (1915-16 p. 109).
3. NajCOa.
Preparation 1116. Gordon et al. Trypsinized Heart
Remove the serum from the blood Solution (Tanner)
(1)
clot and allow to drain. Constituents:
(2) Pass the clot thru a wire gauze. 1. Water 1000.0 cc.
(3) Boil and divide finely again. 2. Heart, beef 500.0 g.
(4) Place one liter of this
semi fluid ma- Preparation
terial in a two liter flask. (1) Free fresh bullock's heart from fat
(5) Add 20.0 g. anhydrous
Na2C03, 5.0 g. and blood vessels.
trypsin, and 15.0 to 20.0 cc. chloro-
(2) Mince (1) very finely and weigh.
form. (3) To each 0.5 kilo add 1 liter of water,
(6) Incubate for 15 days with a second and make faintly alkaline to litmus
addition of 3.0 g. trypsin on the by the addition of 20.0% KOH
tenth day. Chloroform may be solution.
added on the fifth and tenth days. (4) Heat slowly to 75 to 80C. for
(7) Make strongly acid by the addition 5 minutes.
of 50.0 to 75.0 cc. HCl. (5) Cool to 37C. and add 1.0% of liquid
(8) Steam in the water bath to drive off
trypsine comp., and keep at 37 for
the chloroform. 2.5 to 3 hours.
(9) Adjust to pH = 7.4 to 7.5.
(6) When trypsinizing is finished test for
(10) Tube. peptone with copper sulphate and
Sterilization: Sterilize in the autoclave. KOH (Method not given).
Use: The author reported that this blood (7) Render slightly acid by the addition
clot digest may be added in from 5.0 to of glacial acetic acid.
10.0% to melted North's or other agar for (8) Bring slowly to boil and boil for
growth of H. influenzae, pneumococcus 25 minutes.
and streptococcus. When sodium oleate (9) Leave over night in a cool place.
is added H. influenzae grows very
vigor- Siphon off the clear liquid in the
(10)
ously. morning.
Reference: Spray (1927 p. 14). (11) Make faintly alkaline to litmus.
Sterilization: Sterilize in an autoclave at
1115. Loffl's Trypsinized Blood Solution
118 for one hour on each of two days.
Constituents: Use: General culture medium.
1. Water. Reference: Tanner (1919 p. 45).
2. Blood.
Preparation 1117. Harvey's Trypsinized Heart Solution
(1) Allow 9 liters of blood to flow into a
liter of 0.8% ammonium oxalate. Constituents:
Mix well. 1. Water 400.0 cc.
(2)
Centrifuge at 3000 revolutions per 2. Heart, ox 1
(3)
minute. 3. NaCl 1.0 g.
This yields about 6 liters of plasma. 4. CaCli 0.5 g.

(4)
Preparation (2) Add 500.0 g. to 1000.0 cc. water.

(1) Mince finely an average sized ox (3) Make faintly alkaline to litmus.
heart. (4) Heat slowly to 75C.
(2) Add to 400.0 cc. tap water. (5) Allow to cool to 45C.
(3) Heat slowly to 75C. (6) Add trypsin solution to 1 per cent
(4) Allow to cool to 45C. and 35.0 cc. chloroform.
(5) Add trypsin solution to per cent.
1 (7) Place in a loose stoppered bottle.
Note: e.g., Liq. trypsin Co. (A (8) Keep 10 days at 37C. with daily
(and H)). shaking.
(6) Place in incubator 21 hours. Note: The reaction must be
(7) Test for peptone by the Biuret test. frequently tested and made faintly
Note: Add 1.0 cc. 5 per cent alkaline to litmus.
copper sulphate to 5.0 cc. trypsin (9) Add, at the end of this time,
digest, followed by 5.0 cc. N/1 po- trypsin solution again to 1 per
tassium hydro.xide. Note the color cent.
change. If the color is pink, pep- (10) Keep a
further period of 10 days
tonization is complete; if bluish at 37C. without shaking:
purple incomplete. Note: The reaction must be
(8) Make faintly acid to litmus with frequently tested and made faintly
4 per cent acetic acid. alkaline to litmus.
(9) Boil 15 minutes. (11) Make faintly acid to litmus with
(10) Allow the solid matter to settle. 4 per cent acetic acid.
Note: Or simply strain thru cloth. (12) Boil 15 minutes.
(11) Pour off the supernatant fluid. (13) Allow the solid matter to settle
(12) Add 1.0 g. sodium chloride and 0.5 g. by placing in the ice chest over
calcium chloride. night.
(13) Make faintly alkaline to litmus. (14) Pour off the supernatant fluid or
(14) Steam 45 minutes. filter thru well-wetted, thick, filter
(15) Bring up to original volume. paper.
(16) Adjust the reaction. (15) Make the reaction 1.2 per cent
(17) Steam 30 minutes. acid to phenolphthalein.
(18) Clarify by the addition of egg (16) Steam 45 minutes.
albumin. (17) Filter.
(19) Tube. (18) Sterilize in the steamer or auto-
Sterilization: Sterilize in the autoclave or clave.
steamer. Reference: Harvey (1921-22 p. 114).
ported good growth of meningococci and
1118. Distaso's Trypsinized Serum Solution
similar organisms when pea extract or Constituents

serum was added. 1. Distilled water 500.0 cc.
Variants: The author gives the following 2. Serum (beef of sheep) 500.0 cc.
variants Preparation
(a)|Add 25.0% of sterile rabbit or horse (1) Mix equal parts of water and sheep or
serum. beef serum.
(b) Add 5.0% of a sterile pea extract pre- (2) Digest sterile (1) for 24 hours at 60C.
pared by steaming a mi.xture of 50.0 g. with a pancreatic extract from a hog
pea flour and 100.0 g. NaCl in in the presence of chloroform. Acti-
1000.0 cc. distilled water for 30 vate the extract with an extract of
minutes, filtering thru paper and the upper portions of the small in-
sterilizing. testines.
(c) The medium was prepared as fol- (3) Filter on paper.
lows from horse heart, omitting the (4) Tube.
NaCl and CaCh. Sterilization: Sterilize (1) at 120 for 15
(1) Mince finely fat-free horse heart. minutes. Sterilize (4) method not given.
Use: General culture medium. Substitute Preparation

for bouillon. Author stated that sugars (1) Add 1.5 liters of boiling water to
might be added to this medium if desired. 1000.0 g. of meat, cut in pieces the
Harvey used a similar medium to demon- thickness of a finger.
strate the indol test. (2) Apply heat until the mixture boils
Variants again.
(a) The author gave a more complete (3) Remove the pieces of meat.
method of the preparation as follows: (4) Pass the meat thru a grinding ma-
(1) Chop the pancreas of a hog into chine.
small pieces and extract with (5) Place the liquid in a large mouthed
chloroform in 9 times its volume of flask, add 1.5 g. dehydrated soda
distilled water for 25 hours. and when the liquid is lukewarm add
(2) Filter on glass wool, pressing the 3.0 g. pancreatin and 15-20.0 cc.
meat free from the liquid. chloroform (and toluol).
(3) Add ether to preserve the extract (6) Seal the flask and mix well.
and store in a sealed flask. (7) Add the ground meat (4) and shake
(4) Mix beef or sheep serum with an once more.
equal volume of water. (8) The meat should be thoroly covered
(5) Boil (4) until it assumes a milky with liquid. If not add water.
appearance. (9) Incubate for 5 days at 20 or 2 days
(6) Mix 15volumes of (3) with 100 at 37. Shake daily.
volumes and digest over night
of (5)
(10) Acidify slightly by the addition of
at 60C. on a salt water bath. HCl.
(7) Filter. (11) Filter thru paper.
(8) Tube Wash the residue on the filter paper
(12)
(9) Sterilize at 120 for 15
minutes.
until the volume of the filtrate is
(b) Harvey mixed one part serum with
3 liters.
2 parts tap water and boiled until the
(13) Filter again after shaking thoroly.
mixture was milky. Then added
(14) Boil for at least 10 minutes.
15.0 cc. ofpancreatic extract to
(15) Dilute to 10 to 50 liters.
100.0 cc. of the mixture and digested
(16) Prepare a saturated solution of crude
at 60C.for 24 hours. Following
salt (not purified) rich in calcium
filtrationthru paper and distribution,
the medium was sterilized by heating
and magnesium.
at 120C. for 15 minutes. This me- (17) Dissolve 5.0 g. of K2HPO4 and small
knife point of K2HPO4 in a little
dium was used for the detection of
distilled water. Filter.
indol production.
(c) Harvey used equal parts serum and (18) Add (17) to 100.0 cc. of filtered (16).
water boiled as above and added (19) Add 20.0 cc. of (18) to each liter of
10.0% pancreatic extract. After di- (15).
gestion at 60C. over night, the me- (20) Filter.

dium was filtered thru thick filter (21) Boil.
paper, tubed and sterilized in the (22) Make slightly alkaline.
autoclave. Sterilization: Not specified.
References: Distaso (1916 p. 600), (1917 Use: General culture medium. Klimmer
p. 253), Harvey (1921-22 pp. 81, 117). reported that for bacteria producing pig-
ment dilute the solution to 50 liters in
1119. Hettinger's Trypsinized Meat step (15), for usual laboratory work to
Solution (Klimmer) 30 liters and to 10 liters if an extremely
Constituents: good culture medium is desired.
1. Water. Variants
2. Meat. (a) Park, Williams and Krumwiede give
3. K2HPO4. the following method of preparation
4. NaCl. of the Hettinger medium. The phos-
5. K3PO4. phates are omitted.
(1) Free 750.0 g. meat from fascia and (24) Dilute the stock broth as desired
cut in finger-thick pieces. for According to Hottinger
use.
(2) Drop (1), piece by piece, into it may
be diluted 10, 20 or more
1500.0 g. of boiling water, stirring times. Excellent results were ob-
constantly. tained, however, by diluting 1 part
(3) Boil up strongly and take from stock broth with 1 part water,
fire. (b) Park, Williams and Krumwiede pre-
(4) Take out the meat and put thru pared a similar medium as follows:
a chopping machine. (1) Add 300 to 500.0 g. of fat-free
(5) Cool the water to 37C. and add chopped meat to 1000.0 g. of water
1.5 g. NaoCOs per liter. to which 0.4% Na-iCOj has been
(6) Put the chopped meat in flasks added.
(2 liter Erlenmeyer) 550.0 g. in (2) Soak over night.
each flask. (3) Heat at 80 C.
(7) Add (5) (the water) at 37C. to the (4) Cool to 38C. and add 15.0 cc. of
flask, filling them up to the narrow liquid trypsin.
neck. (5) Keep at 38C. for 5 hours stirring
(8) Add 3.0 g. of pancreatin, 10.0 cc. frequently. If kept over night at
chloroform and 10.0 cc. toluol to this temperature add 10.0 cc. of
each flask. toluol (or thymol crystals).
(9) Cork tightly. (6) Add normal HCl to neutralize
(10) Shake thoroly. (indicator not specified).
(11) Incubate at 37 over night. (7) Boil 7 minutes.
(12) Shake the next morning and add (8) Strain.
more pancreatin unless the fluid (9) Adjust the reaction.
shows a yellow color, and particles (10) Boil 30 minutes.
of meat look smaller, indicating (11) Filter.
digestion. (method not given).
(12) Sterilize
(13) Digest for 4 or 5 days at room References: Klimmer (1923 p. 175), Park,
temperature or for 2 or 3 days in Williams and Krumwiede (1924 pp. 118,
the incubator. Shake the flasks 119).
each day. The meat should be in
1120. Peckham's Trypsinized Beef Solution
a finely divided mass, giving off a
very offensive odor, when digestion Constituents
is complete. 1. Water 1000.0 cc.
(14) The medium may be stored in the 2. Beef 500.0 g.
ice box, after acidifying to litmus 3. NaCl 5.0 g.
by the addition of HCl, if neces- 4. Trypsin 4.0 g.
sary. (Hottinger's statement.) Preparation
(15) As soon as the digestion is com- (1) Place 500.0 g. of finely chopped beef
plete decant the liquid thru cheese which is as old as can be obtained
cloth. (This process gives better from the shops, in order that it be
results according to the author.) free from muscle sugar, in 500.0 cc.
(16) Add an equal amount of water of water.
to the residue. (2) Make slightly alkaline with sodium
(17) Shake (16) thoroly. carbonate.
(18) Allow to settle and again decant. (3) Place in a water bath and raise tem-
(19) Place the meat on the cheese cloth perature to 40C. and add trypsin.
and allow to drain. (4) After an hour the mixture must be
(20) Boil the filtrate a few minutes. again made alkaline with sodium
(21) Filter thru absorbent cotton and carbonate.
paper until clear. (5) Allow to digest only from one to Ih
(22) Autoclave at 15 pounds pressure hours, or traces of indol may be
for 30 minutes. detected.
(23) Store as stock broth. (6) Boil and strain thru gauze.
(7) Filter thru wet filter paper when Sterilization: Filter (12) thru a Berkefeld
cold to remove fat. and a Doulton candle in series.
(8) Add and enough water to bring
salt Use: Cultivation of B. tetani and other
the volume to 1 liter. anaerobic organisms. Author reported
(9) Adjust so that 20 to 30.0 cc. of a that B. sporogenes was inhibited.
decinormal NaOH solution will be Variants: Harvey prepared a similar me-
required to bring one liter of the dium as follows:
medium the
to neutral point of (1) Mince finely fat-free beef or rabbit
phenolphthalein. flesh.
(10) Clarify and filter. (2) Add 500.0 g. to 1000.0 cc. water con-
Sterilization: Method not given. taining 5.0g. sodium carbonate.
Use: Indol production. (3) Place in incubator 20 hours.
Reference: Peckham (1897 p. 554). (4) Make faintly alkaline to litmus.
(5) Add trypsin solution to 2 per cent.
1121. Harvey's Trypsinized Meat and (6) Place in the incubator for a further
Kidney Solution 20 hours.
(7) Filter thru well-wetted, thick filter
Constituents
paper.
1. Water 1000.0 cc.
Meat 500.0 g. (8) Make faintly acid to litmus.
2.
Kidney, rabbit (9) Steam or boil 45 minutes.
3.
Preparation
paper.
(1) Mince meat.
finely, fat-free lean
(11) Bring the volume up to 1000.0 cc.
(2) Add 500.0 g. to 1000.0 cc. tap water.
Heat the mixture 20 minutes at a (12) Make neutral to phenolphthalein.
(3)
temperature not exceeding 50C. (13) Steam 30 minutes.
(14) Filter, while hot, thru well-wetted
(4) Skim on the surface.
off fat floating
thick filter paper. Occasionally it
(5) Raise the temperature rapidly to
boiling point.
may be necessary to filter thru a
Doulton candle.
(15) Distribute the filtrate into test
(7) Make faintly alkaline to litmus.
tubes.
(8) Add trypsin solution to 2 per cent.
Place in incubator in an open vessel, (16) Add just before use l/16th part of
(9)
4 days.
fresh sterile rabbit kidney and use as
soon as possible after the addition of
the kidney.
paper.
(11) Bring the volume to 1000.0 cc. by
1122. Celozzi's Placenta Digest Blood
(12) Make neutral to phenolphthalein at
Solution
room temperature.
(13) Store the sterile medium in sterile Constituents
flasks under layer of paraffin to 1. Ringer-Locke solution 1000.0 cc.
which sodium formate has been 2. Placenta 500.0 g.
added to the extent of 1 per cent of 3. Glucose (0.5%) 5.0 g.
the total volume of the medium. 4. Glycerol (2.0%) 20.0 g.
(14) Test the sterility of the medium 5. Blood
before use by anaerobic culture. Preparation
Incubate at least 7 days. (1) Thoroly wash fresh placenta and pass
(15) Add before use l/16th part of fresh thru a meat chopper.
sterile rabbit kidney to 5.0 cc. me- (2) Mix two parts by weight of Ringer-
dium. With kidney added, incuba- Locke solution with one part (1).
tion may be aerobic. Use as soon (3) Adjust the reaction to that of human
as possible after the addition of the blood and add 1.0% pancreatin and
kidney. 0.5% chloroform.
(4) Digest at 40 to 42 C. for 24 hours. Preparation

(5) Filter thru cloth and paper, and (1) Add 100.0 g. commercial casein to
adjust the reaction to the correct 500.0 cc. boiling water containing
alkalinity. 0.8% anhydrous NaaCOj.
(6) Steam for one hour, filter and add (2) Raise the temperature to the boiling
0.5% glucose and 2.0% glycerol. point.
(Agar and gelatin may be prepared (3) Add 500.0 cc. of cold water.
from this medium.) (4) Make faintly alkaline to litmus.
(7) Add 3 drops of blood to supply vita- (5) Coolto45C.
mines just before use. (6) Add 400.0 cc. of pancreatic extract.
Sterilization: Method not given. (7) Incubate 4 hours at 37C.
Use: Used in human microparasitology. (8) Raise the temperature to the boil-
The addition of blood may be
unnecessary ing point.
with subcultures. Author claimed good (9) Filter thru thick
wet clean cloth.
results with anaerobes as well as aerobes. (10) Filter thru thick wet filter paper.
Reference: Celozzi (1918 p. 291). Taken (11) Bring up the volume to 1000.0 cc. by
from (1919 p. 147). the addition of water.
(12) Add 2.5 g. NaCl and 0.125 g. CaCl,.
1123. Douglas Trypsin Broth (Hartley) (13) Adjust the reaction.
Constituents: (14) Steam 30 minutes.
1. Water 250.0 cc. (15) Filter while hot thru paper.
2. Meat, horse 150.0 g. (16) Disolve 1.0% of one of the added
3. XazCOa (0.8% soln.) 250.0 cc. nutrients in sterile (15).
Preparation (17) Distribute to a depth of one inch in
(1) Mi.x 150.0 g. of lean minced horse small test tubes containing fer-
meat with 250.0 cc. tap water and mentation tubes.
heat at 80C. in a steamer. (18) Cover with 0.25 inch of liquid
(2) Add 250.0 cc. of an 0.8% NajCOa paraffin.
(anhydrous) and cool to 45^0. (19) Add 3 drops of egg albumin mixed
(3) Add 5.0 cc. of chloroform and 5.0 cc. with bouillon (see variant 848 for
of Cole and Onslow's pancreatic preparation) to each tube. (This
extract. serves as an indicator.)
(4) Incubate at 37C. for 6 hours shaking (20) Place the tubes in boiling water for
frequently. 20 minutes.
(5) Add 40.0 cc. normal HCl and heat in Sterilization: Sterilize (15) in the auto-
the steamer for 30 minutes. clave.
(6) Cool and filter. Use: Determine fermentation of sugars by
(7) Adjust to pH 8.0. anaerobic organisms.
(8) Distribute as desired. Added nutrients: Author added 1.0% glu-
Sterilization: Pass steam thru the auto- cose or any other desired carbohydrate,
clave for one hour then raise the pressure alcohol, etc.
slowly to 10 pounds and turn off the Reference: Harvey (1921-22 p. 111).
steam. For sterilization of larger quan-
tities (one liter in a flask) maintain the
1125. Teruuchi and Hida's Trypsinized
pressure at 10 pounds for 30 minutes. Casein Solution
Use: Preparation of diphtheria to.xin. Constituents:
Reference: Hartley (1922 p. 482). 1. Water 1000.0 cc.
2. Casein 100.0 g.
1124. Harvey's Basal Trypsinized Casein
3. NaCl (0.5%) 5.0 g.
Solution
Preparation
Constituents: (1) Dissolve 100.0 g. of pure casein in
1. Water 1000.0 cc. 1 liter of 0.8% NazCOj (anhydrous).
2. Casein 100.0 g. (2) Add 5.0 to 10.0 g. of pancreatin
3. NaCl 2.5 g. (Gehe and Co.) and shake with
4. CaCl2 0.125 g. chloroform.
(3) Place for 3 to 5 days in the incubator, (6) Digest at 39 C. for 10 days, with
shaking occasionally. daily shaking and addition of more
(4) Each day test the mixture for trypto- toluol if necessary.
phane, by adding a few drops of (7) Add per liter, 100.0 cc. 7.5 per cent
acetic acid to a sample and then hydrochloric acid.
adding bromine water. (8) Steam 20 minutes.
(5) When the tryptophane content has (9) Filter thru well-wetted, thick filter
reached the maximum and the paper.
tjTOsin has separated out as white (10) Make the reaction nearly neutral
clumps, heat the fluid for a short to litmus with 5% sodium hy-
time at 80 C. droxide.
(6) Filter and neutralize with a few cc. (11) Preserve as stock tryptic broth or
of HCl. stock "tryptamine."
(7) Heat moderately in a vacuum if (12) use "tryptamine" 1:
Dilute for
possible and evaporate to a syrupy water 2 = tryptamine bouillon.
thickness. (Possibly may be fil- (c) Harvey prepared a similar medium as
tered again.) follows:
(8) Place in a mortar and knead it with (1) Add very gradually 200.0 g. casein
alcohol (amount not specified). to 1000.0 cc. boiling water contain-
(9) Dry in a vacuum desiccator. ing 20.0 g. anhydrous sodium
(10) This yields a light yellow colored carbonate.
powder, which dissolves quite easily (2) Allow to cool to 45C.
and clear in water. The yield is (3) Add pancreatin 3.0 g. or pan-
nearly quantitative. creatic extract 50.0 cc, chloroform
(11) Add 0.5% NaCl and 1.0% NajCO, to 15.0 cc
a 5.0% watery solution of (9). (4) Place in incubator 5 days, shaking
Sterilization: Not specified. vigorously each day to break up
Use: Enrichment of cholera vibrio, toxin clumps.
production by Proteus vulgaris and gen- (5) Add again pancreatin 3.0 g. or
eral culture medium. pancreatic extract 50.0 cc.
Variants (6) Place in incubator again for 10
(a) Berthelot prepared a pancreatic ca- days.
sein digest, and dissolved 20.0 g. of (7) Add 400.0 cc. N/1 hydrochloric
it in a liter of water. The medium acid.
was sterilized with ether, method not Note: Or 400.0 cc. pure con-
given, and used to produce toxin by centrated hydrochloric acid di-
Proteus vulgaris. luted with 350.0 cc water.
(b) Harvey prepared a "tryptamine" (8) Steam 30 minutes.
medium as follows: (9) Filter, while hot, thru well-wetted,
(1) Prepare a suspension in a well thick filter paper.

stoppered bottle: casein 1; dis- (10) Add 120.0 cc. N/1 sodium hy-
tilled water 10. droxide to the filtrate.
(2) Shake well to break up clumps. (11) Adjust reaction.

(3) Adjust the reaction if necessary (12) Dilute for use 1/3 with 0.5 per cent
with the help of cresol red. sodium chloride.
Note: The optimum reaction (13) Sterilize in the autoclave or
for the tryptic digestion of casein steamer.
is about pH = 8.1 at which point References: Teruuchi and Hida (1912
cresol red indicator solution gives p. 572), Berthelot (1914 p. 916), Harvey
a reddish violet color and phenol- (1921-22 p. 116).
phthalein remains colorless.
1126. Zipfel's Trypsinized Casein Solution
(4) Add per liter: Pancreatic extract
60.0 cc, toluol 5.0 cc. Constituents:
(5) Shake to mix. 1. Distilled water 1000.0 cc.
2. Plasmon (sodium caseinate). 500.0 g. (21) Filter and add H2SO4 to the filtrate
3. KH2PO4 5.0 g. so that it is 5.0% H2SO4.
4. Magnesium phosphate 0.3 g. (22) To separate the cystin, precipitate

5. Ammonium lactate fractionally with HgS04 in that one
Preparation adds first HgS04, carefully, until
(1) Mix "plasmon" (sodium
500.0 g. of just a conglomerate precipitate of
caseinate) with 2^ liters of lukewarm cystin mercury sulphate is formed.
2.0% Na2C03 solution. (23) Allow to stand a few hours and filter,
(2) Add 25 liters of distilled water. (24) Precipitate again with HgS04.
(3) Allow to soak and place in a large (25) Allow to stand, filter, wash with
flask. 5.0% H2SO4 as in (16) and add the
(4) Soak 10.0 g. of trypsin in a little moist precipitate to distilled water.
water and add to (3). (26) Decompose the precipitate with H2S
(5) Place a layer of toluol about 1 cm. as in (18)and (19).
high to keep down bacterial growth. (27) Filter and evaporate to dryness.
(6) Place at 37C. for several days shak- (2S) This brown colored mass may be
ing occasionally. crystallized in hot water several
(7) From time to time test for trypto- times or pure tryptophane may be
phane with acetic acid and bromine obtained.
water. The maximum amount of (29) To obtain pure tryptophane add lead
tryptophane is reached after about carbonate (about 10.0% of the used
10 to 20 days. albumin, therefore about 50.0 g.) to
(8) Remove the toluol and heat to 80 C. the filtrate (27) and heat for about
to coagulate any undigested al- 30 minutes in a water bath. Am-
bumin. monia is given off.
(9) Add 100.0 g. talcum and shake (30) After cooling precipitate the lead
thoroly. by passing thru H2S.
(10) Allow to stand several hours until (31) Filter and evaporate the filtrate to
it is rather clear. dryness.
(11) Tyrosin and some cystin will settle (32) After several crystallizations with
out during the cooling. dilute alcohol one obtains pure
(12) Filter. silvery rectangular platelets of tryp-
(13) Add H2SO4 to the clear filtrate until tophane.
it is 5.0% H2SO4. (33) Dissolve 0.3 g. of (32), 5.0 g. of
(14) Add a 10.0% solution of HgS04 in secondary potassium phosphate, 0.3
5%, H2SO4 to (13). A lemon colored g. of Magnesium phosphate and
precipitate of a union of HgS04 and ammonium lactate (amount not
tryptophane is formed. given) in 1 liter of distilled water.
(15) Allow to stand for 24 hours and (34) Distribute in 10.0 cc. lots in tubes.
filter. Test the filtrate to see that it Sterilization: Sterilize on 2 successive days
is free from tryptophane by adding in a steam sterilizer.
more HgS04 to it. Use: Indol production. Preparation of
(16) Wash the precipitate with 5.0% pure tryptophane.
H2SO4 until the wash water no longer Variants: The author added glucose and
gives a red coloration with Millon's glycerol, amounts not given.
reagent. Reference: Zippel (1912-13 p. 572).
(17) Add the damp precipitate to 500.0 g.
1127. Bacto Tryptophane Broth
of distilled water.
(Dehydrated)
(18) Decompose the precipitate by heat-
ing and pass in H2S. Constituents
(19) Pass in CO2 to drive out the excess 1. Distilled water 1000.0 cc.
of H2S. 2. Hydrolyzed casein 1.0% 10.0 g.
(20) The liquid contains tryptophane and (Bacto tryptophane broth dehydrated)
cystin while the precipitate con- Preparation
tains HgS. (1) Prepare a 1.0% solution of Bacto
Tryptophane Broth in hot distilled Sterilization: Sterilize in autoclave at 10

water. pounds for 10 minutes.
(2) If sterilized at 15 pounds pressure for Use: To study requirements of strepto-
20 minutes pH = 6.8. cocci and pneumococci. Author reported
Sterilization: Sterilize at 15 pounds pres- that the organisms grew in impure casein
sure for 20 minutes. medium and not in pure casein.
Use: Indol production. Medium permits Variants: The author prepared a pure
a strong positive indol test in 12 hours. casein digest by precipitating a solution
Reference: Digestive Ferments Co. (1925 of commercial casein in Na2C03 three
p. 13). times by acetic acid. Then washed pre-
cipitate in alcohol and ether. Treat this
1128. Bacto Peptonized Milk (Dehydrated) purified casein as in steps (2), (3), (4) and
(5) above.
Constituents
Reference: Mueller (1922 pp. 318, 319).
2. Bacto Peptonized Milk 1130. Cole and Onslow's Trypsinized Casein
(Dehydrated) 15.0 g. Solution
Preparation:
Constituents:
(1) Dissolve 15.0Bacto Peptonized
g.
Milk (Dehydrated) in 1000.0 cc. dis-
2. Casein 200.0 g.
tilled water.
for 20 minutes at 15
3. NaCl (0.5% soln.) 2000.0 cc.
(2) If sterilized
Preparation
pounds pressure pH = 6.5zt.
(1) Obtain the fresh pancreas of a pig,
Sterilization: Sterilize at 15 pounds pres-
free it from fat as far as possible
and weigh.
Use: Cultivation of lacto-bacillus group.
medium (2) Mince (1) finely and add 3 times its
Variants: Add 0.1% Bacto agar to
weight of distilled water, and its own
for maintaining stock cultures.
weight of strong alcohol.
Reference: Digestive Ferments Co. (1925
(3) Shake the mixture well in a large
p. 14).
bottle, and allow to stand for 3 days
at room temperature, shaking occa-
1129. Mueller's Trypsinized Casein Solution
sionally.
Constituents: (4) Strain thru muslin and filter thru a
1. Water 1000.0 cc. large folded filter paper.
2. NaCl (1.0%) 10.0 g. (5) Measure the filtrate and add 1.0 cc.
3. MgS04 (0.04%) 0.4 g. of concentrated HCl for each liter
4. CaCl, (0.02%) 0.2 g. of the filtrate.
5. K2HPO4(0.2%) 2.0 g. (6) Allow to stand for a few days and
6. Glucose (0.2%) 2.0 g. filter off the sediment.
7. Phenol red (0.02% solution). 80.0 cc. (7) The may be stored in a stop-
filtrate
8. Casein pered bottle, without the addition

Preparation of an antiseptic. If the extract is to
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. be used at once it is not necessary

(2) Digest a solution of commercial casein toadd the HCl.
with Fairchild's trypsin at 37C. for (8) Add 20.0 g. of anhydrous NazCOa to
2 weeks. one liter of tap water, and boil in a 2
(3) Boil. liter flask or large evaporating basin.
(4) Filter. Twelve cubic centimeters of (9) When (8) is boiling, transfer the
the filtrate represents about 0.5 g. of vessel to a boiling water bath, and
casein. This is an impure casein gradually dust in 200.0 g. of casein
digest. (Laitproto j^6 for bacteriological
(5) Mix 12.0 cc. of (4) with 13.0 cc. of purposes from Casein Limited).
water and add 25.0 cc. of (1). Stir well to avoid the formation of
(6) Adjust to pH = 8.0. lumps.
(10) Transfer to a Winchester quart and Sterilization: Method not given.

wash out the mixing vessel with a Use: To study bacterial nutrition.
liter of cold tap water. Add this Reference: Berman and Rettger (1918
wash water to the casein solution. pp. 383, 384).
(11) Cool to 40C. and add 50.0 cc. of (7)
1132. Norris' Trypsinized Caselnogen
and 15.0 cc. of chloroform.
Solution
(12) Shake well, stopper with a loose cork,
and incubate at 37 to 40C. Shake Constituents
the bottle each day to break up 1. Water 1000.0 cc.
lumps. 2. Caseinogen 100.0 g.
(13) Add 50.0 cc. more of (7) after 5 days Preparation
and allow to stand for 10 more days. (1) Digest 100.0 g. (10.0%) caseinogen in
The material is digested 15 days in an 0.8% aqueous solution of washing
all. soda with a 0.5% pancreatic extract
(14) Shake well and transfer to a large at 37 C. for 24 hours.
add 400.0 cc. of normal
3 liter flask, (2) Concentrate the products of diges-
HCl (or 40.0pure concen-
cc. of tion on the water bath yielding a paste
trated HCl diluted with 360.0 cc. of the consistency and color of
water) Shake, steam for 30 minutes
. Liebig's meat extract.
and filter. (3) Add100.0 g. (10.0%) of 2 to 100.0 cc.
(15) Add 120.0 cc. of normal NaOH. tap water.
(16) Adjust to pH =
7.35. (4) Adjust the reaction to +1.0.
(17) This is a stock broth solution. Sterilization: Method not given.
(18) To prepare tryptic broth dilute (17) Use: General culture medium. The author
with twice its volume of 0.5% NaCl. suggested that the term "trypsinoid" be
Adjust the reaction to pH = 7.35. employed for the paste. Brewer's yeast
Sterilization: Sterilize on 3 consecutive and fresh yeast were treated in exactly
days rather than in the autoclave. the same manner but the casein product
(Details not given.) gave the best results.
Use: Substitute for peptone. General cul- Variants
ture medium. (a) The paste from (2) may be further
Variants: The author dissolved 2.0 g. of dried in a desiccator over H2SO4,
glucose or dulcitol in 40.0 cc. of a 0.04% yielding a brittle resinous mass which
phenol-sulphone-phthalein solution in a may be powdered.
liter of the medium as prepared above. (b) The author dissolved(2) in physio-
The medium was tubed in 3.0 cc. quanti- logical salt solution instead of tap
ties and sterilized in steam on each of 3 water.
successive days for 20 minutes each day. Reference: Norris (1920 p. 706).
Reference: Cole and Onslow (1916 pp. 10,
1133. Cannon's Trypsinized Casein Solution
1012).
(Norton and Sawyer)
1131. Berman and Rettger's Trypsinized
Constituents
Casein Solution
Constituents: 2. Casein (c.p.) 20.0 g.
1. Water 1000.0 cc. 3. Asparagin 5.0 g.
2. Casein 4. Ammonium lactate 5.0 g.
3. Beef extract 2.5 g. 5. K2HPO4 2.0 g.
4. NaCl 5.0 g. 6. MgS04 0.2 g.
(1) Digest a solution of casein with com- (1) Add 20.0 g. casein to 250.0 cc. distilled
mercial trypsin at 45C. for several water and make alkaline to phenol-
hours. phthalein with Na2C03.
(2) Dissolve 2.5 g. beef extract and 5.0 g. (2) Add 0.5 g. trypsin and allow to digest
NaCl in 1000.0 cc. of (1). for 6 hours.
(3) Autoclave. (Time not specified.) (5) Add 6.4 cc. 5.0% sodium hydroxide
(4) Add asparagin, ammonium lactate, for every 100.0 cc. of 0.5% hydro-
K0HPO4 and MgS04. chloric acid used.
(5) Make up to 1 liter and adjust to +1 (6) Stir well and filter thru folded filter
phenolphthalein. paper.
Sterilization: Final sterilization not speci- (7) Shake up with a little toluol.
fied. (8) Make the reaction less acid by the
Use: To show production of indol by cautious addition of 10.0% sodium
bacteria. hydroxide.
Reference: Norton and Sawyer (1921 (9) Store in a stoppered bottle in a cool,
p. 473). dark place.
(10) Mince finely ox heart or human
1134. Duval and Harris' Tryptic Digest
placenta.
Solution
(11) Add 500.0 g. to 1000.0 cc. tap water.
Constituents: (12) Make faintly alkaline to litmus.
1. Serum, egg albumin, liver or (13) Heat slowly to 75C. and maintain
placenta 200.0 cc. at this temperature 10 minutes.
Preparation (14) Cool to 37C.
human serum,
(1) 200.0 cc. of fresh sterile (15) Add 1.0 or 2.0% of (9) to (14).
egg albumin, or the equivalent in (16) Keep at 37C. for 4 hours. If the
grams of liver or placenta tissue are digestion is extended to 6 hours or
placed in a flask and 20.0 cc. of a longer it is necessary to add chloro-
1.0% physiological salt solution of form or toluene. Control the prog-
added.
sterile trypsin ress of digestion by Biuret and
(2) Digest for 5 days at 37 C., changing tryptophane tests.
the reaction to neutral as occasion (17) Make faintly acid to litmus with
demands. glacial acetic acid.
(3) Heat at 70C. for one hour. (18) Raise slowly to the boiling point.
Sterilization: Filter thru a Berkefeld filter. (19) Boil gently 15 minutes.
Use: Cultivation of leprosy bacilli. The (20) Filter.
author stated that the same materials (21) Adjust the reaction.
might be autolyzed under sterile condi- (22) Add 0.2% di-potassium phosphate.
tions at 40C. for 2 weeks or hydrolyzed (23) Steam 20 minutes.
with a culture of Finkler and Prior's (24) Clarify and filter.
vibrio or some other bacterial proteoly- Sterilization: Sterilize in the autoclave or
zer for 24 to 36 hours, and then neu- steamer.
tralized with normal NaOH. Sterilize as Use: General culture medium.
indicated above. Reference: Harvey (1921-22 p. 114).
Reference: Duval and Harris' (1911 p. 168).
1136. Stickel and Meyer's Tryptic Digest

1135. Harvey's Tryptic Digest Solution
Solution
Constituents
or placenta 1. Distilled water 1000.0 cc.
2. Heart (ox),
(human) 500.0 g. 2. NaCl 5.0 g.
3. K2HPO4 2.0 g. 3. CaCl2 0.1 g.
(1) Free fresh hog pancreas from fat as 5. CaHP04 2.0 g.
far as possible. 6. K2HPO4 2.0 g.
and weigh. 7. Heart, beef or placenta,

(2) Mince finely
(3) Add 3.0 cc. 0.5% hydrochloric acid human 500.0 g.
for every gram of the minced pan- Preparation

creas. (1) Remove fat from beef heart and chop
the mixture at intervals for finely, or rinse human placenta in
(4) Stir
30 minutes. water and pass thru a meat chopper.
(2) Mix 500.0 g. beef heart or human (6) Transfer to large bottles and steam
placenta with 1000.0 cc. water. for 10 minutes at 100C. to stop
(3) Make faintly alkaline to litmus digestion.
using N KOH or N Na-.COs. (7) Cool to 80C. and make faintly alka-
(4) Heat slowly to 70-80 for 5-10 line to litmus using 2N KOH or
minutes. 2 normal Na2C03.
(5) Cool to 37C. and add 1.0% pan- (8) Cool to 37C. and add 1.0% pan-
creatic extract or "Bacto" trypsin. creatic extract or "Bacto" trypsin.
(6) Incubate at 37C. for 2-5 hours. (9) Keep the mixture at 37C. for 3 to
(7) Control the progress of digestion by 10 hours depending on the reaction
repeated Biuret and tryptophane of the trypsin and the digestion
tests. In case it is necessary to desired. Control the process by
extend the digestion over a period of repeated tests for tryptophane.
6 hours add chloroform or toluene. (10) When trypsinizing is sufficiently ad-
(8) When the process is sufficiently ad- vanced render reaction slightly acid
vanced, render slightly acid with <vith glacial acetic acid, and bring
glacial acetic acid and boil slowly slowly to boiling point for 10
for 15 minutes. ininutes.
(9) Either filter or decant the clear (11) Filter thru paper or keep in cool
fluid which results on placing the place over night and decant the
digest over night in a cool place. clear liquid in the morning.
(10) Adjust the reaction as desired. (12) Add K2HPO4 and adjust the reaction
(11) Dissolve 2, 3, 4, 5 and 8 in (10). faintly alkaline or to the desired
(12) Heat for 15-30 minutes in the H-ion concentration.
steamer at 100 C. (13) Heat in steamer at 100 for 15
(13) Filter again if necessary. minutes.
Sterilization: Sterilize at 100C. on 3 con- (14) Correct reaction and filter thru
secutive days if not to be used at once. paper.
Use: General inexpensive culture medium. (15) Distribute in receptacles used for
Reference: Stickel and Meyer (1918 p. 80). culture.

Sterilization: Sterilize at 100C. for 30 min-
1137. Stickel and Meyer's Digest Solution utes on 2 successive days or at 10 pounds
Constituents Use: General inexpensive culture medium.
1. Water, tap 4000.0 cc. Variants: The authors prepared a medium
2. Liver, placenta or blood as indicated above steps (1) thru (7).
clot 400.0 g. The remainder of the preparation was as
3. Stomach, hog 400.0 g. follows:
4. K2HPO4 8.0 g.
(8) Cool to 37C add 1.0% pancreatic
Preparation extract or 40.0 g. trypsin, 8.0 g.
(1) Wash clean and mince fine 5 or more K2HPO4, 4.0 g. CaCOs, and 1.0% of a
large pig's stomachs. 24 hour old broth culture of B. sac-
(2) Mince an equal amount of clean char olyte.
pig's or beef liver, cheap fat free (9) Incubate at 37C. for 12-18 hours and
beef, placenta or blood clots. control the digestion by tryptophane
(3) Mix 400.0 g. of (1), 400.0 g. of one tests and the removal of carbohy-
of (2) and 40.0 g. of Baker Chemical drates by the gas formation in fer-
Co. HCl in 1000.0 cc. of tap water mentation tubes.
at 50 C. (10) When the digest is sugar free steam
(4) Incubate at 50C. for 18 to 24 hours. 15 minutes.
(5) Make a Biuret and tryptophane test. (11) Adjust to desired reaction and steam
When both are + the digest is yel- another 15 minutes.
lowish green and contains very (12) Filter thru paper.
little undigested debris. (13) Distribute as desired.
(14) Sterilize at 100 for 30 minutes on 2 (3) Exactly neutralize with 10.0% H2SO4
successive days or 10 pounds for and test for absence of both Ba and
15 pounds in the autoclave. SO4 ions.
References : Stickel and Meyer (1918 p. 80), (4) Concentrate to thick syrup in
Harvey (1921-22 p. 101). vacuum.
(5) Dilute to a final solids content of
1138. Frieber's Digest Extract Solution
2.0% with distilled water.
Constituents: (6) Treat gelatin in the same manner as
1. Water 1000.0 cc. gliadin (1) thru (5) above.
2. Physiological salt solution.. 3000.0 cc. (7) Dissolve 3, 4, 5, 6, 7 and 8 in 500.0 cc.
3. Fibrin of (6).
4. Beef extract, Liebig's 5.0 g. (8) Mix equal (7) and (5).
parts
5. NaCl 5.0 g. (9) Adjust to pH
from 8.0 to 8.2.
Preparation : (10) Steam 15 minutes and check the
(1) Prepare Frieber's Fibrin Digest reaction.
Solution by digesting fibrin with pep- (11) Distribute as desired.
sin and HCl (see medium 1110). Sterilization: Heat at 115C. for 20 minutes.
(2) Add 5.0 g. Liebig's beef extract, Use: Cultivation of Bad. diphtheriae for
5.0 g. NaCl and 7.0 cc. of normal soda toxin production.
solution to 1000.0 cc. of (1). Reference: Davis and Ferry (1919 p. 232).
(3) Neutralize to litmus and boil.

1140. Robinson and Rettger's Hydrolyzed
(4) Distribute in a flask having a closely
Edestin Casein Solution
fitting glass stopper.
(5) Cool to 40C. Constituents

(6) Add 0.2 g. Grubler's trypsin, 10.0 cc. 1. Water 1000.0 cc.
chloroform and 5.0 cc. toluol. 2. Casein 30.0 g.
(7) Shake thoroly and place in the incu- 3. Edestin 30.0 g.

bator for 24 hours. 4. Lactalbumin 30.0 g.
(8) Filter thru a damp filter. 5. Dextrose 0.5 g.
(9) Dilute 1 part (8) with 3 parts physio- Preparation

logical salt solution. (1) Boil 50.0 g. of casein with 10.0% HCl
(10) Distribute in 5.0 cc. lots. under a condenser until the
reflex
Sterilization: Sterilize in the steamer for solution no longer responds to the

one hour. Biuret test.
Use: To detect indol production. (2) Evaporate on the water bath until
Reference: Frieber (1921 p. 427). nearly all the HCl is removed.
(3) Neutralize the remaining acid with
1139. Davis and Ferry's Hydrolyzed Gliadin NaOH. This solution is of dark
Solution known
brown color and is as "Ca-
Constituents: sein C."
1. Distilled water 1000.0 cc. (4) Treat edestin and lactalbumin in
2. Gliadin 20.0 g exactly the same manner as casein as
3. Tryptophane 0.4 g in steps (1) thru (3).
4. Tyrosine 0-75 g (5) Dissolve 3.0% (3), 3.0% edestin

5. Cystine 0-2 g product and 3.0% lactalbumin prod-
6. NaCl 2.5 g uct in water.
7. K2HPO4 1-5 g (Q') Adjust the reaction to neutral to
8. MgS04 0.25 g litmus.

9. Gelatin 20.0 g (7) Distribute in 20.0 cc. lots in 150.0 cc.
Preparation Erlenmeyer flasks.
Hydrolyze gliadin with 25.0% HoSO. Sterilization: Sterilize at 12 to 14 pounds

(1)
for 24 hours on sand bath. Tem- pressure for 15 minutes.
perature not given. Use: Cultivation of B. diphtheriae and
(2) Add Ba(OH) 2 until alkaline. Filter study toxin production. Authors re-
ported that the cultures were nearly Ab. Containing organs of flowering plants
non-toxic. or their derivatives.
Variants: The authors added 0.1% sterile Bi. Leguminous plants specified.
glucose or lactose to the sterile medium. Ci. Commercial extracts used.
Reference: Robinson and Rettger (1917 Buchanan's Basal Legume Extract
p. 364). Solution 1156
Buchanan's Salt Legume Extract
SUBGROUP I-C. SECTION 15 Solution 1157
C2. Commercial extracts not used.
Liquid media or basal solutions not con- Di. Leaves and stems employed.
taining digests; containing plant deriva- Buchanan's Basal Clover Infusion
tives of unknown chemical composition. Solution 1158
Ai. Containing yeast derivatives. Buchanan's Vetch Infusion Solution 1159
Bi. Basal solutions; employed with the D2. Flowers employed.
addition of other nutrients. Wilhelmi's Clover Flower Infusion
Henneberg's Basal Yeast Infusion Solution 1160
Solution 1141 D3. Pods employed.
Bj. Complete media. Reed and Cooley'a Bean Pod Infu-
Ci. Containing additional organic carbon of sion Solution 1161
known chemical composition. D4. Seeds or their derivatives employed.
Di. Additional carbon supplied as carbo- Maze's Sucrose Bean Infusion Solu-
hydrates. tion 1162
Heinemann's Glucose Yeast Infu- DeRossi's Glucose Bean Infusion
sion Solution 1142 Solution 1163
Gassner's Lactose Yeast Infusion Kaufmann's Jequirity Infusion Solu-
Solution 1143 tion 1164
Korf's Sucrose Yeast Infusion Solu- Tanner's Pea Flour Infusion Solu-
tion 1144 tion 1165
Bottger's Nitrate Yeast Infusion Stutzer's Basal Legume Seed Infu-
Solution 1145 sion Solution 1166
Dj. Additional carbon supplied as alcohols. B2.* Non-leguminous plants or their deri-
Janke's Alcohol Yeast Infusion Solu- vatives specified.
tion 1146 Ci. Tubers or roots employed.
Bertrand's Sorbitol Yeast Infusion Di. Containing potato or derivatives.
Solution 1147 Smith's Potato Infusion Broth 1167
Ci. Not containing additional organic car- Eisner's Hydrochinone Potato Infu-
bon of known chemical composition. sion Solution 1168
Thoinot and Masselin's Yeast Infu- Robertson and Davis' Potato Infu-
sion Solution 1148 sion Solution 1169
Aj. Containing bacteria or derivatives. Lubinski's Glycerol Potato Infusion
Stoklasa's Arabinose Azotobacter Solution 1170
Solution 1149 Berthelot's Vegetable Infusion Solu-
A3. Containing fungi (fleshy) or their tion 1171
derivatives. Migula's Potato Juice 1172
Lanken and Meyer's Fungus Infu- Winogradsky's Gypsum Root Solu-
sion Solution 1150 tion 1173
A4. Containing peat, moss, etc., or their Da. Not containing potato or derivatives.
derivatives. Tanner's Carrot Infusion Solution. 1174
.
Ampola and Garino's Nitrate Peat Robertson and Davis' Carrot Infu-
Solution 1151 sion Solution 1175
Revis' Peat Infusion Solution 1152 Cs.t Leaves or stems employed.
Brussoff's Iron Peat Infusion Solu- Di. Hay or straw.
tion 1153
Schmidt's Hydrolyzed Peat Solution 1154 * See Bj next page.
Schmidt's Peat Solution 1155 tSee C3 and C4 next page.
Migula's Hay Infusion Solution 1176 Di. Extracts or infusions of grains (not
Winogradsky's Ferric Hydroxide including malts, beer worts, etc.).
Hay Infusion Solution (Molisch). 1177 Harvey's Wheat Flour Solution 1207
Jensen's Nitrate Straw Solution. . . . 1178 Omeliansky's Flax Stem Solution.. 1208
Wolbach and Binger's Glucose Hay Speakman and Phillips' Maize Mash
Infusion Solution 1179 Solution 1209
Sherman's Soil Hay Infusion Solu- D2. Malt extract, beer wort, beer, etc.,
tion 1180 employed.
D2.* Cabbage. El. Beers used.
Smith's Cabbage Infusion Solution. 1181 Lafar's Beer Solution 1210
Conrad's Glucose Cabbage Infusion Waterman's Sucrose Beer Solution. 1211
Solution 1182 E2. Malts used.
Will's Cabbage Juice 1183 Wurtz's Malt Infusion Solution... 1212
D3. Other leaves or stems. Beijerinck's Malt Wort Solution... 1213
Reed and Cooley's Basal Spinach Peklo's Malt Infusion Solution 1214
Infusion Solution 1184 Bokorny's Malt Infusion Solution. 1215 .
Sucksdorff's Tea Infusion 1185 Bacto Malt Extract Broth (Dehy-

Bushnell's Asparagus Solution 1186 drated) 1216
Tausz and Peter's Leaf Infusion Reddish's Malt Extract Solution. . . 1217
Solution 1187 Ej. Beer worts used.
Miiller-Thurgaw's Grape Vine Infu- Park, Williams and Krumwiede's
sion Solution 1188 Basic Beer Wort Solution 1218
Malenkovic's Wood Infusion Solu- Peklo's Beer Wort Solution 1219
tion 1189 Nakazawa's Beer Wort 1220
Olitsky's Tomato Infusion 1190 Peklo's Tartrate Beer Wort Solution 1221
C3. Fruits or their derivatives employed. Will's Asparagin Beer Wort Solution 1222
Di. Extracts or infusions used. Ds. Juices of grains employed.
Schardinger's Prune Solution 1191 Owen's Cane Juice 1223
Sucksdorff's Coffee Infusion 1192 Owen's Sulphured Cane Juice Solu-
Tanner's Fruit Infusion Solution. 1193 . . tion 1224
Turner's Quince Seed Infusion Sherman's Corn Juice 1225
Solution 1194 D4. Silage employed.
D2. Fruit juices used. Buchanan's Silage Infusion Solution 1226
El. Grapes and wines specified. B3. A variety of plant materials specified.
Miiller-Thurgau and Osterwalder's Reith's Plant Infusion Solution 1227
Grape Juice 1195 Reith's Blood Cell Plant Infusion. 1228 .
Schukow's Grape Must Solution 1196

Perold's Wine Medium 1141. Henneberg's Basal Yeast Infusion
1197
Solution
E2. Currants specified.
Bierberg's Currant Must Solution. . 1198 Constituents:
MiJller's Currant Must Solution 1199 1. Yeast infusion (3.0%) 1000.0 cc.
E3. Other juices or mixture of juices spec- Preparation
ified. (1) Preparation of 3.0% yeast infusion
Ottolenghi's Melon Juice Solution. 1200 . not given.
Bierberg's Cherry Must Solution.. 1201 (2) Dissolve one of the added nutrients
Miiller-Thurgau and Osterwalder's in (1).
Pear Juice Solution 1202 Sterilization: Method not given.
Giltner's Fermented Cider 1203 Use: Cultivation of lactic acid bacteria.
Zikes' Nitrate Fruit Must Solution. 1204 Other investigators have used similar
Schukow's Fruit Must Solution 1205 media for the cultivation of a variety of
D3. Fruit used. bacteria.
Bachman's Mince Meat Medium 1206 Added nutrients and variants:
C4. Grains or their derivatives employed. (a) The author added 20.0% glucose,
maltose or sucrose to the basal
* See D3. solution.
(b) Henneberg prepared a 10.0% yeast (e) Besson prepared the medium as
infusion solution and added 2.0 to follows:
5.0% of any desired carbohydrate, (1) Boil 100.0 g. of beer yeast in
alcohol, etc. 1000.0 cc. of water.
(c) Bobilioff-Preisser added one of the (2) Filter thru paper.
following materials to yeast infusion: (3) Add 5.0% of glucose or sucrose.
dextrin glucose (4) Add a small amount of phosphoric
galactose maltose acid and then add sufficient lime
levulose raffinose water to give a slightly alkaline
lactose sucrose reaction.
The media were sterilized in the (5) Heat for 5 minutes at 116 to IH^C.
steamer. (6) Filter.
(d) MUller-Thurgau and Osterwalder pre- (7) Tube.
pared the basal solution as follows: (8) Sterilize at 115C.
(1) Add water (amount not given) to References: Henneberg (1903 p. 8), Bobi-
1000.0 g. pressed yeast and boil. lioff-Preisser (1916 p. 387), Muller-
(2) Filter. Thurgau and Osterwalder (1918 p. 2),
(3) Dilute the filtrate to 10 liters by the Besson (1920 p. 35).
addition of water.
(4) Acidify by the addition of 1.0% 1142. Heinemann's Glucose Yeast Infusion
malic acid. (Do not add acid here Solution
when adding an acid as a carbon
source.) Constituents:
(5) Distribute in 100.0 cc. lots. 1. Water 2000.0 g.
(6) Sterilize(Method not given). 2. Yeast 1.0 lb.

3. Dextrose (10.0 to
(7) Add one of the following materials
Glucose 2.0% or 4.0% 15.0%) 200.0 to 300.0 g.
Levulose 1.5% or 4.0% Preparation

Galactose 4.0% (1) Boil one pound of pressed yeast or
Sucrose 5.0% or 4.0% 1 liter of washed yeast with 2 liters of
Maltose 4.0% water for one hour.
Malic acid 4.5% or 2.0% (2) Filter the solution until clear.
Potassium malate (3) Do not adjust the reaction.
(acid) 10.0% (4) Dissolve 10.0 to 15.0% dextrose in (3).
Ethyl malate 10.0% Sterilization: Sterilize in the Arnold on 3
Calcium malate (acid) 10.0% consecutive days.

[2.2% or 4.7% Use: General culture medium.
Citric acid or 2.0% or Reference: Heinemann (1905 p. 130).
I
4.0%
Lactose 4.0% 1143. Gassner's Lactose Yeast Infusion
Raffinose 1.0% Solution
1-arabinose 15.0%
Xylose 10.0% Constituents:
a-methyl glucoside. 2.0%
. .
1. Water 18,000.0 cc.
Glycerin 1.0% 2. Yeast, Brewers.... 10,000.0 cc.

ammonium malate 10.0%
. . .
3. Lactose 0.3% or 0.5%
(8) Sterilize once more (method not Preparation
given). (1) Place about 10 liters of brewers yeast
The media were used to study fer- in a flask and wash with water.
mentation by Bacterium mnnnito- Allow to stand for 30 minutes and
poeum, Bacterium intermedium, pour off the liquid.
Bad. Gayoni and mannitol bacteria (2) Repeat the washing process until the
from wine. The authors also used wash water is no longer brown but
the basal solution without any slightly turbid. Washing 5 times is
additions. usually sufficient.
(3) After pouring off the last water add ness of alcohol or ether have dis-
18 liters of water to the remaining appeared.
washed yeast cells. (15) Prepare a 10.0% solution of (14) in
(4) Boil in the autoclave or steamer as in distilled water.
the preparation of meat bouillon. (16) Mix about 10.0% of (15) with 10.0%
(5) Allow to stand for a suitable length of (9).
of time and remove the liquid from (17) Distribute in 150.0 cc. lots in fer-
the sediment or filter thru filter paper. mentation flasks.
(6) Add 0.3 or 0.5% lactose to the filtrate Sterilization: Sterilize in streaming steam.
or supernatant fluid. Use: To study fermentation by yeast.
(7) Adjust to slightly alkaline to litmus. Waterman used a similar medium to
Sterilization: Not specified. study the inversion of sucrose by bacteria.
Use: General inexpensive culture medium. Variants
Variants The author used the medium with
: (a) Syr(e prepared a similar medium as
constituents of double strength, with or follows:
without 0.5% NaCl. (1) Wash brewers yeast in a decanta-
Reference: Gassner (1916-17 p. 311). tion funnel with distilled water
until the wash water, to which
1144. KorfE's Sucrose Yeast Infusion some yeast has been added, boiled
Solution
and filtered, will give no reaction
Constituents: with Fehling's solution.
1. Water 2000.0 to 3000.0 cc. (2) Boil 1000.0 g. of the washed yeast
2. Yeast 1000.0 g. with 2 liters distilled water for
3. Sucrose. 2 hours.
Preparation (3) Filter.
(1) Mix 1000.0 g. of yeast with water and (4) Boil the filtrate once more.
decant. Continue this until the (5) Filter.
liquid is colorless and does not re- (6) Estimate the nitrogen content by
duce Fehling's solution. Kjeldahl's method and dilute so
(2) Pour the yeast in a porcelain funnel that the nitrogen content is
provided with a sieve plate. 0.0448%.

(3) Wash repeatedly with distilled (7) Weigh 100.0 g. of dried sucrose,
water. prepared as indicated above.
(4) Place the yeast in a large flask with Step (10) thru (14) in a liter volu-
2 or 3 liters of water and boil for metric flask.
30 minutes. (8) Pipette 100.0 cc. of (6) in the flask
(5) Allow to settle. with the sugar.
(6) Filter. (9) Add distilled water to make
(7) Boil the filtrate once more. 1.0 liter volume.
(8) Filter. (10) Distribute into 150.0 cc. lots, plug
(9) Determine the weight and
specific with cotton and gum, and sterilize
dilute to a suitable specific weight in streaming steam.
(1.0033 g.). (11) Shake often during cooling to satu-
(10) Dissolve 1000.0 g. of white sugar rate the medium with air.
candy in 500.0 g. distilled water by (b) Henneberg added 10.0% sucrose to a
heating on the water bath. 1.0 to 15.0% yeast infusion solution.
(11) Filter while hot in the hot water The reaction was made neutral with
funnel into a large porcelain dish. soda. The medium was used for the
(12) Add 2 liters of warm absolute alco- cultivation of lactic acid bacteria.
hol, stirring continually. (c) Will added 6.0% sucrose to yeast
(13) Pour the cool alcohol from the sugar water (preparation not given).
and wash the sugar with absolute (d) Waterman added 4.0% sucrose to
alcohol and then with ether. yeast water (preparation not given)
(14) Dry at 60C. until all traces of damp- to study the inversion of sucrose by
B. racens, B. Pasteurianum, B. aceli, 4. CaClj 0.1 g.

B. melanogenum and B. xylunum. 5. MgS04 0.06 g.
He reported that after 3 days gener- 6. Sorbitol (5.0%) 50.0 g.
ally there was little or no reaction Preparation
with Fehling's solution. (1) Preparation of yeast infusion not
References: Korff (1898 p. 532), Syree given. should contain about 5.0 g.
It
(1899 p. 12), Henneberg (1903 p. 8), Will of dissolved material per liter.
(1908 p. 387), Waterman (1913 p. 455). (2) Dissolve 2, 3, 4 and 5 in (1).
(3) Add 5.0% sorbitol to (2).
1145. Bottger's Nitrate Yeast Infusion Sterilization: Not specified.
Solution Use: Production of sorbose by Bacterium
xylinum, Mycoderma aceli.
Constituents:
Reference: Bertrand (1898 p. 397).
1. Yeast water 1000.0 cc.
2. Sucrose (10.0%) 100.0 g. 1148. Thoinot and Masselin's Yeast
3. KNOs 0.2, 0.5, 1.0, 2.0 or 4.0% Infusion Solution
Preparation
Constituents:
(1) Preparation of dilute yeast water not
specified.
1. Water 1000.0 cc.
Add 10.0% cane sugar to (1). 2. Yeast 100.0 g.

(2)
Preparation
(3) Add 0.2, 0.5, 1.0, 2.0 or 4.0% KNO3
to (2). (1) Add 100.0 g. of yeast to 1000.0 cc. of
water.
(2) Mi.x slowly.
Use: To study toxicity of nitrates. Author
(3) Boil in the autoclave or on a water
reported that the presence of nitrates had
little effect.
bath.
Variant: Author used 0.17, 0.41, 0.83, 1.67 (4) Make slightly alkaline by the addi-
tion of soda.
or 3.33% NaNO, instead of KNO3.
Reference: Bottger (1921 (5) Filter.
p. 224).
1146. Janke's Alcohol Yeast Infusion
115 for 15 minutes.
Solution
Constituents: Variants
1. Water 900.0 cc. (a) Henneberg used a 2.5, 5.0 or 10.0%
2. Yeast (pressed) 100.0 g. yeast water solution but did not give
3. Alcohol (3.0%) 30.0 cc. the method of preparation. He culti-
Preparation vated lactic acid bacteria.
(1) Boil 100.0 g. of pressed yeast with (b) Heinemann prepared the medium
900.0 cc. water. as follows:
(2) Filter.
(1) Boil one pound of pressed yeast or
(3) Add 3.0% by volume of absolute alco- one liter of washed yeast with
hol after sterilization. 2 liters of water forone hour.
Sterilization: Method of sterilization of (2) Neutralize to phenolphthalein.
(2)not given. Filter the solution until clear.
(3)
Use: Cultivation of acetic acid bacteria the Arnold on 3 con-
(4) Sterilize in
found in beers. secutive days.
Reference: Janke (1916 p. 6). (c) Gassner prepared a similar medium
as follows:
1147. Bertrand's Sorbitol Yeast Infusion
(1) Place about 10 liters of brewers
Solution
yeast in a flask and wash with
Constituents: water. Allow to stand for 30 min-
1. Yeast infusion 1000.0 cc. utes and pour off the liquid.
2. KH2PO4 0.1 g. (2) Repeat the washing process until
3. Sodium phosphate 0.1 g. the wash water is no longer brown
but slightly turbid. Washing 5 (4) Decant the supernatant fluid.

times is usually sufficient. (5) Make neutral to litmus.
(3) After pouring off the last water add (i) Klimmer used a medium prepared as
18 liters of water to the remaining follows:
washed yeast cells. (1) Dilute 10 liters of Brewers yeast or
(4) Boil in the autoclave or steamer as beer yeast with 20 liters of water.
in the preparation of meat bouillon. (2) Allow to stand for 0.5 to 11 hours,
(5) Allow to stand for a suitable length remove the water and add fresh.
of time and remove the liquid from Repeat the process until the wash
the sediment or filter thru filter water is slightly turbid.
paper. (3) Make up the volume of the yeast
Adjust the filtrate or supernatant
(6) to 18 liters.
fluid to a slight alkaline reaction (4) Boil in the autoclave or steamer.
using litmus as an indicator. (5) Allow to settle.
(7) Sterilization not specified. (6) Decant or filter.

(d) Besson prepared the medium as (7) Use the filtrate as meat peptone and
follows: peptone or nutrose.
(1) Boil 100.0 g. of beer yeast in (8) The solution may be evaporated to
1000.0 cc. of water. dryness.
(2) Filter thru paper. (9) The filtrate may be used by the
(3) Distribute as desired. addition of 0.5% NaCl and making
(4) The reaction is slightly acid, but slightly alkaline.
may be made slightly alkaline if (j) Cunningham prepared a medium as
desired. follows:
(5) Sterilize at 115C. (1) Place 1000.0 g. of fresh pressed
(e) Dopter and Sacquepee used a me- yeast in a double walled pot and
dium prepared in the following mix thoroly with 1000.0 cc. of water.
manner: (2) Steam for one hour.
(1) Dialyse yeast in 6 times their (3) Filter thru paper.
weight of water. (Time not given.) (4) Tube in 5.0 cc. quantities.
(2) Boil, stirring constantly. (5) Sterilize by intermittent steaming.
(3) Filter. References: Thoinot and Masselin (1902

(4) Distribute. p. 29), Henneberg (1903 p. 8), Heinemann
(5) Sterilize. (1905 p. 130), Gassner (1916-17 p. 311),
(f) Harvey added 75.0 g. pressed yeast Besson (1920 Dopter and Sacquepee
p. 34),
to 1000.0 cc. of water. Further prep- (1921 p. 121), Harvey (1921-22 p. 120),
aration not given. Klimmer (1923 p. 170), Cunningham
(g) Harvey prepared a medium as (1924 p. 103).
follows
1149. Stoklasa's Arabinose Azotobacter
(1) Add 100.0 g. yeast to 1000.0 cc.
Solution
water.
(2) Boil 10 minutes. Constituents
(3) Filter thru well-wetted, thick filter 1. Distilled water 1000.0 cc.
paper. 2. d-arabinose 1-0 g.
(4) Adjust the reaction. 3. MgCl2 0.5 g.

(5) Distribute into flasks or test tubes. 4. Iron sulphate 0.1 g.
(6) Sterilize in the autoclave or 5. Azotobacter culture.

steamer. Preparation
(h) Harvey prepared a similar medium (1) Dissolve 2, 3, and 4 in 1.
as follows: (2) Distribute in 250.0 cc. lots in Fern-
(1) Prepare: Baker's yeast 1; water 5. bach fermentation tubes.

(2) Boil 20 minutes, with vigorous (3) Adjustment of reaction not specified
stirring. (4) Add an azotobacter membrane from
(3) Place in a tall glass vessel 24 hours. mannitol culture to each flask.
Sterilization: Sterilize thoroly (method not References: Lanken and Meyer (1921
given). p. 511), Wiegert (1922-23 p. 110), Klim-
Use: Decomposition of organic nitrogen mer (1923 p. 204).
products by Bacillus mycoides.
1151. Ampola and Garino's Nitrate Peat
Solution
1150. Lanken and Meyer's Fungus Infusion Constituents:

Solution 1. Water 1000.0 cc.
2. Peat 20.0 g.
Constituents: NaNOs
3. 3.2 g.
1. Water 1000.0 cc.
Preparation: (1)Mix 2 and 3 with 1.
2. Fungus, dry 25.0 g.
3. NaCl 5.0 g.
Use: To study denitrification. Authors
Preparation reported that the nitrates were not
(1) Gather the fungus in large amounts reduced.
and remove all dirt from them. Reference: Ampola and Garino (1897
(2) Run thru a meat grinding machine. p. 309).
(3) Dry the chopped fungus in flat plates
and then rub to a fine powder which 1152. Revis' Peat Infusion Solution
is dried and stored.
Constituents:
(4) Add 25.0 g. of (3) to 1000.0 g. water 1. Ammonium hydrate.
and allow to stand at room tempera- 2. Peat.
ture for 24 hours. 3. CaCOa.
(5) Filter. The filtrate should be clear. Preparation:
If not, add a teaspoonful of filter (1) Exhaust peat with dilute ammonium
cell, shake thoroly and filter again,
hydrate (Details of method not
(6) Add 5.0 g. NaCl, and make alkaline given).
to litmus by the addition of 10.0% Neutralize the alkaline fluid with
(2)
soda solution standing for 20 minutes. phosphoric acid.
Sterilization: Sterilize on 3 successive days and tube.
(3) Filter
for ^ hour in the steamer. Add CaCOs (amount not given).
(4)
Use: Substitute for meat extract medium. Sterilization: Not specified.
Authors used anthrax staphylococci, Use: To study stability of physiological
colon-typhoid group and pyocyaneus, and properties.
reported better growth than on media Reference: Revis (1910 p. 175).
containing peptone. Lactarius turpis,
Mordschwamm, Lactarius turminosus 1153. Brussoff's Iron Peat Infusion Solution
white Reizker, Lactarius rufus Riibling Constituents:
and Lactarius vuleruis Wolfschwamm were 1. Water.
the fungi used. 2. Peat.
Variants 3. Iron.
(a) Wiegert prepared a similar medium Preparation
as follows: (1) Pour water (amount not given) over
(1) Boil 50.0 g. fungus with
dried finely divided peat in a beaker.
500.0 cc. water until the fungus is (2) Heat in the autoclave under 2 atmos-
softened. pheres pressure for 30 minutes.
(2) Add 2.0 g. of NaCl. (3) Pour off the dark brown extract and
(3) Evaporate until a thick extract is press the peat free from liquid.
formed. (4) Distribute the extract in high cylin-
(4) Add 1.0, 2.0 or3.0% of this extract drical glasses to a depth of 4 or 5 cm.
instead of meat extract in the (5) After adding a suitable amount (exact
preparation of nutrient media. amount not specified) of water, cover
(b) Klimmer used 50.0 g. dried fungus the glasses with glass plate cover
instead of 25.0 g. gls
(6) Add 2 or 3 pieces of sterile iron 1156. Buchanan's Basal Legume Extract
filings to sterile (5) under aseptic Solution
conditions.
Constituents:
Sterilization: Sterilize (5) in streaming
1. Water 1000.0 cc.
steam for 6 hours. Sterilize 2 or 3 pieces
2. Legume extract (Park Davis & Co.).
of iron filings dry heat at 150 for one
Preparation
hour.
(1) Boil 1000.0 cc. of one of Park Davis &
Use: Cultivation of Ferribacterium duplex.
Company's standard legume fluid ex-
Author reported that after 5 or 10 days a
tract until all the alcohol has evapo-
slight yellow membrane was formed.
rated. The legume extracts used
Reference: Brussoff (1916 p. 549).
were those of clover, tolu, catechu,
physostigma, red clover blossom,
1154. Schmidt's Hydrolyzed Peat Solution
glycyrrhiza, kino, baptisia, senna and
Constituents scoparius.
1. Water 1000.0 cc. (2) Make up to 1000.0 cc. by the addition
2. Peat 20.0 g. of water.
3. Salts. (3) Filter.
Preparation (4) Prepare 10.0, 5.0, 1.0 and 0.1% solu-
(1) Add 1000.0 g. of 0.3% HCl to 20.0 g. tions of these extracts with water.
of peat and hydrolyze for 1 hour at In case of the clover extract, do not
2.5 atmospheres pressure. dilute. In case of the glycyrrhiza
(2) Filter. prepare only 1.0 and 0.1% solutions.
(3) Neutralize and add an excess of (5) Add one of the added nutrients to
CaCOs. extract solutions.
(4) Add the usual salts (amounts or kinds Sterilization: Sterilize for 20 minutes on
not given). The salts are to be nitro- each of 3 successive days in streaming
gen free. steam.
Sterilization: Not specified. Use: To study gum formation by Bacillus
Use: To study nitrogen assimilation by radicicola. The author reported that
soil forms. Author reported that azoto- the extract of catechu, physostigma, kino
bacter formed as a scum on the surface of and scoparius inhibited growth. The
the medium in several days. remaining legume extracts favored growth
Reference: Schmidt (1920 p. 283). and gum production.
1155. Schmidt's Peat Solution sucrose or 1.0% maltose to the extracts
Constituents prepared as indicated in step (4) above.
1. Water 1000.0 cc. Reference: Buchanan (1909 p. 391).
2. NH4CI (0.25%) 2.5 g.
K2HPO4 (0.05%) 1157. Buchanan's Salt Legume Extract
3. 0.5 g.
Solution
4. CaCOs (2.0%) 20.0 g.
5. Peat 20.0 g. Constituents
Preparation 1. Water 1000.0 cc.
(1) Dissolve 2, 3 and 4 in 1. 2. Legume extract (Park Davis & Co.).
(2) Add 20.0 g. of peat to (1). Preparation
Sterilization: Not specified. (1) Boil 1000.0 cc. of one of Park Davis &
Use: Cultivation of and horse manure
soil Company's standard legume fluid
forms. Azotobacter. Author reported extract until all the alcohol has
that azotobacter formed a membrane on evaporated. The legume extracts
the surface after several days. used were those of clover, tolu, ca-
Variants: The author used moss instead of techu, physostigma, red clover blos-
peat. som, glycyrrhiza, kino, baptisia,
Reference: Schmidt (1920 p. 284). senna and scoparius.
(2) Make up to 1000.0 cc. by the addition the bacteroids, either unmodified or upon
of water. the addition of asparagin or peptone.
(3) Filter. Added nutrients: The author added one of
(4) Heat distilled water containing 0.2% the following:
KH2PO4 and 0.01% MgS04 to boiling. KH2PO4 0.2%
(5) Cool (4) and filter. MgS04 0.1%
(6) To each 100.0 cc. of (5) add 0.1%, Ammonium phosphate 0.5%
1.0%, 2.0%, 5.0% or 10.0% of one of Asparagin 1.0%
(3). (Filtered extract solution.) Sodium asparaginate 1-0%
(7) Incubate at room temperature for one Peptone 0.1, 0.5, 1.0,
week to test sterility. 2.0 or 5.0%
for 20 minutes on
Sterilization: Sterilize [Sucrose 2.0%
each of 3 successive days in streaming \ [0.005, 0.01,
steam. [KNO3 i0.05, 0.1, 0.2
Use: To study gum formation by Bacillus [0.5 or 1.0%
radicicola. Author reported that tolu, Glucose 2.0%
red clover blossoms, glycyrrhiza, kino, Reference: Buchanan (1909 pp. 62, 386,
baptisia and senna favored production. 391).
Cathechu, physostigma, scoparius and
kino in high concentrations were un-
1159. Buchanan's Vetch Infusion Solution
suitable. Constituents:
Variants: The author added 1.0% maltose. 1. Water 2000.0 cc.
Reference: Buchanan (1909 pp. 72, 386). 2. Vetch stems and leaves 200.0 g.
Preparation:
1158. Buchanan's Basal Clover Infusion
(1) Boil 200.0 g. of dry vetch stems and
Solution
leaves in 2 liters of water.
Constituents: (2) Filter.
1. Water 4000.0 cc. (3) Concentrate to ^ its volume by heat.
2. Clover leaves and stems (4) Add several times its volume of
{Trifolium pratense) 200.0 g. alcohol.
Preparation: (5) Filter.
(1) Extract 200.0 g. of the leaves and (6) Evaporate the filtrate to dryness.
stems of clover (Trifolium pratense) (7) Wash the precipitate with absolute
with 4 liters of boiling tap water. alcohol, several times and evaporate
(2) Filter until clear. to dryness.
(3) Add one of the added nutrients to (2). (8) Dissolve each of these extracts in
(4) Heat to boiling. 100.0 cc. of distilled water.
(5) Cool and filter. (9) Filter.
(6) Incubate for one week at room tem- (10) The first of these is called alcoholic
perature to test sterility. extract and the second the aqueous
for 20 minutes on
Sterilization: Sterilize extract.
each of 3 successive days in streaming (11) Prepare 0.01, 0.1, 0.2 or 1.0% solu-
steam. tions of the aqueous extract in dis-
Use: To study gum formation by Bacillus tilled water and 0.1, 0.5, 1.0, 2.0 or
radicicola, and cultivation of Bacillus 5.0% solutions of the alcoholic ex-
radicicola bacteroids. Author reported tract in distilled water.
that clover extracts alone favored the (12) Tube.
production of gum. Peptone, and to a (13) Incubate for one week at room tem-
less degree, asparagin and sodium aspara- peratures to test sterility.
ginate inhibited the production of gum. Sterilization: Sterilize in streaming steam
Nitrates except in large amounts did not for 20 minutes on each of 3 successive
effect growth and gum production. days.
Clover extract did not constitute a Use: To study gum formation by Bacillus
favorable medium for the development of radicicola and cultivation of Bacillus
radicicola bacteroids. The author re- Sterilization: Method not given.

ported that the aqueous and alcoholic Use: Cultivation of Heterosporium vari-
extracts in low dilutions tended to favor abile.
the production of gum. Both the alco- Reference: Reed and Cooley (1911-12
holic and aqueous extracts of the vetch p. 50).
served as suitable nutrients for growth of
B. radicicola when in proper concentra- 1162. Maze's Sucrose Bean Infusion
tion. There was little bacteroid pro- Solution
duction. Constituents:
Variants: The author specified the use of 1. Water 1000.0 cc.
V. Faba stems or leaves. 2. Beans (white)
Reference: Buchanan (1909 pp. 71, 386). 3. Sucrose (2.0%) 20.0 g.
4. NaCl (1.0%) 10.0 g.
1160. Wilhelmi's Clover Flower Infusion
Preparation
Solution
(1) Infuse white beans in water for
Constituents: 30 minutes at 100 C.
1. Distilled water 1500.0 cc. (2) Add 2.0%, sucrose, 1.0% NaCl and a
2. Clover flowers trace of sodium bicarbonate to (1).
3. Glucose 75.0 g. (Filtering of (1) not specified.)
(1) Fill a 2 liter flask with clover flowers Use: Cultivation of nitrogen fixing bac-
and add 1500.0 cc. distilled water teria.
and 75.0 g. glucose. Variants
(2) Sterilize for 20 minutes in the auto- (a) Tanner, Besson, and Dopter and
clave at 120 C. Sacquepê prepared the medium as
(3) Add sterile distilled water to restore follows:
to 1500.0 cc. (1) Macerate 50.0 to 60.0 g. haricot
(4) Filter. beans in a liter of water for several
(5) Add 25.0% HCl until litmus paper is hours in the cold.
turned red (about 5.0 cc). (2) Boil for 30 minutes.
(6) Allow to stand a day and again adjust (3) Pour on a coarse sieve.
(about 1 or 2 cc acid will be required)
.
(4) Collect the liquid.
(7) A slight acidity will be reached after (5) Add 1.0% NaCl, 2.0% sucrose and
4 or 5 days, then observe the reaction a pinch of sodium bicarbonate.
of the extract for 2 or 3 days. (6) Boil and filter thru paper.
(8) Add the required amount of HCl (best (7) Tube.
growth takes place when there is (8) Sterilize at 115C.
about 5.5 or 5.8% pure hydrochloric (b) Harvey prepared the medium as
acid material present. follows:
Sterilization: Sterilization is effected in (1) Mince finely haricot beans.
step (3) above. (2) Add 250.0 g. to 1000.0 cc. distilled
Use: Isolation of Saccharovnjces guttulatus water or clear tap water.
from intestines and stomach. Author (3) Heat the mixture 20 minutes at a
reported that bacteria did not develop in temperature not exceeding 50 C.
this acid medium. (4) Raise the temperature to boiling
Reference: Wilhelmi (1898 p. 358). point.
1161. Reed and Cooley's Bean Pod Infusion Add 1.0 cc. 1.0% sodium bi-
(6)
Solution
carbonate.
Constituents: (7) Add 10.0 g. sodium chloride.
1. Water. (8) Steam 45 minutes.
2. Bean pods (Phaseolus vulgaris). (9) Bring the volume up to 1000.0 cc.
Preparation: (1) Cook green pods of Pha- by the addition of distilled water.
seolus vulgaris in water (amount not (10) Filter while hot thru thick filter
given). paper.
(11) Add 20.0 g. sucrose. 2. Pea flour 100.0 g.

(12) Distribute into test tubes. 3. NaCl 100.0 g.
(13) Sterilize in the steamer or auto- Preparation
clave. (1) Add 100.0 g. of pea flour and 100.0 g.
References: Maze (1897 p. 45), Tanner NaCl to 1 liter distilled water.
(1919 p. 58), Besson (1920 p. 35), Dopter (2) Mix and steam, stirring constantly
and Sacquepee (1921 p. 121), Harvey for 30 minutes.
(1921-22 p. 119). (3) Allow to settle.
(4) Filter.
1163. de Rossi's Glucose Bean Infusion
Solution
Use: Peptone substitute. The author re-
Constituents: ported that this solution was especially
1. Water 1000.0 cc. designed to replace Witte's peptone.
2. Beans 10.0% 100.0 g. The infusion should, preferably, be made
3. Glucose 2.0% 20.0 g. fresh each time it is used.
Preparation Reference: Tanner (1919 p. 57).
(1) Prepare a bean infusion using 10.0%

1166. Stutzer's Basal Legume Seed Infusion
beans.
Solution
(2) Dissolve 2.0% glucose in (1).
Use: To study nitrogen fixation by bacteria 1. Distilled water 1000.0 cc.
from nodules of leguminous plants. 2. Pea seeds 10.0 g.
Reference: de Rossi (1909), (1910 p. 271). or
clover seeds {Trifol hy-
1164. Kaufmann's Jequirity Infusion bridum) 10.0 g.
Solution
or
Constituents: bean seeds (Vicia Faba) 10.0 g.
1. Water 1000.0 cc. or
2. Jequirity seeds 100.0 g. lupine seeds 10.0 g.
(1) Grind 100.0 g. Jequirity seeds in a (1) Grind pea seeds, clover seeds {Trifol
mortar. hybridum), bean seeds {Vicia Faba)
(2) The peeled or shelled seeds now or lupine seeds and pour 1 liter of
weight about 80.0 g. water over each 10.0 g. of one of the
(3) Add to 1000.0 cc. of water. ground seeds.
(4) Boil in a steam sterilizer for about (2) Boil for 1 to 2 hours, adding the water
two hours. lost by evaporation.
(5) Cool and filter. (3) Filter.
(6) Reaction is neutral or slightly alka- (4) Dilute or concentrate the filtrate as
line. desired so that the ratio of pea seed
(7) Distribute into test tubes. to water be 100, 75, 50, 40, 30, 20, 10,
Sterilization: Sterilize in the usual manner 5, 4, 3, 2 or 1.0 g. per liter. Best
(method not given). results are obtained however using
Use: General culture medium. Author re- 10.0 g. peas per liter water as in (1).
ported that the medium supported the (5) Dissolve one of the added nutrients
growth of Bacillus pyocT/aneus and many in (4).
other organisms. Sterilization: Not specified.
Variants: The author aded 6.0% glycerol Use: The study the cultivation of Pisum
or 6.0% glycerol with 2.0% peptone. sativum, Vica Faba, Trifoliuin pratense
Reference: Kaufmann (1891 pp. 65, 68). and Trifolium incarnatum. Author re-
ported that generally the addition of
1165. Tanner's Pea Flour Infusion Solution
carbohydrates and asparagin favored the
Constituents: growth of the bacteroids. Oil seemed to
1. Distilled water 1000.0 cc. have little effect.
Added nutrients and variants: (2) Add a little distilled water (amount
(a) The author added one of the fol- not given).
lowing :
(3) Allow to stand in the cold for
Asparagin 2.0% 24 hours.
Glucose 1.0, 5.0 or 10.0% (4) Press thru a towel.
Sucrose 1.0, 5.0 or 10.0% (5) Sterilize until there is no more co-
starch 2.0% agulum formed (time not specified).
flour 2.0% (Filtration not specified.)
inulin 2.0% (b) Tanner prepared a similar medium as
oil small amount follows:
(b) The author prepared an infusion from (1) Wash and grind a few potatoes.
clover seeds in the following manner: (2) To 30.0 g. of (1) add a liter of dis-
(1) Heat 3.0 to 4.0 g. of clover seed in tilled water.

water on a water bath for several (3) Allow to stand in the ice box over
hours. night.
Pulverize the soft seeds in a rub-
(2) (4) Filter.
bing dish. (5) Boil the filtrate.
(3) Heat again for an hour. (6) Filter again if necessary.
(4) Add distilled water until 1000.0 cc. (7) Tube.
are obtained (1.0 g. K2HPO4 may (8) Sterilize (method not given).
be added). (9) If the reaction is too acid, adjust
(5) Distribute in 10.0 cc. lots. as usual.
(6) Sterilize 3 times in the usual way. (c) Besson prepared the medium as
(Method not given.) follows:
(c) The author added 0.1% K2HPO4 to an (1) Clean and grate potatoes.
infusion prepared as indicated in the (2) Soak 30.0 g. of (1) in 1000.0 cc.
basic solution using 2.5 g. Vicia Faha water for 4 hours.
per liter and added 0.0 or 1.0 g. (3) Decant the liquid.
K0HPO4. (4) Boil.
(d) The author used the basic solution (5) Filter.
without any additions. (6) Reaction may be neutralized if
Reference: Stutzer (1900 pp. 898, 903, 908). desired.

1167. Smith's Potato Infusion Solution
(8) Sterilize in the autoclave or
Constituents: steamer.
1. Water 1000.0 cc. (d) Dopter and Sacquepee prepared a
2. Potato 500.0 g. similar medium as follows:
Preparation (1) Grate potatoes.
(1) Pass clean, pared potatoes rapidly (2) Macerate 10.0 to 20.0 g. of (1) with
thru a grating machine. 1 liter of water for two hours.
(2) Throw immediately into double the (Temperature not specified.)
weight of distilled water. (3) Strain thru a cloth.
(3) Place on a water bath and quickly (4) Bring to a boil.
raise the temperature to 55C. Keep (5) Filter thru paper.
at this temperature for an hour, stir- (6) Distribute.
ring frequently. (7) Sterilize in the autoclave.
(4) Filter. (e) Robertson and Davis studied the
Sterilization: Sterilize in the steamer. influence of vitamines on bacterial
Use: General culture medium. Author growth. They reported that the me-
cultivated plant parasites. dium did not give continued growth
Variants of yeast. The medium was prepared
(a) Peklo cultivated plant actinomyces as follows:
on a medium prepared as follows: (1) Grate potato fine and cover with
(1) Prepare a pulp from raw potatoes. sterile distilled water.
(2) Allow to stand at room tempera- 5. MgS04 0.2 g.

ture for 18 hours. 6. K2HPO4 1.0 g.
(3) Pass thru a Berkefeld filter. 7. NaCl 5.0 g.
(4) Adjustment of reaction not given. 8. Potato
(5) Add sterile (method of sterilization Preparation:
not given) physiological salt solu- (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1 by
tion to (3). boiling 3 minutes.
(f) Park, Williams and Krumwiede pre- (2) Restore original volume with sterile
pared the medium as follows: distilled water.
(1) Run white potatoes thru the grater (3) Adjust reaction to pH = 7.4.
or chopping machine. (4) Tube.
(2) Mix 1.0 pound of (1) with water. (5) Grate potato fine and cover with
(3) Soak over night. sterile distilled water.
(4) Heat to boiling. (6) Allow to stand at room temperatures
(5) Press thru cheese cloth. for 18 hours.
(6) Add one egg per liter. (7) Pass thru a Berkefeld filter.
(7) Autoclave one hour. (8) Add various amounts of (7) to each
(8) Filter thru cotton (very tedious). tube of sterile (4).
(9) Store in flasks and sterilize in the Sterilization: Autoclave (3) at 20 pounds
autoclave for 30 minutes at 15 pressure for 30 minutes.
pounds pressure. Use: To study influence of vitamines on
References: Smith (1905 p. 42), Peklo (1910 bacterial growth. Authors reported that
p. 551), Tanner (1919 p. 60), Besson (1920 yeast grew very luxuriantly.
p. 35), Dopter and Sacquepee (1921 Reference: Robertson and Davis (1923
p. 120), Robertson and Davis (1923 p. 154), p. 154).
Park, Williams and Krumwiede (1924
1170. Lubinski's Glycerol Potato
p. 122).
Infusion Solution
1168. Eisner's Hydrochinone Potato Constituents:

Infusion Solution 1. Water 1500.0 cc.
2. Potato 1000.0 g.
Constituents:
3. Glycerol (4.0%) 60.0 g.
1. Potato water.
Preparation
2. Hydrochinone.
(1) Wash potatoes clean and cut them in
Preparation
small pieces.
(1) Preparation of potato water not
(2) Boil (1) for 3 or 4 hours under a free
given.
flame, or in a steamer, in 1500.0 cc.
(2) Donot adjust the reaction.
of water.
(3) Add hydrochinone, amount not given.
(3) Filter.
Sterilization: Method not given. The me-
(4) Add 4.0% glycerol to the filtrate.
dium turns brown during sterilization.
(5) Neutralize (may be used without
Use: Differentiation of coli and typhoid
neutralization).
bacteria. Author reported that B. coli
reduced the brown color. Typhoid bac-
teria gave no color change.
Variants
Reference: Eisner (1896 p. 28).
(a) Harvey prepared the medium as
follows:
1169. Robertson and Davis' Potato
(1) Grate finely washed peeled po-
Infusion Solution
tatoes.
Constituents: (2) Add 1000.0 to 2000.0 cc. water.
1. water
Sterile distilled 1000.0 cc. (3) Heat the mixture 20 minutes to a
2. Asparagin (Merck) 3.4 g. temperature not exceeding 50C.
3. CaClj 0.1 g. (4) Raise to boiling temperature.
4. Glucose 20.0 g. (5) Boil 10 minutes.
(6) Pour the mixture on a clean, thick (9) Allow to stand for 24 hours in the
cloth. cold or better on ice.
(7) Collect the fluid which drains thru (10) Filter thru paper.
the cloth together with that ob- (11) Distribute as desired.
tained by squeezing the cloth. Sterilization: Sterilize at 115 for 20
(8) Filter the fluid collected thru minutes.
thick filter paper. Use: General culture medium for sapro-
(9) Add the filtrate to an equal quan- phytes and pathogenic forms.
tity of distilled water. Variants: Tanner gave the following
(10) Steam 60 minutes. method of preparation:
(11) Add glycerol to 4 per cent. (1) Peel the potatoes.
(12) Mix well. (2) Wash the carrots and turnips.
(13) Filter. (3) Cut (1) and (2) in small pieces and
(14) Distribute into test tubes. place in cold water.
(15) Sterilize in the autoclave or (4) Boil 4 hours.
steamer. (5) Strain.
(b) Harvey prepared a medium similar (6) Make weakly alkaline.
to the above as follows: (7) Heat in autoclave at 120C.
(1) Grate finely washed, peeled po- (8) Allow to stand in a refrigerator
tatoes. over night.
(2) Add 2 parts 4 per cent glycerol to (9) Filter thru paper.
1 part potato gratings. (10) Final sterilization not given.
(3) Boil. References: Berthelot (1917 p. 131), Tan-
(4) Filter. ner (1919 p. 46).
(5) Use the filtrate as potato extract.
1172. Migula's Potato Juice
References: Lubinski (1895 p. 126), Harvey
(1921-22 pp. 118, 119). Constituents:
1. Potato.
1171. Berthelot's Vegetable Infusion
Preparation:
Solution
(1) Pass peeled potatoes thru a sieve, or
Constituents: grind real fine.
1. Water 4000.0 cc. (2) Press the juice thru a linen towel.
2. Potato 300.0 g. (3) Distribute in tubes.
3. Carrots 150.0 g. Sterilization Boil for 20 minutes on each of
:
4. Turnips 150.0 g. 3 successive days in a steamer.

Preparation Use: General culture medium.
(1) Peel the potatoes. They may be Reference: Migula (1901 p. 20).
left whole or cut in two.
1173. Winogradsky's Gypsum Root Solution
(2) Wash the carrots and turnips.
(3) Mix (1) and (2) in 4 liters of cold Constituents:
water. 1. Water 3000.0 to 5000.0 cc.
(4) Bring to boil and boil for 4 hours in 2. Gypsum
order to reduce the volume of water 3. "Butomus-Rhizonus"
one-fourth. Preparation
(5) Filter thru a fine towel. (1) Cut a "Butomus-Rhizomus," freshly
(6) Bring the filtrate to 3 liters volume removed from the ground, into several
either by further boiling or adding pieces.
distilled water. (2) Place the pieces containing mud or
(7) Add 10.0% soda solution to neutral- slime into a deep jar, containing 3.0
ize boiling (6). The reaction may to 5.0 liters of water.
be adjusted to a slight alkalinity to (3) Add a few grams of gypsum.
litmus. Sterilization: Not specified.
(8) Heat in the autoclave at 120 for Use: Enrichment of sulphur bacteria.
30 minutes. Author reported that H2S was formed
after 5 to 7 days. The slime at the bot- (d) Draper gave the following method of
tom became black. The liquid became preparation. The medium was used
opalescent and surface 3 to 6 weeks a for the cultivation of Oidium albi-
membrane of sulfur formed on the surface. cans.
Reference: Winogradsky (1888 p. 11). (1) Chop raw carrots into fine particles
and allow to extract in sterile dis-
1174. Tanner's Carrot Infusion Solution tilled water at about 10C. for
Constituents: 48 hours.
1. Distilled water 1000.0 cc. (2) Filter.
2. Carrots 30.0 g. (3) Tube.

Preparation (4) Autoclave for 15 minutes at 15
(1) Wash and grind a few carrots. pounds pressure.
(2) To 30.0 g. of (1) add a liter of dis- References: Tanner (1919 p. 60), Besson
tilled water. (1920 p. 35), Dopter and Sacquepee (1921

(3) Allow to stand in the ice box over p. 120), Robertson and Davis (1923
night. p. 154), Draper (1924 p. 635).
(4) Filter.
1175. Robertson and Davis' Carrot Infusion
(5) Boil the filtrate.
Solution
(6) Filter again if necessary.
(7) Tube. Medium identical with medium 1169 but
(8) If the reaction is too acid, adjust carrot used instead of potato.
as usual.
1176. Migula's Hay Infusion Solution
Use: General culture medium. Constituents

Variants 1. Water 3000.0 cc.
(a) Besson soaked the carrots for 4 hours 2. Hay 100.0 g.
instead of over night, as in step (3) Preparation
above. (1) Pour 3 liters of water over 100.0 g. of
(b) Dopter and Sacquepee prepared the hay and allow to stand for 24 hours
medium as follows: in a warm place.
(1) Grate carrots. (2) Press thru linen.
(2) Macerate 10.0 to 20.0 g. of (1) with (3) Neutralize and make slightly alkaline
1 liter of water for 2 hours. (Tem- to litmus.
perature not specified.) (4) Boil for one hour.
(3) Strain thru a cloth. (5) Cool to 40C. and add the whites of
(4) Bring to a boil. 3 eggs.
(5) Filter thru paper. (6) Boil and filter.
(6) Distribute. (7) Distribute in 100.0 cc. lots in flasks.

(7) Sterilize in the autoclave. Sterilization: Steam on 3 successive days
(c) Robertson and Davis studied the for one hour each day.
influence of vitamines on bacterial Use: General culture medium.
growth. They prepared a medium Variants: The following methods of prep-
as follows: aration have been suggested by various
(1) Grate carrots fine, and cover with investigators
sterile distilled water. (a) Heinemann.
(2) Let stand at room temperatures for (1) Macerate 10.0 g. of chopped hay in
18 hours. 1000.0 g. water in the water bath
(3) Pass thru a Berkefeld filter. for 3 hours.
(4) Adjustment of reaction not given. (2) Filter.
(5) Add to (3) sterile (method of (3) Sterilize in the autoclave for 10
sterilization not given) physio- minutes at 120C.
logical salt solution. Amounts not (b) Linde cultivated Cladothrix in a
specified. light yellow completely transparent
They reported that the medium did hay infusion, or in a coffee brown
not give continued growth of yeast. hardly transparent medium. The
media were employed without any (5) Boil 10 minutes,

additions, or made slightly alkaline (6) Adjust reaction,
by the addition of NaoCOs. (7) Filter.
(c) Tausz and Peter. (8) Distribute into flasks or test tubes.
(1) Wash grass that has been piled up (9) Sterilize in the autoclave or
for a few days and only slightly steamer.
decomposed, with a little water to References: Migula (1901 p. 20), Heine-
remove the dirt. mann (1905 p. 129), Linde (1913 p. 386),
(2) Boil with a little water. Tausz and Peter (1919 p. 509), Tanner
(3) Cut the grass into small pieces and (1919 p. 57), Besson (1920 p. 35), Dopter
boil for 20 minutes with 3.5 liters and Sacquepee (1921 p. 120), Harvey
of water, (1921-22 pp. 120, 121).
(4) Filter. The filtrate is dark brown.
(5) Neutralize by the addition of sev- 1177. Winogradsky's Ferric Hydroxide Hay
eral drops of KOH. Infusion Solution (Molisch)
(6) Sterilize by steaming on each of 3
Constituents:
successive days for 30 minutes.
1. Water.
They cultivated Bacterium alipha- Hay.
2.
ticum, Bacterium aliphaticum lique-
3. Ferric hydroxide.
faciens, paraffin bacteria.
Preparation
(d) Tanner, Besson, and Dopter and
(1) Place a handful of macerated hay
Sacquepee. that has been well extracted with
(1) Macerate 15.0 to 20.0 g. of finely
water in a glass cylinder 50 cm. high.
chopped hay in 1000.0 g. of water
(2) Sprinkle some freshly precipitated
for one or two hours.
ferric hydroxide on the hay,
(2) Boil a few minutes.
(3) Fill the cylinder with well water.
(3) Filter. Sterilization: Not specified.
(4) Tube. Use: Cultivation of iron bacteria, Chlamy-
(5) Sterilize at 115C.
dothrix {Leptothrix) ochracea.
(6) The infusion may be neutralized if
Reference: Molisch (1910 p. 32).
the reaction is a little acid.
Besson, and Dopter and Sacquepee Straw Solution

1178. Jensen's Nitrate
treated straw in exactly the same
manner, Constituents:
(e) Harvey. 1. Water 100.0 cc.

2. Straw 5.0 g.
(1) Prepare dried chopped hay 10; dis-
tilled water 1000, in a well-stop- 3. NaN03 1.0 g.
pered bottle. Preparation: (1) Mix 2 and 3 with 1.
Heat to 70C. Sterilization: Not specified.

(2)
(3) Close tightly with stopper. Use: To study denitrification by straw
Heat 3 hours at 60C. on a water forms. The author reported that glycerol
(4)
bath, aided in the reduction of nitrates.
(5) Steam 60 minutes. Variants: The author added 2.0% glycerol.

thru thick filter paper. Reference: Jensen (1897 p. 691).
(6) Filter
(7) Distribute into test tubes.
1179. Wolbach and Singer's Glucose Hay
(8) Sterilize in the autoclave or
Infusion Solution
steamer.
(f) Harvey. Constituents:
(1) Mince finely hay or straw. 1. Water 1000.0 cc.
(2) Add 15 to 20.0 g. to 1000.0 cc. water. 2. Hay 120.0 g.
(3) Heat the mixture at a temperature 3. Glucose 10.0 g.
not exceeding 50C. Preparation

(4) Raise the temperature rapidly to (1) Cut 120.0 g. hay fine and soak in 1
boiling point. liter of water for | hour. Final reac-

tion being about 0.2% acid to plienol- 1182. Conrad's Glucose Cabbage Infusion
phthalein. Solution
(2) Decant the water and add 1.0% Constituents:
glucose. 1. Water 200.0 cc.
(3) Adjust the reaction to neutral to 2. Cabbage 100.0 g.
phenolphthalein. 8. Glucose 3.0%
(4) Tube after sterilization. 4. NaCl 0.5%
Sterilization: Sterilize by filtration thru Preparation:
Chamberland F filter.
(1) Prepare an extract of cabbage (Weiss-
Use: Cultivation oi Spirocheta elusa (free kraut abkochung) in the ratio of 1:2.
living). Author reported that the me- Method not given.
dium was clouded by growth in about (2) Add 3.0% glucose and 0.5% NaCl
48 hours. Medium not so satisfactory if to(l)._
sterilized by repeated steaming or in the (3) Place in a large flask.
autoclave. Sterilization: Sterilize on 3 successive days
Reference: Wolbach and Binger (1914 p. 9). in the steamer.
1180. Sherman's Soil Hay Infusion Solution

Use : To study fermentation by sauerkraut
bacteria {Bacterium brassicae acidae).
Constituents: Reference: Conrad (1897 p. 65).
2. Soil 100.0 g. 1183. Will's Cabbage Juice
3. Hay infusion (1.0%) 190.0 cc. Constituents:
4. CaCOs small excess 1. Cabbage juice.
Preparation Preparation: (1) Method of obtaining cab-
(1) Boil 1 and 2. bage juice not given.
(2) Filter clear. Sterilization: Method not given.
(3) Add 3. Use: Cultivation of mycoderma. Fred and
(4) Add 4 in small excess. Peterson studied the effect of different
Sterilization: Method not given. NaCl concentrations on the growth of
Use: Cultivation of protozoa, flagellates, lactic acid producing bacteria. They re-
ciliates and amoebae. ported that the higher the salt concentra-
Reference: Sherman (1916 p. 42). tion the less acid produced, 4.0% was the
1181. Smith's Cabbage Infusion Solution critical point. Yeast grew well at 4.0%
Constituents:
or higher NaCl concentrations, m '
Variants: Fred and Peterson added 0.0, 1.0,

1. Distilled water.
2.0, 4.0, 5.0 or 6.0% NaCl to cabbage
2. Cabbage leaves. juice.
Preparation: Boil a few cabbage leaves
(1) References Will (1910 p.
: 3), Fred and Peter-
in distilled water. son (1922 p. 266).
Use: Cultivation of Pseudomonas cam- 1184. Reed and Cooley's Basal Spinach
pestris (Pammel). Author reported that Infusion Solution
organisms grew very well in the medium Constituents:
producing a heavy clouding and a copious 1. Water 500.0 cc.
yellow precipitate. B. cloacae gave gas 2. Spinach 100.0 g.
from this medium. In litmus medium, Preparation
the culture continued to be red for several (1) Steep 100.0 g. of young spinach leaves
days. Then the litmus lost its color ex- in 500.0 cc. water.
cept in the very top layer where it came (2) Filter.
in contact with air. (3) Neutralize.
Variants: The author
added sufficient (4) Add H2SO4 to make slightly acid to
Trommsdorf's litmus to color the solution litmus.
a pale red. Re-sterilize. (5) Dissolve 1.0% of one of the added
Reference: Smith (1897 p. 479). nutrients in (4).
Sterilization: Not specified. by steaming for 30

Use: Cultivation of Heterosporium vari- minutes on each of 3 successive days.
ahile. Use: Cultivation of Bacterium alipha-
Added nutrients: The author added 1.0% ticum, Bacterium aliphaticum liquefaciens,
NH4NO3 or1.0% peptone. paraffin bacteria.
Reference: Reed and Cooley (1911-12 Variants : The author gave the following
p. 50). method of preparation.
(1) Pour boiling water over 150.0 g. fresh
1185. Sucksdorff' s Tea Infusion green maple leaves and then cut into
Constituents small pieces.
1. Water 1000.0 cc. (2) Boil with 3.5 liters of water for
2. Tea 10.0 g. 30 minutes.
Preparation (3) Filter or clarify by allowing the ma-
(1) Place 10.0 g. tea in a liter of boiling terial to stand. The filtrate is
water. green. Filtration proceeds slowly.

(2) After 5 minutes heating filter half the (4) Neutralize by the addition of KOH
tea into a clean flask. Continue heat- until phenolphthalein is colored
ing the other half for 5 minutes and slightly red.
then filter into a clean flask. (5) Flask and plug.
(3) Distribute into clean non-sterile (6) Sterilize by steaming for 30 minutes
beakers and place in an open window. on each of 3 successive days.
Sterilization:Do not sterilize. Reference: Tausz and Peter (1919 p. 49).
Use: To study the type of organisms that
1188. Miiller-Thurgau's Grape Vine
will grow in tea. The author reported
Infusion Solution
that schizomycetes grew better in tea
than coffee. Molds, however, grow bet- Constituents:
ter in coffee (1192) than tea. 1. Water 1000.0 cc.
Reference: Sucksdorff (1886 p. 368). 2. Grape vine (stems) 100.0 g.
3. Sucrose 50.0 g.
1186. Bushnell's Asparagus Solution Preparation:
Constituents (1) Prepare an infusion of 100.0 g. of
1. Tap water 300.0 cc. grape vine (stems) in 1000.0 cc. of
2. Asparagus 100.0 g. water, method not given.
Preparation: (1) Suspend asparagus in (2) Add 5.0% sucrose to (1).
water (method not given). (3) Dissolve 1.0%, Ca3(P04)2 and 0.5%
Sterilization: Autoclave at 20 pounds for MgS04 in (2).
30 minutes. Sterilization: Method not given.
Use: To study changes (chemical) produced Use: Cultivation burning fungus of
of red
by anaerobes, B. sporogenes group. grape vines. Pseudopeziza tracheiphila

Reference: Bushnell (1922 p. 381). Author reported that fungus grew luxu-
riant at first; then began to turn brown
1187. Tausz and Peter's Leaf Infusion and to show different degeneration
Solution
products.
Constituents: Variants: The author omitted the
1. Water 3500.0 cc. Ca3(P04)2 and MgS04 and added 5.0%
2. Leaves, decomposed 70.0 g. bouillon.
Preparation Reference: Miiller-Thurgau (1903 p. 82).
(1) Boil 70.0 g. of decomposed leaves in

1189. Malenkovic's Wood Infusion Solution
3.5 liters of water for about 40 min-
utes. Constituents
(2) Filter. 1. Water 1000.0 cc.
(3) Neutralize by the addition of several 2. KNOa...., 1.5 g.
drops of KOH. 3. K:HP04 1.5 g.
(4) Distribute in flasks and plug. 4. NH4H2PO4 1.0 g.

5. MgS04 0.5 g. (3) Filter.

6. Wood (4) Tube.
Preparation: (5) Sterilize inthe autoclave or steamer.
(1) Dissolve 2, 3, 4 and 5 in 1. References: Schardinger (1907 p. 161), Tan-
(2) Extract 1000.0 cc. wood with (I). ner (1919 p. 57).
(Method not given.)
1192. Sucksdorff's Coffee Infusion
Use: Cultivation of Coniophora cerebella Constituents:
{Corticium putaneum). Author reported 1. Water 1000.0 cc.
good growth. 2. Coffee 50.0 g.
Reference: Malenkovic (1906 p. 409). Preparation
(1) Pour 1 liter of boiling water over
1190. Olitsky's Tomato Infusion
50.0 g. coffee.
Constituents: (2) Boil for 2 to 3 minutes.
1. Distilled water 250.0 cc. (3) Filter thru a double filter into a clean
2. Tomato plants 80.0 g. flask.
Preparation (4) Distribute into clean non-sterile
(1) Select 80.0 g. of fresh young stems, beakers and place in an open window.
leaves and shoots of a healthy tomato Sterilization: Do not sterilize.
plant. Use: To study the type of organisms that
(2) Cut in small pieces and grind by hand develop in coffee. Author reported that
to a soft pulp in a large mortar. molds grew well in the coffee, while
(3) Thoroughly mix with 250.0 cc. of schizomycetes grew better in tea (1185).
sterile distilled water, having a pH Reference: Sucksdorff (1886 p. 368).
between 5.3 and 5.4.
1193. Tanner's Fruit Infusion Solution
(4) Centrifuge at a high speed for one
hour. Constituents:
(5) Medium discarded if not crystal 1. Water 1000.0 cc.
clear, or reaction does not lie within 2. Fruit (dried) 50.0 to 100.0 g.
5.3 and 6.0 on the Sorensen scale. Preparation
Sterilization: Pass the supernatant fluid (1) Macerate 50.0 to 100.0 g. of dried
through two sterile Berkefeld filters size fruit in a liter of water for several
N. Incubate at 7 days at 28-30C. hours.
Use: Cultivation of the active agent of (2) Stew (1) in the water (time not
mosaic of tobacco and tomato. given).
Reference: Olitsky (1925 p. 131). (3) Pass thru a coarse sieve.
(4) Boil.
1191. Schardinger's Prune Solution
(5) Filter.
Constituents: (6) Tube.
1. Water 200.0 cc. Sterilization: Sterilize at 115C.
2. Prunes 500.0 g. Use: Cultivation of molds.
3. CaCOs 10.0 g. Variants: Harvey used bananas, prunes or
Preparation: (1) Sterilize 200.0 cc. water, raisins and proceeded as follows:
500.0 g. of pitted prunes and 10.0 g. (1) Mince finely bananas, prunes or
CaCOa. raisins.
Sterilization: Given in step (1) above. (2) Add 75.0 g. to 1000.0 cc. of water.
Use: Cultivation of Bacillus ?nacerans and (3) Heat the mixture 20 minutes at a
used as a general culture medium. temperature not exceeding 50C.
Variants: Tanner prepared a similar me- (4) Raise the temperature to boiling
dium as follows: point.
(1) Macerate 50.0 g. of prunes in 1 liter (5) Boil 10 minutes.
of distilled water. (6) Strain the fluid thru a thick, clean
(2) Allow to stand in the ice chest until cloth and squeeze out the residue of
next morning. the fluid.
(7) Adjust the reaction. (a) Used tartaric acid instead of malic.
(8) Filter thru well-wetted, thick filter (b) Used 0.3% tartaric acid and 0.3%
paper. malic acid instead of malic acid
(9) Distribute into flasks or test tubes. alone.
References: Tanner (1919 p. 59), Besson (c) Heinemann prepared a similar me-
(1920 p. 36), Harvey (1921-22 p. 119). dium as follows:
(1) Dilute wine must with 4 times its
1194. Turner's Quince Seed Infusion
weight of water.
Solution
(2) Dissolve 0.5% ammonium tartrate
Constituents: in (1).
1. Distilled water. (3) Macerate in the water bath for
2. Quince seed. 1 hour.
(1) Boil quince seeds in distilled water. (5) Sterilize in the Arnold for 3 con-
(2) Pass the thick glutinous mass thru a secutive days.
sieve. References: Schukow (1896 pp. 608, 610),
(3) Dilute with distilled water to obtain Heinemann (1905 p. 129).
the desired consistency.
1197. Perold's Wine Medium
Use: Cultivation of Euglena. Constituents
Reference: Turner (1917 p. 239). 1. Spatburger wine.
Preparation: (1) Neutralize completely fer-
1195. Miiller-Thurgau and Osterwalder's
mented Spatburger wine of an acidity of
Grape Juice
9.15 g. wine acid per liter, to 5.0% acetic
Constituents acid by the addition of 21.28% KOH.
1. Grape juice. Sterilization: Method not given. After
Preparation: (1) Preparation of grape juice sterilization the medium had the follow-
not given. ing composition:
Sterilization: Not specified. Total acid 5.0% acetic acid.
Use: To study fermentation by Bacterium Alcohol content 8.5% by volume.
mannitopeum, Bacterium intermedium, Volatile acid 0.39% acetic acid.
Bad. Gayoni, Mannite bacteria from Use: Cultivation of bacteria found
in wine.
winft. Bacterium gracile, Micrococcus Variants
acidovorax, Micrococcus variococcus (a) Miiller-Thurgau and Osterwalder
Reference: Miiller-Thurgau and Oster- used red or white wine. Wine may
walder (1918 p. 17). or may not be sterilized. Used to
study fermentation of Bacterium
1196. Schukow's Grape Must Solution
mannitopoeum Bacterium interme-
Constituents: dium, Bact. Gayoni mannitol bac-
1. Water 300.0 cc. teria from wine.
2. Grape must 100.0 cc. (b) Tanner and Besson neutralized the
3. Malic acid (9.0 to wine before sterilization.
10.0%) 36.0 to 40.0 g. References: Perold (1909 p. 18), Muller-
Preparation Thurgau and Osterwalder (1918 p. 19),
(1) Dilute one part grape juice with 3 Tanner (1919 p. 57), Besson (1920 p. 36).
parts water.
1198. Bierberg's Currant Must Solution
(2) Add 9.0 to 10.0% of malic acid to (1).
(3) Distribute into fermentation flasks Constituents:
(sealed with H2SO 4). 1. Water 4000.0 cc.
Sterilization: Sterilize in a steamer. 2. Currant must 1000.0 cc,
Use: To study the utilization of acid by 3. NH4CI 20.0 or 40.0 g.
yeast. Preparation
Variants: The author gave the following (1) Add 1 liter of currant must (800
variants Oechsle) to 4 liters of water.
(2) Dissolve 20.0 or 40.0 g. of NH4CI short time, however, the alkali was
in (1). neutralized and development stopped.
Sterilization: Not specified. Melon juice was superior to cucumber
Use: To study fermentation by yeast. juice.
Author reported that the addition of Variants : The author substituted cucumber
ammonium salts seemed to hasten the juice for melon.
COj production. The higher amount of Reference: Ottolenghi (1911 p. 370).
salts gave a little more CO2 at first.

Over a long period of time there was little 1201. Bierberg's Cherry Must Solution
difference in the amount of CO 2 formed.
Constituents
1. Water 2000.0 cc.
variants
2. Cherry must 1000.0 cc.
(a) Used 20.0 or 40.0 g. of ammonium
3. Sucrose
phosphate instead of NH4CI.
Preparation
(b) Omitted the NH4CI.
(1) The cherry must primarily should
Reference: Bierberg (1909 p. 18).
have a must weight of 66 Oechsle
1199. Miiller's Currant Must Solution and 22.9 Prs. M. acid.
(2) Dilute 1 liter of (1) with 2 liters of
Constituents:
water.
1. Water 1200.0 cc.
(3) Add sucrose to (2) until the must
2. Currant juice 400.0 cc.
weight is 140 Oechsle.
3. Sucrose 200.0 g.
Preparation
Use: To study fermentation by yeast.
(1) Add 400.0 cc. of currant juice to
The author reported that the addition of
1200.0 cc. water.
ammonium salts seemed to hasten the
(2) Dissolve 200.0 g. sucrose in (1).
CO2 production.The higher amount of
salts gave a more CO2 at first.
little
Use: To study fermentation by yeast.
Over a long period of time there was
The author reported that after three and
little difference in the amount of CO2
a half months wine was formed. The
formed.
type of wine formed varied little whether
the juice was taken from normal or
variants:
diseased currants.
(a) The addition of 20.0 g. or 40.0 g. of
Reference: Muller (1909 p. 157).
ammonium phosphate.
1200. Ottolenghi's Melon Juice Solution (b) The addition of 20.0 or 40.0 g. of
NH4CI.
Constituents:
Reference: Bierberg (1909 p. 18).
1. Melon juice 1000.0 cc.
2. NaoCOa (3.0% soln.) (10.0%). 100.0 cc.
1202. Mtiller-Thurgau and Osterwalder's
Preparation
Pear Juice Solution
(1) Method of obtaining melon juice not
specified. Maturity of melons not Constituents
specified. 1. Pear juice.
(2) Neutralize the juice with 3.0% Preparation: (1) Preparation of pear juice
Na.COa and add 10.0%. not given.
(3) Flask. Sterilization: Not given.
(4) Sterilize in the autoclave. Use: To study fermentation by Bacterium
(5) Filter. mannitopoeum, Bacterium, intermediutn,
Sterilization: Sterilize in the autoclave Bad. Gayoni, mannitol from wine.
following filtration. Variants: The authors specified that malic
Use: Enrichment media for cholera acid or alcohol might be added to the
vibrio. The author reported that in pear juice.
melon juice the cholera vibrio developed Reference: MuUer-Thurgau and Oster-
much like in peptone water. After a walder (1918p. 48).
1203. Giltner's Fermented Cider (c) Apple must of 20.0 Balling.

(d) Chrzaszcz cultivated Physarum
Constituents:
leucophaeum (amoba) on grape must
1. Cider.
or pear must {Monilia fructigenes)
Preparation
References: Schukow (1896 p. 609),
(1) Inoculate unfermented cider with
Chrzaszcz (1902 p. 433).
Sacch. ellipsoideus, and allow to
proceed until the evolution of gas 1206. Bachmann's Mince Meat Medium
ceases.
Constituents:
(2) Filter.
1. Water 900.0 cc.
(3) Tube or flask as desired.
2. Chopped apple 1
Sterilization: Pasteurize. Time and tem-
3. Raisins 1 cup
perature not specified.
4. Butter. \ lb.
Use: Cultivation of acetic acid bacteria.
5. Vinegar 1 tbsp.
6. Sugar 1 cup
7. Steak, hamburger ^ lb.
1204, Zikes' Nitrate Fruit Must Solution
8. Cinnamon 1 tsp.
Constituents 9. Cloves 1 tsp.
1. Prune must 10. Nutmeg 1 tsp.
2. NaNOa 1.0, 5.0 or 10.0% 11. Mace 1 tsp.
Preparation 12. NaCl 1 tsp.
(1) Preparation of prune must not given. Preparation
(2) Add 0.0, 1.0, 5.0 or 10.0% of NaNOs (1) Mix 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 in
to (1). enough water to make 900.0 cc.
Sterilization: Not specified. (2) Cook in the steamer for 30 minutes.
Use Volutin production by yeast. Oidium
:
(3) Distribute in 6 inch test tubes and
lactis. Author reported that the pres- fill them half full.
ence of nitrate tended to decrease the Sterilization: Sterilize in the autoclave.
number of yeast. Use: To study effect of spices on growth
Variants: The author used raisin must of Clostridium botulinum. Author re-
instead of prune must. ported that Clostridium botulinum grew
Reference Zikes (1922 p. 33).
: readily in the medium.
Variants: The author used beef suet in-
1205. Schukow's Fruit Must Solution
stead of butter.
Constituents: Reference: Bachmann (1923 p. 237).
1. Grape must.
1207. Harvey's Wheat Flour Solution
Preparation
(1) Prepare grape must of 16.8 Balling. Constituents:
(2) Distribute into fermentation flasks 1. Water 1000.0 cc.
(sealed with H2SO4). 2. Glucose 15.0 g.
Sterilization: Sterilize in a steamer. 3. KNO3 1.0 g.
Use: To study utilization of acid by yeast. 4. MgS04.. 0.5 g.

Author reported that after 85 days 0.112 g. 5. Wheat flour 150.0 g.
of acid were utilized from grape must, Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
0.25 g. acid from currant must, and 0.05 g. (Further preparation not given.)
acid from apple must. Acid determined Sterilization: Not specified.
by titration using litmus as an indicator. Use: General culture medium.

Used by Chrzaszcz for the cultivation Reference: Harvey (1921-22 p. 119).
of amoba.
1208. Omeliansky's Flax Stem Solution
Variants: Author used the following in-
stead of grape must: Constituents:
(a) Currant must of 14.4 Balling. 1. Distilled water 1000.0 cc.
(b) Currant must of 14.4 Balling diluted 2. Ammonium phosphate 1-0 g.
with an equal volume of water. 3. Potassium phosphate 1-0 g.

4. MgS04 0.5 g. (a) Heat 50.0 cc. of Lager beer in Erlen-

5. NaCl trace meyer flasks on 3 successive days for
6. Flax stems 20 minutes. Add 2.0 cc. alcohol
7. Chalk before inoculation.
Preparation (b) Heat 100.0 cc. of light Lager beer in
(1) Dissolve 2, 3, 4 and 5 in 1. Erlenmeyer flasks on 3 successive
(2) Cut the flax stems in pieces about days for 30 minutes. Add 5.0 cc. of
7.0 cm. long and tie in little bunches. alcohol before inoculation.
(3) Place these bunches in a long necked (c) Dilute 50.0 cc. light Lager beer that
flask holding about 300.0 cc. has been boiled to remove the alco-
(4) Shake a little chalk in the bottom of hol with 75.0 cc. water. Heat 3
the flask. times for 25 minutes each and add
(5) Fill the flask with (1). each flask.
5.0 cc. alcohol to
Sterilization: Not specified. (d) Heat dark beer in a steamer 3 times
Use: To study changes produced in flax minutes each.
for 25
stems by pectin and cellulose fermenters. (e) Heat dark beer in 125.0 cc. lots in
Reference: Omeliansky (1904 p. 35). Erlenmeyer flasks 3 times for 25 min-
utes each. Add 5.0 cc. alcohol.
1209. Speakman and Phillips' Maize Mash Dilute 50.0 of dark beer with
(f) cc.
Solution
25.0 cc. water and heat 3 times for
Constituents: 20 minutes each. Add 2.0 cc. of
1. Water 3000.0 cc. alcohol to each flask.
2. Maize 10.0 g. (g) Janke prepared a similar medium as
Preparation: Prepare a 3.0%
(1) maize follows:
mash (method not given). (1) Distribute Austrian double malt
Sterilization: Sterilize in the usual manner beer in 10.0 cc. lots in Freuden-
(details not specified). reich flasks.
Use: Cultivation of B. granulobacter- on each of 3 successive
(2) Sterilize
'pectinovorwn, B. volutans, B. mesen- days for 20 minutes in the steamer.
tericus. (3) The alcohol content remains over
Reference: Speakman and Phillips (1924 2.2% by volume.
p. 186). (h) Janke prepared a Lager beer medium
as follows:
1210. Lafar's Beer Solution
(1) Evaporate Lager beer to ^ its
Constituents: original volume by steaming.
1. Water. (2) Add water to its original volume.
2. Lager beer. (3) Distribute in 10.0 cc. lots in Freu-
3. Alcohol. denreich flasks.
Preparation (4) Sterilize on 3 successive days in the
(1) Boil lager beer to remove the alco- steamer.
hol and make up the loss by the addi- (5) Add 3.0% by volume of absolute
tion of water. alcohol.
(2) Distribute in 125.0 cc. lots in Erlen- References: Lafar (1895 pp. 132, 140),
meyer flasks, plug the flasks with cot- Janke (1916 p. 5).
ton, and place a paper cap on each
1211. Waterman's Sucrose Beer Solution
flask.
(3) Before use, add 6.0 cc. alcohol to each Constituents

flask of sterile (2). 1. Beer 1000.0 cc.
Sterilization : Sterilize on 3 successive days 2. Sucrose 40.0 g.
in streaming steam for 25 minutes. Preparation: (1) Dissolve 40.0 g. of sucrose
Use: To study lactic acid production by in beer.
Bacterium Pasteurianum and B. aceti H. Method not specified.
Sterilization:
Variants: Lafar prepared the medium as Use Cultivation of vinegar bacteria, myco-
:
follows: derma, B. Pasteurianum, B. rancens,

B. aceti, Acetobacter melanogenum, B. (3) Strain thru muslin.

xylinum. (4) Steam 30 minutes.
Reference: Waterman (1913 p. 452). (5) Filter.
(6) Sterilize 20 minutes at 100C.,
1212. Wurtz's Malt Infusion Solution
3 days.
Constituents: (e) Harvey added 25.0 cc. of the solution
1. Water 1000.0 cc. obtained from variant (d) to a liter
2. Malt. 100.0 g. of water. Further preparation not
Preparation given.
(1) Grind 103.0 g. of malt. (f) Harvey added 100.0 g. ofmalt to a
(2) Add (1) to 1000.0 cc. of water. liter ofwater. Further preparation
(3) Heat for one hour between 55-58C. not given.
(not above 58). References: Wurtz (1897 p. 46), Thoinot
(4) Boil. and Masselin (1902 p. 30), Tanner (1919
(5) Filter. pp. 57, 58), Bezanôn (1920 p. 115), Bes-
Sterilization: Sterilize at 115 for 15 son (1920 p. 34), Dopter and Sacquepee
minutes. (1921 p. 120), Harvey (1921-22 pp. 112,
Use: Cultivation of yeast, molds and 113).
trichophyta.
Variants
1213. Beijerinck's Malt Wort Solution
(a) Thoinot and Masselin prepared a sim-
ilar medium from malt dust as Constituents
follows: 1. Water, ditch 1000.0 cc.
(1) Wash 100.0 g. of malt dust with 2. Malt wort 3.0 cc.
water. 3. NasCOs 1.0 g.
(2) Boil in a liter of water. 4. Mohr's salt (ammonium
(3) Filter. ferrous sulphate) 0.2 g.
(4) Distribute as desired. Preparation:
(5) Sterilize at 115 for 15 minutes. (1) Add 2, (about 10 Balling) 3, and 4 to
(b) Tanner prepared a similar medium 1 liter of ditch water.
as follows: (2) A precipitate of calcium and ferro-
(1) Place 250.0 g. of malt into a flask. phosphate and carbonate forms.
(2) Add 1000.0 cc. of distilled water (3) Shake well so the precipitate is
heated to 70C. to (1), and close thoroly distributed, and distribute
with a rubber stopper. the mixture into a flask or a beaker.
(3) Place in a water bath at 60 C. for Fill the flask full so that there is little
1 hour. surface exposed to air or cover the
(4) Strain thru cheese cloth into clean beaker with a tight fitting glass plate.
flasks. (4) Place in the incubator about 25 to
(5) Heat in an Arnold for 30 minutes. 30 C.
(6) Filter thru paper. Sterilization: Not specified.
(7) Tube in 10.0 cc. quantities. Use: To study the reduction of sulphates
an Arnold.
(8) Sterilize in by Spirillum desulfuricans Author re- .
(c) Dopter and Sacquepee prepared a ported that at the end of 5 days there was
medium in the same manner as vari- 35 mg. H.iS formed per liter.
ant (a) above, but used only 50.0 g. Reference: Beijerinck (1895 p. 54).
of malt dust instead of 100.0 g.
(d) Harvey cultivated yeast, molds and
1214. Peklo's Infusion Solution
trichophyta on a medium prepared
as follows: Constituents
(1) Prepare: Crushed malt 1 part; dis- 1. Water to 1480.0 cc.
tilledwater at 70C. 4 parts. 2. Malt
(2) Keep 60 minutes at 60C. in a flask 3. K2HPO4 8.0 g.
closed with a rubber cork. 4. K2CO3 6.0 g.
Preparation (2) If sterilized for 20 minutes at 15

(1) Add and thoroly mix malt with dis- pounds pressure pH =
4.7d=.
tilled water. Sterilization: Sterilize in the usual manner.
(2) Boil for 1 hour and allow to stand at Use: Cultivation of yeast.
60C. for 24 hours. Variants: A more concentrated solution
(3) Boil again for one hour. may be employed if desired.
(4) Filter. Reference: Digestive Ferments Co. (1925
(5) Dilute the filtrate by the addition of a p. 14).
little distilled water.
(6) After several sterilizations (times or 1217. Reddish's Malt Extract Solution
method not specified) and filtering ofi
Constituents:
the formed precipitate, distribute in
sterileErlenmeyer flasks.
2. Malt extract 100.0 g.
(7) To 700.0 cc. of (6) add 780.0 g. of dis-
Preparation
tilled water, 8.0 g. K2HPO4 and 6.0 g.
(1) Dissolve 100.0 g. dry extract of malt
of K2CO3.
in 900.0 cc. distilled water.
(8) The reaction is strongly alkaline.
(2) Make to 8 Kaiser (saccharometer)
Sterilization decreases the alkalinity
by adding distilled water (approxi-
somewhat but the reaction still re-
mately 100.0 cc).
mains alkaline.
(3) Adjust the reaction to +1.5 (1.5%
Sterilization: Sterilize (7); method not
agar may be added and dissolved).
given.
(4) Autoclave for 15 minutes at 15 pounds
pressure.
(5) Filter thru folded filter paper.
(6) Tube.
1215. Bokorny's Malt Infusion Solution
Constituents 10 minutes or 10 pounds for 15 minutes.
1. Water 500.0 cc. Use: Substitute for beer wort.
2. Malt infusion 500.0 cc. Reference: Reddish (1919 p. 6),
3. Maltose (2.0%) 20.0 g.
Preparation 1218. Park, Williams and Krumwiede's
(1) Preparation of malt infusion not Basal Beer Wort Solution
given.
Constituents
(2) Mix (1) with equal parts of water and
1. Beerwort.
add 2.0% maltose.
Preparation
(1) Obtain hopped beerwort from the
brewery.
Variants: The author used the following:
(2) Autoclave.
(a) Undiluted malt infusion without any
(3) Cool.
maltose.
(4) Filter.
(b) Dilute malt infusion with 3 times
(5) Dissolve 2.0% of one of the added
its volume of water and add 1.0%
nutrients in the filtrate.
maltose.
(6) Tube.
Sterilization: Sterilization not specified.
1216. Bacto Malt Extract Broth Use: Cultivation of yeast and molds.
(Dehydrated) Added nutrients: The authors added 2.0%
of any desired carbohydrate, alcohol, etc.
Constituents:
Reference: Park, Williams and Krumwiede
(1924 p. 134).
2. Malt e.xtract, dehydrated,
Bacto 15.0 g.
1219. Peklo's Beer Wort Solution
Preparation:
(1) Dissolve 15.0 g. Bacto dehydrated Constituents
malt extract in 1000.0 cc. of distilled 1. Distilled water 450.0 cc.
water. 2. Beer wort 550.0 cc.
3. K2HPO4 8.0 g. teria in hopped lager beer wort of

4. K0CO3 6.0 g. 12.75 Balling.The medium was
Preparation sterilized in the steamer.
(1) Mix 550.0 cc. of wort beer unhopped (c) Klimmer, and Park, Williams and
and 450.0 cc. of distilled water. Krumwiede boiled and filtered beer
(2) Dissolve 3 and 4 in (1). wort before sterilization. They cul-
Sterilization: Not specified. tivated yeast and molds in the
Use: Cultivation of plant actinomyces. medium.
Variants: The author used the following References: Nakazawa (1909 p. 530),
solutions: Heinze (1906 p. 647), Janke (1916 p. 6),
(a) 1. Distilled water. . 200.0 cc. Klimmer (1923 p. 207), Park, Williams
2. Beer wort 800.0 cc. and Krumwiede (1924 p. 134).
3. K2HPO4 15.0 g.
4. K2CO3 2.0 g. 1221. Peklo's Tartrate Beer Wort Solution
5. MgCOs 0.0 or 1.8 g.
Constituents:
or
MgS04 0.0 or 1.8 g.
(b) 1. Distilled water. 350.0 cc.
.
3. K2HPO4 6.0 g.
2. K2HPO4 6.0 g.
4. K2CO3 4.5 g.
3. K2CO3 4.5 g.
5. Calcium tartrate 24.0 g.
Preparation
(c) 1. Distilled water. 200.0 cc.
.
(1) Mix 350.0 cc. distilled water and

450.0 cc. beer wort.
3. NaaCOs 10.0 g.
Dissolve 3 and 4 in (1).
(2)
(d) 1. Distilled water . . 200.0 cc.
(4) To each lot add 1.5 g. of calcium
3. NaaCOs 8.0 g.
tartrate.
4. K2HPO4 0.0 or 15.0 g.
K2CO3 0.0 or 4.0 g.
(e) 1. Beer wort 1000.0 cc.
Will used a similar medium to cultivate
2. NasCOs 20.0 g.
mycoderma.
(i) 1. Distilled water. 200.0 cc.
Variants: Will added 1.0% ammonium tar-
.

trate to hopped beer wort of 10.0% B. to
3. Na.COs 2.0 g.
cultivate mycoderma.
4. K0HPO4 16.0 g.
Reference: Peklo (1910 p. 492), Will (1910
5. K2CO3 4.0 g.
p. 3).
References: Peklo (1910 pp. 472, 482, 507,
509, 514, 551). 1222. Will's Asparagin Beer Wort Solution
1220. Nakazawa's Beer Wort Constituents:
Constituents 1. Beer wort, hopped 1000.0 cc.
1. Beer wort, unhopped.

2. Asparagin (0.7%) 7.0 g.
Preparation: (1) Prepare a 12.0%, unhopped Preparation: (1) Add 0.7% asparagin to
beer wort. hopped beer wort of 10.0% B.

Sterilization: Not specified. Sterilization: Method not given.
Use: Cultivation of yeast, molds, acetic Use: Cultivation of Mycoderma.

acid bacteria, etc. Reference: Will (1910 p. 3).
Variants
1223. Owen's Cane Juice
(a) Heinze used undiluted beer wort to
study nitrogen assimilation of green Constituents:
algae, chlorella, chlorothecium and 1. Cane Juice 1000.0 cc.
stichococcus. He reported that ni- Preparation: (1) Distribute cane juice in
trogen was not assimilated. flasks.
(b) Janke cultivated acetic acid bac- Sterilization: Not specified.
Use: To determine effect of B. saccharalis Preparation

on composition of cane sugar. (1) Boil 500.0 g. of fresh silage for
Variants 30 minutes with 1 liter of tap water.
(a) The author added 1.0% CaCOj to (2) Filter. Wash the silage on the filter
each flask. with hot water until a liter of the
(b) The author sterilized raw sugar cane decoction is secured.
juice of a pH about 5.0 on each of 3 (3) Filter.
successive days at 30C. This me- (4) Distribute in 50.0 cc. lots.
dium was used to study the produc- Sterilization: Not specified.
tion of gum levan. Use: Cultivation of Monascus purpurens
Reference: Owen (1916 p. 242), (1923 from silage.
p. 434). Reference: Buchanan (1910 p. 101).
1227. Reith's Plant Infusion Solution

1224. Owen's Sulphured Cane Juice
Solution Constituents:
Constituents:
2. Wheat 100.0 g.
1. Sugar cane juice 1000.0 cc.
Preparation
2. SO2 (gas)
(1) Soak 100.0 g. wheat in 300.0 g. of tap
Preparation
water for 18 hours.
(1) Pass SO 2 gas into raw sugar cane
(2) Autoclave 30 minutes at 15 pounds.
juice until 10.0 cc. of the juice require
(3) Strain thru cheese cloth.
5.9 cc. of 0.1 normal NaOH to cause
Adjust to pH = 7.5.
(4)
neutralization.
(5) Tube.
(2) pH range from 3.8 to 6.9.
Sterilization: Sterilize on 3 successive days.
Use: General culture medium. Author
Use To study production of levan by levan
:
reported that the media did not support

forming bacteria isolated from raw
the growth of Pfeiffer's bacilli unless red
sugar. Bacillus levaniformans
blood cells were added, see med. 1228.
Variants The author added milk of lime to
:
Variants : The author treated rye, beans,

the above medium until the acidity was
peas, or hemp in exactly the same man-
reduced from 5.9 cc. 0.1 normal NaOH ner as wheat above.
to 0.5 cc. NaOH.
Reference: Reith (1923 p. 245).
1228. Reith's Blood Cell Plant Infusion
1225. Sherman's Corn Juice
Enrich medium 1227 by the addition of
Constituents: 2.0% red cells of horse blood.
1. Corn juice. Use: Cultivation of Pfeiffer bacillus.
Preparation Gives good growth, but not indol reaction.
(1) Obtain green corn plants in about Reference: Reith (1923 p. 245).
tosseling stage.
(2) Pressure applied to get juice. SUBGROUP I-C. SECTION 16
(3) Heat in autoclave a few minutes. Liquid media or basal solutions not con-
(4) Filter thru filter paper. taining digests, but containing ingredients
(5) Tube. of animal origin of unknown chemical
Sterilization: Method not given. composition.
Use: To show acid production by silage Ai.* Only one constituent of unknown
organisms. chemical composition present.
Reference: Sherman (1916 p. 449). Bi. Materials of animal origin, exclusive of
infusions and extracts employed.
1226. Buchanan's Silage Infusion Solution
Ci. Containing animal tissues, cells or
Constituents their derivatives.
2. Silage 500.0 g. See page 358 for A2 and page 357 for B2
Di. Brain or other organ employed. Davis' Serum Medium 1257

Harrass' Glucose Liver Medium 1229 Marmier's Serum Solution 1258
Harrass' Glucose Brain Medium . . . 1230 F2. Containing additional organic carbon.
Moon's Brain Medium 1231 Boeck's Glucose Serum Solution 1259
Weiss' Brain Medium 1232 Sinton's Glucose Serum Solution. 1260. .
Zotta's Brain Infusion Solution 1233 Toyoda's Glucose Serum Solution. 1261 .
Harvey's Heart Medium 1234 Marbais' Lactose Serum Solution... 1262

Dj. Fish employed. Hiss' Inulin Serum Solution 1263
Harde and Hauser's Fish Medium. . 1235 Legroux's Formol Serum Solution. 1264 .
D3. Bone, horn, etc. employed. Marzinowsky's Citrated Blood

Stoklasa's Bone Meal Solution 1236 Solution 1265
Remy and Rosing's "Hornspahne" Griffith's Citrated Blood Solution. 1266 .
Solution 1237 Di* Blood or its derivatives employed.

Cunningham's Hornmeal Solution. . 1238 El. Whole blood used.
D4. Meat employed. Fi. Citrated.
Migula's Meat Medium 1239 Besson's Citrated Blood 1267
Nevins' Glucose Veal Medium 1240 Rogers' Citrated Blood (Stitt) 1268
Do. Egg or derivatives employed. F2. Not citrated.
El. Whole egg employed. Buchner's Blood Solution 1269
Hueppe's Egg Medium 1241 Davis' Blood Medium 1270
Robertson's Egg Solution (Kahn) . . 1242 Klimenko's Laked Blood Solution. . 1271
Harvey's Egg Solution 1243 Wurtz and Soppington's Diluted
E2. Egg yolk employed. Blood Solution 1272
Nastiukoff's Egg Yolk Solution 1244 E2. Defibrinated blood used.
Harvey's Egg Yolk Solution 1245 Bass, Foster and Johns' Glucose
E3. Egg albumin employed. Blood Solution 1273
Fi. Basal solutions; employed with the Besson's Defibrinated Blood 1274
addition of other constituents. Row's Defibrinated Blood Solution
Hollande and Fumey's Basal Al- (Harvey) 1275
bumin Solution 1246 Carpano's Defibrinated Blood
Waksman's Basal Albumin Solution . 1247 Solution 1276
F2. Complete media. Ej. Blood derivatives used.
Kent's Glycerol Albumin Solu- Waksman's Basal Fibrin Solution 1277
. .
tion 1248 Stoklasa's Glucose Fibrin Solution 1278

. .
Barthel's Albumin Solution 1249 Beijerinck's Sucrose Fibrin Solution

Bainbridge's Albumin Solution 1250 (Percival) 1279
Castellani's Lactose Albumin Solu- Fischer's Blood Meal Solution 1280
tion (Stitt) 1251 Remy and Rosing's Blood Albumin
Hogue's Ovomucoid Medium (Heg- Solution 1281
ner and Becker) 1252 Klimmer's Hemoglobin Solution 1282
C2.* Containing animal fluids. D3. Bile employed.
Di. Serum employed. Meyerstein's Bile Medium 1283
El. Basal solutions; employed with the Ottolenghi's Nitrate Bile Solution.. 1284
addition of other materials. Jackson's Lactose Bile Solution 1285
Hiss' Basal Serum Solution 1253 Meyerstein's Glycerol Bile Solution. 1286
Leuch's Basal Serum Solution D4. Other body fluids employed.
(Klimmer) 1254 Thoinot and Masselin's Aqueous
E2. Complete media. Humor Medium 1287
Fi. Not containing additional organic Chira and Noguchi's Glucose Ascitic
carbon. Fluid Medium 1288
Lorrain-Smith's Alkaline Serum Sinton's Glucose Body Fluid
Solution (Heinemann) 1255 Solution 1289
Klein's Basal Alkaline Serum Solu- C3. Containing animal secretions or ex-
tion (Leuch) 1256 cretions.
Martin, Pettit and Vaudremer's Di.f Secretions employed.
Serum Solution 1256a
* See D3 and D4.
* See C3 next column. t See page 357 for Da
El. Skim or whole milk used. Doerr's Mannitol Nutrose Solution. 1318
Fi. No indicators added. F2. Nutrose not specified.
Tanner's Milk Powder Solution 1290 Whittaker's Lactose Caseinogen
Brown and Howe's Milk Solution. . 1291 Solution 1319
Boekhout's Sucrose Milk Solution . . . 1292 Waksman's Basal Casein Solution.. 1320
Reinsch's Milk Medium 1293 Seliber's Basal Casein Solution
Fj. Indicators added. (Harvey) 1321
Gi. Litmus or its salts used. Laxa's Lactic Acid Casein Solution. 1322
Hiss' Basal Litmus Milk Solution. . 1294 Baginsky's Casein Solution 1323
Tanner's Azolitmin Milk Powder Harvey's Alkaline Casein Solution. 1324
Solution 1295 D2. Excretions employed.
Bacto Litmus Milk (Dehydrated) 1296 . . El. Urine used.
Calandra's Picric Acid Litmus Milk Geilinger's Basal Urine Solution... 1325 .
Solution 1297 Bokorny's Sucrose Urine Solution.. 1326

Park and Krumwiede's Litmus Gly- Besson's Urine Medium (Tanner)... 1327
cerol Milk Solution 1298 E2. Feces used.
Smith's Litmus Milk Solution 1299 Burri and Stutzer's Nitrate Feces
Gj. Litmus or its salts not used. Solution 1328
Sherman and Albus' Indicator Milk Dimitroff's Fecal Infusion 1329
Solution 1300 B2. Containing extracts or infusions of
Harvey's Brom Cresol Purple Milk materials of animal origin.
Solution 1301 Ci. Infusions employed.
Harvey's China Blue Rosolic Acid Di. Meat (beef or veal) infusion used.
Milk Solution 1302 El. Containing no additional organic
Bacto Purple Milk (Dehydrated)... 1303 material.
E2. Whey used. Dunham's Meat Infusion Solution.. 1330
Fi. Prepared from a commercial powder. Stutzer and Hartleb's Nitrite Infu-
Bacto Whey Broth (Dehydrated). 1304
. . .
sion Solution 1331
F2. Not prepared from a commercial E2. Containing additional organic material.
powder. Besson's Glucose Infusion Solution. 1332
Gi. Casein precipitated by an enzyme. Harvey's Starch Beef Infusion 1333
Stutzer and Hartleb's Whey Beijerinck's Urea Meat Infusion... 1334
Solution 1305 D3. Blood (blood clot) infusion used.
Emile-W'eil's Litmus Whey Solu- Klimmer's Blood Infusion 1335
tion 1306 Szasz's Blood Clot Infusion 1336
Durham's Litmus Whey Solution. 1307 . .
Dj. Infusions of special tissue used.
Go. Casein not precipitated by an enzyme. Wêllman's Placenta Infusion 1337
Giltner's Sour Whey 1308
Moon's Brain Infusion 1338
Bronfenbrenner, Davis and Mori-

Wolbach and Saiki's Liver Infusion. 1339 .
Brieger's Thymus Gland Infusion

shima's China Blue Rosolic Acid
(Besson) 1340
Whey Solution 1309
Nencki, Sieber and Wyznikiewicz's
Jouan's Litmus Whey Solution 1310
Salivary Gland Infusion 1341
Petruchsky's Litmus Whey Solution
Graham-Smith's Heart Infusion. 1342 . . .
(Grimbert and Legros) 1311

Robertson and Davis' Heart Infu-
E3. Casein or its salts used.
sion Medium 1343
Fi. Nutrose specified.
D4. Infusion of other materials used.
Barsikow's Basal Nutrose Solution. 1312
Mereshkowsky's Egg White In-
Purwin & McNutt's Basal Nutrose
fusion 1344
Solution 1313 Proca's Spleen Infusion Solution. 1345 . .
Thoni and Allemann's Nutrose Harde and Hauser's Fish Infusion.. 1346
Solution 1314 Pergola's Mussel Infusion 1347
Ficker and Hoffmann's Cafiine Nu- Besson's Chicken Infusion (Tanner). 1348
trose Solution 1315 Cj.* Extracts employed.
LoefHer's Lactose Nutrose Solution. 1316
Hill's Artificial Milk Medium 1317 See C3 page 358.
Di. Containing no additional organic B2. Not containing animal tissues or

material. cells: containing animal fluids.
Biisgen and Hoflich's Meat Extract Ficker's Sputum Serum Solution.... 1372
Solution (Linde) 1349 Kayser's Blood Bile Solution 1373
Stutzer's Nitrate Meat Extract Noguchi's Blood Serum Solution . . . 1374
Solution 1350 Berman and Rettger's Casein Meat
D2. Additional organic nitrogen supplied. Extract Medium 1375
Zike's Basal Glucose Meat Extract Adams' Coke Milk Medium 1376
Solution 1351
Stutzer's Glucose Meat Extract 1229. Harrass' Glucose Liver Medium
Solution 1352
Constituents:
Hurler's Succinate Meat Extract
1. Water 1000.0 cc.
Solution 1353
2. Liver (veal) 1.0 lb.
Bloch's Basal Asparagin Meat Ex-
3. Glucose (1.0%) 10.0 g.
tract Solution 1354
Preparation
Kappen's Urea Meat Extract Solu-
(1) Grind one pound of calf liver in a
tion 1355
meat grinding machine.
Viehoever's Urea Meat Extract
(2) Add (1) to 1000.0 cc. of water.
Solution 1356
(3) Add 1.0% glucose (or peptone).
Kappen's Cyanamide Meat Extract
(4) Mix thoroly.
Solution 1357
(5) Distribute into sterile Erlenmeyer
C3. Materials other than infusions or
flasks and plug with cotton.
extracts employed.
Sterilization: Sterilize for 1.5 to 2 hours in
Homer's Tryptophane Gelatin
streaming steam.
Solution 1358
Use: Cultivate obligate anaerobes.
Remy and Rosing's Gelatin Solution. 1359
Reference: Harrass (1906 p. 2237).
Standfuss and Kallert's Bone Jelly
Solution 1360
1230. Harrass' Glucose Brain Medium
Berman and Rettger's Gelatin
Solution 1361 Constituents:
A2. More than one constituent of unknown 1. Water 1000.0 cc.
chemical composition present. 2. Brain, veal 1.0 lb.
Bi. Containing animal cells or tissues. 3. Glucose 10.0 g.
Ci. Animal fluids added. Preparation

Di. Blood used. (1) Grind one pound of calf brain in a
Fleming's Blood and MincedMeat meat grinding machine.

Medium 1362 (2) Add (1) to 1000.0 cc. of water.
Dean and Nouat's Blood Egg Me- (3) Add 1.0% glucose (or 1.0% peptone)
dium (Cutler) 1363 to (2).
Bruschettinis Egg Yolk Blood In- (4) Mix thoroly.
fusion Solution 1364 (5) Distribute into sterile Erlenmeyer
D2. Serum used. flasks and plug with cotton.

Noguchi's Serum Tissue Medium. . . 1365 Sterilization: Sterilize for 1.5 to 2.0 hours
Hata's Serum Tissue Medium 1366 in streaming steam.
D3. Ascitic or other serous fluids used. Use : Cultivation of obligate anaerobes.
Noguchi's Ascitic Fluid Tissue Reference: Harrass (1906 p. 2237).
Medium 1367
1231. Moon's Brain Medium
Kligler and Robertson's Ascitic
Fluid Egg Medium (Stitt) 1368 Constituents:
C2. Animal fluids not added. 1. Ringer's solution.
Bacto Egg-Meat Medium (De- 2. Brain, dog.
hydrated) 1369 Preparation
Rettger's Egg-Meat Medium 1370 (1) Obtain brain material from normal
Besredka's Egg Meat Infusion dog under the most careful aseptic
Medium (BezanQon) 1371 precautions.
CULTURE MEDIA FOR CULTIVATION' OF MICROORGANISMS 359
(2) Cover brain material with Ringer's Preparation

solution (see medium 180). (1) Cut up 500.0 g. of fat-free ox or horse
(3) Cover with sterile oil. heart.
Sterilization: Method not given. (2) Add water to just cover the meat.
Use: Cultivation of Negri bodies. (3) Cook thoroly and slowly in a closed
Reference: Moon (1913 p. 233). vessel over a small flame.
(4) Pour off the liquid portion into a
1232. Weiss' Brain Medium beaker.
Constituents: (5) Mince the solid residue very finely.
1. Water 1000.0 cc. (6) Add the minced residue to the liquid
2. Sheep brain 2000.0 g. in the beaker.
Preparation (7) Bring up the weight of the contents
(1) Mix two parts finely ground sheep of the beaker to 1000.0 g.
brain and one part water. (8) Break down any small lumps of meat
(2) Do not adjust the reaction. between the fingers.
Sterilization: Sterilize in the autoclave. (9) Make faintly alkaline to litmus.
Use: To develop B. botulinus spores and to (10) Distribute into test tubes.
study thermal death rates of Clostridia. (11) Cover with a layer of paraffin.
Variants: Tanner and McCrea mixed two Sterilization: Sterilize 45 minutes at 100C.
parts water and one part sheep brain. Use: Cultivation of anaerobes.
The pH normally was from 6.8 to 7.0. Variants
The medium was sterilized in the auto- (a) Stitt gave the following method of
clave at 15 pounds pressure for 30 minutes preparation:
in 125 to 150 cc. lots in 250 cc. Erlenmeyer (1) Grind 250.0 g. of beef heart muscle.
flasks. (2) Suspend (1) in 250.0 cc. of water.
References: Weiss (1921 p. 71), Tanner and (3) Heat slowly to cook the meat
McCrea (1923 p. 270). thoroly.
(4) Add NaOH to make the reaction
1233. Zotta's Brain Infusion Solution
neutral to litmus.
Constituents: Distribute in test tubes.
(5)
1. Saline solution (0.75% the autoclave.
(6) Sterilize in
NaCl) 200.0 cc. (b) Park, Williams and Krumwiede pre-
2. Brain (beef) 100.0 g. pared the medium as follows:
Preparation (1) Mince 8.0 oz. bullock's heart and
(1) Shake 100.0 g. of fresh beef brain grind in a mortar with 8.0 oz. of
freed from its meninges with broken tap water.
glass until a homogenous mixture is (2) Cook (time not given).
formed. (3) Make alkaline.
(2) Add 200.0 cc. of saline solution (method not given).
(4) Sterilize
(0.75% NaCl) to (1) and mix Tube.
(5)
thoroly. References: Harvey (1921-22 p. 94), Stitt
(3) Filter thru gauze. (1923 p. 42), Park, Williams and Krum-
(4) Heat for 20 minutes. wiede (1924 p. 125).
(5) Tube the supernatant liquid.
min-
1235. Harde and Hauser's Fish Medium
utes. Final pH 6.9. Constituents:
Use: Cultivation of Leptomonns pyr- 1. Water.
rhocoris. 2. Fish (whiting).
Reference: Zotta (1923 p. 281). Preparation
(1) Cut the flesh of a whiting fish in about
1234. Harvey's Heart Medium
1 centimeter cubes and place one cube
Constituents: in a test tube.
1. Water 500.0 cc. (2) Add 8.0 cc. of water to each tube
2. Heart, beef 500.0 g. of (1).
Sterilization: Sterilize at 120 for 20 (3) Distribute into small Erlenmeyer

minutes. flasks.
Use: General culture medium. Sterilization: Sterilize in streamingsteam
Variants: Kahn prepared the medium as by means of the fractional method.
follows: Use: To study decomposition of organic
(1) Free fresh cod fish from bone and skin, N materials by soil forms.
chop into small pieces and grind in a Reference: Remy and Rosing (1911 p. 39).
mortar.
1238. Cunningham's Hornmeal Solution
(2) Add 800.0 cc. tap water for each pound
of fish and boil gently for 45 minutes. Constituents
(3) Adjust the reaction to pH = 7.2. 1. Water 1000.0 cc.
Prior to testing mix the fish muscle 2. Hornmeal (1.0%) 10.0 g.
and broth so that one verticle inch of 3. K2HPO4 (0.05%) 0.5 g.
the tissue is present in each tube. Preparation
(4) Autoclave at 15 pounds pressure for (1) Dissolve 2 and 3 in 1.
20 minutes. (2) Distribute in 10.0 cc. lots in 50.0 cc.
(5) The tubes are sealed with a vaseline flasks or tubes.

cap. Sterilization: Sterilize at 1.5 atmospheres
References: Harde and Hauser (1919 p. pressure.
1259), Kahn (1922 p. 174). Use: To study ammonification by soiland
manure forms, Bac. mycoides, Bad.
1236. Stoklasa's Bone Meal Solution ftuorescens, Bad. vulgar e, Bad. coli,
Sarcina lutea, Bac. putrificus.
Constituents:
Reference: Cunningham (1924 p. 149).
2. CaS04 1.0 g. 1239. Migula's Meat Medium
3. MgCl2 0.5 g.
Constituents:
4. FeS04 0.1 g.
1. Water.
5. Bone meal 10.0 g.
2. Meat.
(1) Dissolve 2, 3, and 4 in a liter of
Chop lean beef into small pieces.
(1)
distilled water.
(2) Add (1) to Erlenmeyer flasks until a
(2) Place 10.0 g. of finely ground bone layer about a finger deep is formed.
meal into a flask. (Bone contains Add water just to cover the meat
(3)
19.0% H3PO4, 5.26% Nitrogen and layer.
1.5% fat). Sterilization: Sterilize the flasks for 30
(3) Add 100.0 cc. of (1) and 800.0 cc. of minutes on each of 3 successive days.
water to (2). Use: Differentiation of colon and typhoid
bacilli. Author reported that the colon
Use: To study bone decomposition. bacilli produced a fetid odor while ty-
Variants: The author used 1.0 g. K2SO4 phoid bacilli did not. Holman cultivated
instead of 1.0 g.CaS04. anaerobes, and Besson Vibrion septique
Reference: Stoklasa (1900 p. 529), (1911 in similar media.
p. 472). Variants
(a) Holman cultivated anaerobes from
1237. Remy and Rosing's Horn Shavings
war wounds on a medium prepared as
Solution
follows:
Constituents: (1) Grind fresh finely chopped fat and
1. Water 1000.0 cc. fiber free beef muscle in a mortar.
2. Horn Shavings 37.5 g. (2) Mix (1) with equal parts of water.
Preparation: (3) Heat slowly to boiling with con-
(1) Dry finely divided "Hornspahne." stant stirring.
(2) Add 0.75 g. of (1) to 20.0 cc. sterile (4) Neutralize to rosalic acid or phenol-
water. phthalein.
(5) Tube. (2) Add soda solution to neutralize or

(6) Autoclave for one hour. make slightly alkaline.
(7) Seed by means of a pipette or (3) Sterilize at 115C.
needle, and mix the inoculum (f) Cunningham prepared a similar me-
thoroly with the medium. dium as follows:
(b) Holman prepared a similar medium as (1) Cut up 500.0 g. of fat free bullock's
follows: heart into pieces about the size of a
(1) Free meat from fat and gross fibers, pea.
mince finely. Grind in mortar and (2) Add 500.0 cc. of water.
mix with equal part of water. (3) Steam until cooked.
(2) Heat slowly to boiling, stir (4) Neutralize to turmeric paper (dis-
constantly. tinctly brown).
(3) Neutralize or make slightly alkaline (5) Stir well.
while hot to phenolphthalein. (6) Steam for 30 minutes.
(4) Tube at least 2 inches high and (7) Pour off the fluid from the pieces of
autoclave at 115C. for 30 minutes. tissue.
(5) Just before use, place the tubes in (8) Tube the pieces of heart and just
flowing steam for 30 minutes and cover them with the extract, taking
cool rapidly. care to wash down any pieces of
(6) Seed with a pipette, swab or needle tissue which adhere to the sides of
and mix material with meat the tube.
particles. (9) Sterilize in the autoclave.
(c) Bachmann studied the effect of spices References: Migula (1901 p. 24), Holman
on the growth of Clostridium botu- (1918 p. 125), Bachmann (1918 p. 237),
linurn, using the following medium: Burke (1919 p. 557), Besson (1920 p. 55),
(1) Grind hamburger and add enough Cunningham (1924 p. 166).
water to make 100.0 cc.
1240. Nevin's Glucose Veal Medium
(2) Add one of the following, cloves,
cinnamon, all-spice, ginger, nut- Constituents:
meg, cayenne pepper, white mus- 1. Water 1000.0 cc.
tard, black mustard in 1.0, 2.0 or 2. Chopped veal 1000.0 g.
2.5 g. quantity. 3. NaCl 10.0 g.
(3) Mix thoroly. 4. Glucose 40.0 g.
(4) Distribute into 6 inch test tubes. Preparation
(5) Sterilize in the autoclave. (1) Mix equal parts chopped veal and
(6) Inoculate when cool. water.
He reported that the organisms were (2) Add 0.5% NaCl and 2.0% glucose.
not affected by any of the spices used (3) Adjust reaction to 0.3% to 0.5%
in amounts up to 2.0%. 2.5% alkaline to phenolphthalein.
retarded the growth of some Sterilization: Method not given.
organisms. Use: Cultivation of B. botulinus.
(d) Burke isolated B. botulinus in a Reference: Nevin (1924 p. 228).
medium prepared as follows:
1241. Hueppe's Egg Meditim.
(1) Chop 500.0 g. of beef heart or other
lean beef with 1000.0 cc. of water. Constituents:
(2) Bring slowly to a boil, stirring 1. Eggs.
constantly. Preparation
(3) Neutralize. (1) Wash the eggs thoroly.
(4) Tube. (2) Sterilize the shell by dipping in sub-
(5) Sterilize at 15 pounds pressure for limate solution.
30 minutes. (3) Wash in sterile water and dry with
(e) Besson cultivated Vibrion septique sterile cotton.
on a medium prepared as follows (4) Make opening in the top of the
a fine
(1) Add 500.00 to 600.00 g. of finely egg with an instrument heated to
chopped beef to a liter flask. glowing.
(5) Inoculate thru the hole with a plati- (4) Autoclave at 15 pounds pressure for
num wire or loop. 20 minutes.
(6) Shake well to mix. (5) Some of the albumin will settle to the
(7) Cover the opening with a small piece bottom of the tubes. This does not
of sterile paper. detract from the value of the medium.
(8) Seal completely with a colloidion (6) The tubes are sealed with a vaseline
membrane. cap.
Sterilization: Given under preparation. Reference: Kahn (1922 pp. 175, 192).
1243. Harvey's Egg Solution
Variants: Investigators have used slightly-
different methods in sterilization of the Constituents
shell, and several have specified that the 1. Water 300.0 cc.
egg be thoroly shaken before use. The 2. Egg
differences are not of sufficient impor- Preparation
tance to warrant a separate discussion for (1) Add the contents of one egg to 300.0
each investigator. cc. water.
References: Hueppe (1888 p. 80), Heim (2) Shake to mix.
(1891 p. 430), Hammerl (1894 p. 155), (3) Raise the temperature slowly to
Abel and Draer (1895 p. 65), Roux and boiling point with frequent shaking.
Rochaix (1911 p. 129), Ball (1919 p. 83), (4) Distribute into test tubes.
Besson (1920 p. 54), Harvey (1921-22 Sterilization: Sterilize in the autoclave or
p. 85). steamer.
Use: Cultivation of wound organisms and
1242. Robertson's Egg Solution (Kahn) other anaerobes. Also used to cultivate
Constituents other microorganisms.
1. Water 500.0 cc. Variants Klimmer prepared the medium as
:
2. Egg follows for the cultivation of amoeba:

Preparation (1) Shake the contents of one egg in a
(1) Beat the yolk of one and the whites of flask containing glass beads.
2 eggs in a beaker. (2) Add 300.0 cc. of distilled water to (1).
(2) Add 6.0 cc. of N/ NaOH to (1). (3) Shake thoroly.

(3) Gradually add 500.0 cc. tap water (4) Boil in a water bath for 30 minutes
to (2). shaking occasionally.
(4) Heat very slowly to 95C. keeping the (5) Distribute in 5.0 cc. quantities in
mixture at this temperature for tubes.
about an hour. (6) Sterilize in the autoclave.
(5) Filter thru cotton, wool and muslin. Reference: Harvey (1921-22 p. 85), Klim-
(6) The reaction at this point is about mer (1923 p. 229), Stitt (1923 p. 35),
pH = 8.2. Park, Williams and Krumwiede (1924
(7) Tube. p. 126).
1244. Nastiukoff's Egg Yolk Solution.
Use: Cultivation of spore forming ana- Constituents:
erobes. The tubes were sealed with a 1. Distilled water 1000.0 cc.
line cap. 2. Egg yolk 100.0 cc.
Variants: The author reported better Preparation
growth in a more acid medium (pH = (1) Separate the yolk of egg from the
7.2) prepared as follows: white by Bunge's method (allow the
(1) Add gradually the yolk of one and the whites to roll from a blotting paper
whites of 2 eggs to 500.0 cc. of tap containing the egg).
water. (2) Add 0.5 cc. of a 10% NaOH solution
(2) Titrate the mixture so that the re- and 100.0 cc. of egg yolk to 1000.0 cc.
action is pH = 7.2. distilled water.
(3) Heat very slowly to 95 and keep at (3) Place in a fiask and steam for 2 hours
this temperature for an hour. in a steamer.
(4) Allow to settle for one day. 3. Egg white

(5) Filter. 4. Litmus
(6) Distribute into test tubes. Preparation:
Sterilization: Sterilize in the usual manner (1) Mix the whites of 2 eggs and measure
(method not given). 30.0 cc. into a flask.
Use: Cultivation of diphtheria, cholera, (2) Add 70.0 cc. of a physiological salt
influenza and other pathogenic forms. solution prepared by dissolving 9.0
Variants: Besredka cultivated tubercle in a liter of distilled water.
bacilli on a medium prepared as follows: (3) Shake from time to time.
(1) Remove the yolks from 20 eggs (350.0 (4) After 15 minutes filter on wet ab-
CO.). sorbent cotton.
(2) Add one liter of neutral distilled (5) Add 8.0 cc. of a solution containing
water (neutralize acid if necessary. 4.0 g. soda to 1000.0 cc. distilled
Indicator not specified). water to the filtrate.
(3) Prepare a 1.0% soda solution. (6) Make up the volume of 200.0 cc. by
(4) Add 175.0 cc. of (3) to (2). the addition of physiological salt
(5) Take a small portion of (4) in a solution (see (2) for preparation).
pipette and determine its trans- (7) Heat in the autoclave at 120 for 20
parency. minutes.
(6) Add (3) to (4) in 1.0 cc. lots until the (8) Decant.
greatest amount of transparency is (9) Distribute in test tubes.
obtained, as above.
testing (The (10) Boil 10.0 g. of litmus cubes in 30.0
quantity soda necessary to be
of cc. ethyl alcohol at 80C.
added varies with the yolk.) (11) Decant, filter and save the filtrate.
(7) Dilute (6) until distilled water has (12) Treat the residue on the water bath
been added to the egg yolk in the for 45 minutes with 40.0 cc. distilled
ration of 1:20. In this case dilute to water.
7.0 liters. (13) Filter and save the filtrate.
(8) Distribute in Roux flasks in 50 to (14) Treat the residue with 10.0 or 20.0
150.0 cc. lots. cc. of ethyl alcohol at 80C.
(9) Sterilize at 110C. for 20 minutes. (15) Mix the filtrates and washings.
References: Nastiukoff (1893 *33 and 34), (16) Neutralize the litmus solution by the
Besredka (1921 p. 291). addition of 5.0% H2SO4 solution.
1245. Harvey's Egg Yolk Solution.
nutrients in 25.0 cc. of (16) by
Constituents: heating.
1. Egg yolk. (18) Allow to cool and immerse strips of
Preparation bibulous paper in the solution.
(1) Extract 20.0 g. of egg yolk with 100.0 (19) Suspend the paper until dryness is
cc. of 30.0% alcohol for 4 days at reached.
37C. (20) Mix equal parts of ether and alcohol.
(2) Allow to deposit. (21) Add one part collodion to 9 parts
(3) Decant the supernatant fluid. (20).
Sterilization: Not specified. (22) Dip the paper (19) into (21).
Use: Add 0.25% supernatant fluid to any (23) Allow the paper to dry a second time.
desired medium. The author treated (24) Cut the paper into small rectangles
ascitic fluid in exactly the same manner. 1 by 3 or 4 centimeters.
Reference: Harvey (1921-22 p. 121). (25) Add one or two of the rectangles to
each tube of (9).
1246. Hollande and Fumey's Basal Albumin
Solution.
118C.
Constituents: Use: To study fermentation by dysentery
1. Water 1000.0 cc. bacillus. The paper is turned red if the
2. NaCl 9.0 g. sugar is fermented.
Added nutrients: The authors used any Sterilization: Sterilization of Na^COs solu-
desired carbohydrate, alcohol, etc. tion not given. Sterilize the egg albumin
Reference: Hollande and Fumey (1917 at 125 to 130C.
p. 836). Use: Cultivation of anaerobic bacteria
found in milk. The author added 0.1 cc.
1247. Waksman's Basal Albumin Solution
to 8.0 cc. of Na2C03 solution, and then
Constituents: added the sterile egg albumin. The air
1. Water 1000.0 cc was removed.
2. Glycerol 30.0 g Reference: Barthel (1910 p. 6).
3. K2HPO4 1.0 g
4. KCl 0.5 g 1250. Bainbridge's Albumin Solution
5. MgSOi 0.5 g Constituents:
6. FeS04 0.01 g
7. Egg albumin (powdered).. 5.0 g
2. Egg albumin 1.0 to 5.0 g.
Preparation
3. NaCl 5.0 g.
(1) Dissolve 2, 3, 4, 5, 6 and one of the
4. Na2S04 1.0 to 2.5 g.
added nutrients in 1.
5. CaClz trace
(2) Dissolve powdered egg albumin in
6. Potassium phosphate.. trace
N/10 NaOH, and add to (1).
Preparation
(3) Tube in 10-12 cc. lots.
(1) Dissolve 2 (proteins used were egg
albumin, serum protein and alkali-
minutes.
albumin) 3, 4, 5 and 6 in 1.
,
Use: To study metabolism of actinomy-
(2) Add N/100 H2SO4 until the medium is
cetes.
very faintly alkaline to extremely
sensitive red litmus paper.
of one of the following
(3) After sterilization, distribute in 5.0
NaNOs (NH 4)2804
cc. quantities.
NaN02 (NH4)2C03
Sterilization: Sterilize by passing thru a
Berkefeld filter.
1248. Kent's Glycerol Albumin Solution Use: To study multiplication of bacteria,

in pure protein solutions. The author
Constituents:
reported very slight growth in pure pro-
1. Egg albumin 1000.0 cc.
tein media with any of the organisms
2. Glycerol (33.0%) 333.0 g.
used.
Preparation:
Reference: Bainbridge (1911 p. 343).
(1) Add 33.0% or less glycerol to egg
albumin.
1251. Castellani's Lactose Albimiin Solution
(Stitt)
Use: Cultivation of virus vaccinia. The
author reported that the diplo-bacillus Constituents:
vacciniae grew in a cotton-like mass. 1. Water 1000.0 cc.
The glycerol content tended to reduce the 2. Lactose (1.0%,) 10.0 g.
growth of staphylococci and other con- 3. Egg albumin (10.0%) 100.0 g.

taminating forms. The medium may be Preparation:
coagulated with heat. (Details not (1) Dissolve 2 and 3 in 1.
given.) Sterilization: Method not given.

Reference: Kent (1898 p. 1393). Use Cultivation of intestinal protozoa and
:
amoeba. Used also to replace the water

1249. Barthel's Albumin Solution of condensation in agar slants.
Constituents: Reference: Stitt (1923 p. 51).
1. Egg albumin.
Preparation:
1252. Hogue's Ovomucoid Medium (Hegner
Prepare a 2.0% Na-COs solution.
and Becker)
(1)
(2) Add 8.0 cc. of (1) to Gruber tubes Constituents:
containing sterile egg albumin. 1. Water 600.0 cc.
2. NaCl (0.7%) 4.2 g. of any desired carbohydrate, alcohol,

3. Egg white etc.,added 1.0% of a 5.0% solution of
Preparation Merck's highly purified litmus solu-
(1) Thoroughly shake up the whites of tion, tubed and sterilized for 10
six eggs with glass beads. minutes on each of 3 successive days
(2) Add (1) to 600.0 cc. of 0.7% saline in streaming steam.
solution. (d) Elser and Huntoon prepared the
(3) Cook for 20 to 30 minutes over a boil- medium as follows:
ing water bath, agitating constantly. (1) Mix one part sheep serum with two
(4) Strain thru cheese cloth. parts distilled water.
(5) Filter thru cotton using a suction (2) Add from 3.0 to 4.5 cc. of awatery
pump. solution of Merck's highly sen-
(6) Tube in 5.0 cc. lots. sitized litmus.
Sterilization.: Sterilize at 15 pounds pres- (3) Sterilize (method not given).
sure for 20 minutes. (4) Prepare a 10.0% solution of one of
Use: Cultivation of Trichomonas hominis the following in distilled water:
and Chilomastix mesnili and other in- glucose mannitol
testinal flagellates. galactose dulcitol
Reference: Hegner and Becker (1922 p. 18). levulose inulin
lactose dextrin
1253. Hiss' Basal Serum Solution
maltose sucrose
Constituents: (5) Sterilize (4) at 100C. for 10
1. Water 200.0 cc. minutes.
2. Serum 100.0 cc. (6) Mix (5) and (3) so that the added
Preparation: nutrient be present in 1.0% con-
(1) Mix two parts water with one part centration.
serum and heat to 100C. for some (7) Tube in sterile tubes.
minutes. (8) Incubate for 3 days to detect acci-
(2) Dissolve one of the added nutrients dental contamination.
in (1). (e) Distaso pepared the medium as
Sterilization: Sterilize at 68C. for one hour follows:
on six consecutive days. (1) Dilute one volume of beef or sheep
Use: To study fermentation by highly serum with three volumes of water.
parasitic forms. Hiss used pneumococci (2) Sterilize at 120 for 15 minutes.
and streptococci and reported that (3) Dissolve 1.0% of the desired car-
pneumococci induced coagulum with bohydrate, alcohol, etc., in (2).
glycogen in 24 hours. Streptococci no (4) Sterilize (method not given).
coagulum. Both organisms produced co- (f) Krumwiede, Pratt and Kahn gave the
agulum with other materials. following medium for the cultivation

Added nutrients and variants: of the paratyphoid enteritidis group:
(a) The author added 3.0 g. of one of the (1) To 400.0 cc. sterile distilled water
following: add 100.0 cc. of sterile horse serum,
glycogen galactose ;
5.0 cc. of Andrades indicator and
glucose maltose 25.0% solution of
2.5 cc. of a sterile
lactose sucrose glucose, or any other desired car-
(b) The author mixed one part ox serum bohydrate, alcohol etc., in distilled
with two parts distilled water and water (final concentration of glu-
added 0.1% normal NaOH. Pneu- cose 0.1%o).
mococci caused coagulation after (2) Tube to a depth of 4.0 cc. and steam
several days in this medium while sterilize intermittently on two suc-
streptococci did not. cessive days for 10 minutes. The
(c) Hiss and Russell diluted 100.0 cc. of medium may be prepared from non-
beef serum with 200.0 or 300.0 cc. of sterile materials and sterilized
distilled water, boiled, added 1.0% intermittently on 3 successive days.
(g) Roddy prepared the medium as (4) Add 1.0% of a 5.0% aqueous solu-
follows: tion of litmus to (3).
(1) Mix 300.0 cc. distilled water with (5) Add 1.0% of any desired carbo-
100.0 cc. of beef blood serum. hydrate, alcohol, etc., to (4).
(2) Add 4.0 g. of glucose or any other (6) Tube.

desired carbohydrate, alcohol, etc., (7) Arnold and allow the
Sterilize in the
to (1). steamer to remain uncovered during
(3) Add a saturated aqueous solution the process to avoid over heating.
of litmus to give a blue solution. Length of time of sterilization not
(4) Sterilize in the steam sterilizer for specified.
20 minutes on each of 3 successive (k) Harvey prepared the medium as
days. follows:
(h) Baeslack and Keane used a medium (1) Mix one part clear serum (ox) with
prepared as follows for the cultiva- 3 parts water.
tion of Spirochaeta pallida from tis- (2) Steam for 15 minutes (or heat for 20
sue. The was removed from
tissue minutes at 118C.)
the patient and pushed into the (3) Add sufficient litmus solution to
medium from one-half to two-thirds give a deep blue tint and 1.0% of
the length of the tube. Incubate any desired carbohydrate, alcohol,
at 37C. for 3 to 5 days and remove a etc. The litmus solution may be
portion with a sterile pipette on a omitted. Heat at 118C. in step
slide for dark field e.xamination. (2) when omitting the litmus.
(1) Dilute normal horse serum free (4) Sterilize at 100C. for 20 minutes on
from preservatives with sterile each of 3 successive days, or by
distilled water in the proportion filtration.
of3:l. (1) Gildemeister prepared a medium as
(2) Fill sterile tubes within an inch follows and used it as a substitute for
from the top with (1) and close the nutrose in Barsiekow's medium:
tube with a sterile rubber stopper. (1) Mix 5.0 to 10.0 cc. of beef serum
(3) Heat for an hour in a water bath at with 90 to 95.0 cc. of distilled water.
60 C. (2) Sterilize for one hour in a steamer.
(4) On the following day heat for one
(3) Dissolve 1.0 g. of glucose, lactose or
hour at 70C. and on the third day mannitol in 5.0 cc. of Kubel-Tie-
at 70C. until the medium takes on mann litmus solution.
the consistency of syrup.
(4) Add (3) to (2).
(i) Giltner prepared the medium as
(5) Distribute in tubes.
follows:
(6) on 3 successive days for 15
Sterilize
(1) Dilute beef or sheep serum with
minutes each day.
to 20
three times its volume of distilled
(m) Klimmer used the same basic solution
water.
as Gildemeister in variant (1), but
(2) Heat in the Arnold for 15 minutes.
used 2.0% mannitol instead of 1.0%,
(3) Distribute into desired quantities.
and used 2.0% sucrose and 2.0%
(4) Add 1.0% of any desired carbo-
maltose also as added nutrients,
hydrate, alcohol, etc. and sufficient
(n) Park, Williams and Krumwiede gave
litmus to give a deep purple color.
the following method of preparation:
(5) Sterilize by the fractional method
(1) Dilute serum with two or three
in the steamer.
times its volume of distilled water.
(j) Abbott gave the following method of
preparation: (2) Sterilize in the Arnold.
(1) Mix one part blood serum with 3 (3) Prepare 10.0 or 20.0% solutions of
parts distilled water. any desired carbohydrate, alcohol,
etc.
(3) Heat in an Arnold steamer until the (4) Heat (3) in small containers in the
mixture becomes opalescent. Arnold sterilizer for 30 minutes on 3
successive days. Sterilize the in- Use: To study fermentation.

ulin solution in the autoclave. Added nutrients Klimmer added one of the
:
(5) Add sufficient of (4) to (3) to give a following:

1.0% concentration of the added glucose 10.0 g.
nutrient under aseptic conditions. lactose 10.0 g.
Generally 5.0% glycerol is used maltose 20.0 g.
instead of 1.0%. sucrose 20.0 g.
(6) In routine work with glucose, mannitol 20.0 g.

lactose, sucrose, mannitol, and Reference: Klimmer (1923 p. 211).
dulcitol it is generally sufficient to
1255. Lorrain-Smith's Alkaline Serum
add the sugar to the medium and
Solution (Heinemann)
sterilize in the Arnold for 30 min-
utes on each of 3 successive days. Constituents:
References: Hiss (1901-05 pp. 325, 327), 1. Serum 1000.0 cc.
Hiss and Russel (1903 pp. 289, 295) Elser 2. NaOH (10.0% soln.)
and Huntoon (1909 p. 404), Distaso (1916 (1.0 to 1.5%) 10.0 to 15.0 cc.
p. 600), Roddy (1917 p. 42), Baeslack and Preparation
Keane (1920 p. 392), Giltner (1921 p. 383), (1) Add1.0 to 1.5% of a 10.0% solution
Krumwiede, Pratt and Kahn (1916-17 p. sodium hydrate to blood serum.
of
357), Abbott (1921 p. 143), Harvey (1921- (2) Tube.
22 p. 78), Gildemeister (1921-22 p. 76), Sterilization: Sterilize in the Arnold or
Klimmer (1923 p. 211), Stitt (1923 p. Koch's serum inspissator.
36), Park, Williams and Krumwiede Use: General culture medium.
(1924 p. 123). Reference: Heinemann (1905 p. 128).
1254. Leuch's Basal Serum Solution 1256. Klein's Basal Alkaline Serum
(Klimmer) Solution (Leuch)
1. Water 800.0 cc. 1. Water 400.0 cc.
2. Serum 180.0 cc. 2. Serum 90.0 cc.
3. NaCl (0.5%) 5.0 g. 3. NaOH (15.0%) 10.0 cc.
4. NaOH (15.0% soln.) 20.0 cc. 4. Litmus
Preparation 5. NaCl (0.5%) 2.5 g.
(1) Mix serum with 1 part of a

9 parts Preparation
15.0% NaOH solution, and incubate (1) Mix nine parts serum (horse) with
at37C. for 2 days. one part 15.0% NaOH.
(2) Make only slightly alkaline to litmus (2) Incubate at 37C. for two days.
by the addition of 25% HCl. (3) Make only slightly alkaline to litmus
(3) Dilute one part (2) with 4 parts water by the addition of 25.0% HCl.
and add 0.57o NaCl. (4) Dilute one part serum with four
(4) After sterilization, filter. parts water and add 0.5% NaCl.
(5) Dissolve one of the added nutrients in (5) Sterilize in the autoclave.
50.0 cc. of Kubel and Tiemann's (6) Allow to cool and filter.
litmus solution that has been boiled (7) Boil Kubel and Tiemann's litmus
for 15 minutes. solution for 15 minutes.
(6) Heat (5) in a boiling water bath for (8) Dissolve one of the added nutrients
6 to 8 minutes. in (7)by boiling 6 to 8 minutes.
(7) Filter (6) until clear. (9) Mix (8) and (6) in the ratio of 1:20,
(8) Add (7) to 1000.0 cc. of (4). while both are warm.
(9) Tube. (10) Tube.
Sterilization: Method of sterilization of Sterilization: Sterilize on each of three
(4) not specified. Sterilize the medium successive days for ten minutes each day.
following distribution into tubes on each Use: Detection of typhoid-paratyphoid
of 3 successive days for 10 minutes. and cholera organisms.
Added nutrients: The author used 1.0% (4) A pH = 7.0 gave the best results.
glucose, 1.0% lactose, 2.0% sucrose, 2.0% (5) Cover with a layer of paraffin oil.
mannitol, etc. (f) Stitt cultivated Balantidium coli
Variants: The author suggested the use of intestinal protozoa and amoeba in a
Czaplewski's alkaline serum (10.0 cc. medium prepared as follows:
normal NaOH per 100.0 cc. serum) instead (1)Mix one part inactivated human
of the alkaline serum given above . Czap- blood serum with 16 parts 0.5%
lewski's mixture may be sterilized in the salt solution.
autoclave. (2) The reaction is faintly alkaline to
Reference: Leuchs (1920 p. 1415), Klein litmus.
(1920, p. 297). (3) Place 8.0 cc. in tubes having a
diameter of 10 mm. and a length of
1256a. Martin, Pettit and Vaudremer's
150 mm. giving the medium a depth
Serum Solution.
of about 100 mm.
Constituents: not specified.
(4) Sterilization
1. Physiological salt solution. 1000.0 cc. Inoculate with 0.1 cc. of undiluted
(5)
2. Serum, bovine 100.0 cc. feces containing mucus, by means
Preparation of the tube capillary pipette into
(1) Dilute bovine serum 1 to 10 with the bottom of the tube.
physiological salt solution. References Martin, Pettit and Vaudremer
:
Sterilization: Not given in the abstract. (1917 p. 197), Griffith (1919-20 p. 60),
Use: Cultivation of Spirochaeta ictero- Harvey (1921-22 p. 79), Hogue (1922 p.
haemorrhagiae. 619), Stitt (1923 p. 52).
Variants
(a) Rabbit serum diluted 1:16 proved 1257. Davis' Serum Medium.
to be a better medium. Constituents:
(b) Griffith cultivated Spirochaeta 1. Serum, rabbit.
icterohaemorrhagiae and other or- Preparation
ganisms on a medium prepared by (1) Tube sterile rabbit's blood serum into
diluting one part beef serum with two sterile test tubes.
parts physiological salt solution and Sterilization: Method not given.
heating at 70C. until the mixture Use: Used in uncoagulated form by Davis
became slightly viscous. He covered to cultivate the Ducrey bacillus (chan-
the medium with a thin layer of croid bacillus). Other investigators cul-
paraffin oil after inoculation. tivated spirochetes, pneumococci,
(c) Harvey mixed 5 parts sterile 0.85% streptococci, etc., in similar media.
NaCl solution with one part rabbit Variants
serum heated at 56C. for 30 minutes. (a) Longcope cultivated pneumococci
(d) Harvey mixed 1 part ox serum heated and streptococci in a medium pre-
to 56C. for 30 minutes with 9 parts pared as follows:
sterile 0.85% NaCl solution, or a (1) Collect 20.0 cc. of blood from the
sterile solution obtained by dissolv- arm vein and allow to clot in a cool
ing 9.2 g. NaCl, 0.05 g. NajCOa, place.
0.1 g. KCl, 0.1 g. CaCh and 10.0 g. (2) Draw off the serum after from 24 to
sodium citrate in a liter of water. 48 hours.
This solution is Locke's solution. (3) Use from 2 to 5.0 cc. serum as
(e) Hogue cultivated Spirochaeta eury- culture medium.
grata on a medium prepared as (b) Schereschewsky heated horse serum
follows: at 60C. until it was brought to a
(1) Dilute serum (best results obtained gelatinous consistency, then incu-
with pig serum) 1:4. bated at 37C. until partial autolysis
(2) Tube 0.85%, sterile salt solution in was effected. The medium was used
15.0 cc. lots in sterile tubes. for the cultivation of the syphillus
(3) AddO.3 cc. of (1) to each tube of (2). spirochaete.
369
Wang tubed serum (ox) in 2.5 cc. 1260. Sinton's Glucose Serum Solution
(c)
quantities and heated at 56C. for Constituents:
3 or more successive days until the 1. Serum, horse 1000.0 cc.
serum became syrupy. The medium 2. Glucose (50.0% soln.) 15.0 cc.
was used in the diagnosis of diph- Preparation
theria. (1) Add 1.5 cc. of a 50.0%, sterile glucose
(d) Harvey cultivated spirochaetes in solution to each 100.0 cc. of asepti-
sterile rabbit serum heated 30 min- cally collected horse serum.
utes at 58 to 60C. covered with a Sterilization : Not specified.
layer of sterile paraffin oil. Use: Cultivation of Spirochaeta carteri
References: Davis (1903 p. 405), Longcope causing Indian relapsing fever.
(1905 p. 627), Schereschewsky (1909 p. Reference: Sinton (1923-24 p. 826).
35), Wang (1919 p. 233), Harvey (1921-
1261. Toyoda's Glucose Serum Solution
22 p. 80).
Constituents:
1258. Marmier's Serum Solution. 1000.0 cc.
1. Serum, dog
Constituents 2. Glucose 10-0 g.
1. Water 100.0 cc. 3. Sodium citrate solution

2. Serum (beef) 100.0 cc. 4. Physiological salt solution
(1) Mix equal parts of beef
serum and (1) To each 10.0 cc. of healthy young dog
water. serum add 0.2 cc. of a 50.0%, sterile
(2) Make slightly alkaline by the addition glucose solution and 0.3 cc. of a
of soda. 2.0% sterile sodium citrate in 0.85%
Sterilization: Sterilize at 115 in the co- NaCl solution.
agulator (time not specified). (2) Pipette the serum in small narrow
Use: Anthrax toxin production by anthrax test tubes 5 cm. high and inactivate
bacilli. the serum at 45C. for one hour.
Reference: Marmier (1895 p. 569). Sterilization: Not specified.
Use: Cultivation of Babesia cannis
1259. Boeck's Glucose Serxim Solution. Reference: Toyoda (1913 p. 76).
Constituents: Serum Solution
1262. Marbais' Lactose
2. NaHC03 0.2 g. Constituents:

3. KCl 0.42 g. 1.
Serum 100.0 cc.

4. CaCh 0.24 g. 2.
NaCl 9.0 g. 3. Lactose 8.0 g.

5.
6. Glucose 2.5 g. 4. Litmus
7. Serum (human, sheep or Preparation
250.0 cc. (1) Add one volume of
clear serum (man
horse)
Preparation or animal, preferably horse or sheep)
(1) Dissolve 2, 3, 4, 5 and 6 in 1. to three volumes of neutral distilled
(2) Mix aseptically one part sterile serum water.

(preferably human) with 4 parts (2) Add sufficient lactose to make 2.0 g.
sterile (1). The usual reaction of the lactose per 100.0 cc. of mixture.
mixture is 0.2% alkaline to phenol- (3) Mix well and filter thru paper.
phthalein. (4) Tube in 7 or 8.0 cc. lots.
(3) Tube in 5.0 cc. lots in sterile tubes. (5) Add 0.1 cc. of a sterile litmus solution
(4) Incubate at 37 over night to test prepared according to Besson (refer-
sterility. ence or method not given) to each
Sterilization: Method of sterilization of sterile tube.
(1) not given. Incubate for 24 hours to test sterility.

(6)
Use: Cultivation of Chilomastix mesnili, Sterilization: Sterilize (4) at 120C. for 15
a parasite in the small intestine of man. minutes.
Use: Differentiation of colon-typhoid ba-
Reference: Boeck (1921 p. 159).
cilli. Author reported that typhoid (5) Sterilize (4) at 15 pounds pressure
cause coagulation after 24 hours, color for 15 minutes.
was blue-violet. Paratyphoid A clouded (6) Mix 1 part (5) with 10 parts (3).
the medium, and the color was lilac. (7) Sterilize on each of 3 successive
Paratyphoid B gave a red color after 5 days for 20 minutes at 100C.
or 6 hours incubation, then changed to Reference: Hiss (1901-05 pp. 324, 330),
original color. B. coli coagulated and Harvey (1921-22 p. 112).
decolorized the medium.
1264. Legroux's Formol Serum Solution
Reference: Marbais (1918 p. 602).
Constituents:
1263. Hiss' Inulin Serum Solution 1. Distilled water 1200.0 cc.
2. Serum (beef or horse) 600.0 cc.
Constituents:
3. Formol 1.0 cc.
Preparation:
2. Beef serum 100.0 cc.
(1) Mix 600.0 cc. of beef or horse serum
3. Inulin (pure) (1.0%) 3.0 g.
with 1.0 cc. of commercial formol.
4. Litmus (5.0% soln.) 3.0 cc.
(2) Dilute (1) with 1200.0 cc. distilled
Preparation
water.
(1) Mi.x two parts distilled water with one
(3) Mix well.
part fresh clear beef serum.
(4) The reaction is acid to methyl red.
(2) Add 1.0% pure inulin, and 3.0 cc. of
5.0% solution Merck's highly purified Sterilization: Sterilize in the autoclave at
litmus.
112 to 115 for varying lengths of time
depending on the size of the container.
minutes (if inulin contains heat resisting
Use: Enrichment of meningococci. The
spores, autoclave at 10 to 15 pounds
author reported that the formol kept the
pressure for 15 minutes.)
medium from coagulating during steriliz-
Use: Differentiation of pneumococci and
ation. It also aided in giving a clear
streptococci. Author reported that
medium.
pneumococci. cultures coagulated the
Reference: Legroux (1920 p. 466).
serum, streptococci did not.
Variants : 1265. Marzinowsky's Citrated Blood
(a) Hiss omitted the litmus. Solution
(b) Hiss prepared a similar medium as Constituents:
follows: 1. Water 100.0 cc.
(1) Add 4.0 g. powdered starch to 400.0 2. Sodium citrate (10.0%) 10.0 g.
cc. water, and boil for 30 minutes. 3. Blood, horse
Allow to stand over night. Preparation:
(2) Obtain, next morning, the clear (1) Prepare a 10.0% watery solution of
fluid by pipetting off the water sodium citrate.
from the starch particles. (2) Distribute in 1.5 to 2.0 cc. lots in test
(3) Add one part serum to two parts of tubes.
the supernatant fluid from (2). (3) Add10.0 cc. of horse blood to each
(4) Sterilize at 68C. for 1 hour on 6 tube of sterile (2). The blood is
consecutive days. taken directly from a vein.
(c) Hiss heated the beef serum and water Sterilization: Sterilize (2); method not
at 100C. for several minutes and given.
then added the inulin and litmus. Use: Cultivation of Piroplasma equi and
(d) Harvey prepared the medium as other piroplasma.
follows: Reference: Marzinowsky (1908-09 p. 419).
(1) Mix 1 part ox serum with 3 parts
water. 1266. Griffith's Citrated Blood Solution
(2) Steam 15 minutes. Constituents
(3) Add sufficient litmus solution to 1. Blood, citrated (horse, beef,
give a blue color. rabbit) 100.0 cc.
(4) Prepare a 10.0% solution of inulin. 2. Physiological salt solution... 200.0 cc.
Sterilization: Sterilize (1) by heating at

Preparation
(1) Mix part citrated blood (horse, beef
1 55C.
or rabbit) with 2 parts physiological Use To show influence of sugar on develop-
:
saline. ment of anthrax and other organisms.

(2) Heat until semi-gelatinous. Author reported that 10.0% sugar in-
Sterilization: Not specified. creased the bacterial count.

Use: Cultivation of Spirochaeta ictero- Variants: The author omitted the sucrose
haernorrhagiae . The author inoculated or used 40.0% instead of 10.0%.
the medium with infected heart blood or Reference: Buchner (1890 p. 67).
tissue fragment, and covered the medium

with a thin layer of paraffin oil. 1270. Davis' Blood Medium
Reference: Griffith (1919-20 p. 61).
Constituents
1267, Besson's Citrated Blood
1. Blood, human.
Constituents: Preparation:
1. Water 1000.0 cc.
(1) Prick the extensor surface of the
2. NaCl 8.0 g. thumb just back of the base of the
3. Sodium citrate 15.0 g. nail, the skin having been previously
4. Blood cleansed with soap and water and
Preparation alcohol.
(1) Dissolve 8.0 g. NaCl and 15.0 g. of
(2) Applying a tourniquet at the base of
sodium citrate in 1000.0 cc. of water. the thumb just tight enough to
(2) Mix 20.0 cc. of sterile (1) with 80.0 cc. obstruct the venous flow, a consider-
of blood obtained under aseptic able quantity of blood may be
conditions. obtained.
The blood is drawn up by capillary
(3)
given attraction into a small glass tube,
Reference: Besson (1920 p. 34). one end of which has been drawn out
Blood (Stitt) to a very fine caliber over the Bunsen
1268. Rogers' Citrated
flame, and the whole tube sterilized
Constituents: by the same means.
1. Blood. When about 0.2 cc. of blood has
(4)
2. Sodium citrate (10.0%).
flowed into the tube the capillary end
3. Citric acid.
may be sealed in the flame, and the
Preparation other end plugged with cotton, giving
(1) Place 1.0 or 2.0 cc. of a sterile 10.0%
a miniature culture tube.
sodium citrate slightly acidified with
(5) The medium is nearly always sterile
citric acid into the barrel of a syringe
when obtained in the manner in-
and aspirate splenic blood directly dicated.
into it.
Sterilization: Given in the preparation.
Use: Cultivation of Ducrey bacillus (chan-
Use: Cultivation of Leishmania.
croid bacillus), pneumococci and strep-
Reference: Stitt (1923 p. 52).
tococci.
1269. Buchner's Blood Solution Variants: Stitt reported that pneumococci
and streptococci maintained their viru-
Constituents:
1000.0 cc. lence in a medium prepared as follows:
1. Water
100.0 cc. (1) Obtain rabbit or human blood under
2. Blood
aseptic conditions and preserve
3. Sucrose (10.0%) 100.0 g.
(whole blood) in small test tubes.
Preparation
cane Heat for 30 minutes at 56C. to
(1) Prepare a 10.0% solution of
(2)
(Reaction may be slightly inactivate the blood.

sugar.
References: Davis (1903 p. 405), Stitt (1923
alkaline.)
(2) Add 0.1 volume of blood to sterile (1). p. 44).
1271. Klimenko's Laked Blood Solution Preparation

Constituents: (1) Draw the blood from the patients
Distilled water vein at the bend of the elbow.
1. 900.0 cc.
2. Blood 10.0 cc. (2) Add 0.1 cc. of 50.0% dextrose solution
Preparation: for every 10.0 cc. of blood drawn.
Mix 10 parts blood and 90 (3) Defibrinate the by gently
blood
(1) parts
sterile distilled water. whipping or stirring with a glass rod.
(2) Tube. Avoid air bubbles.
Sterilization: Not specified. (4) After defibrination, plug the tube
Use: Cultivation of whooping cough bacil- with a fresh sterile plug.
lus. Author reported that the organisms (5) The column of blood must be from
grew first on the surface of the medium, one to two inches thick. This gives a
then on the bottom; after several days the column of serum Ho 1 inch thick.
whole tube became turbid and was finally Sterilization: Not specified.
colored dark brown. Use: Cultivation of malarial plasmodia.

Reference: Klimenko (1909 p. 312). Variants
1272. Wurtz and Sappington's Diluted Blood follows:
Solution (1) Add 15.0 cc. aspirated malarial
Constituents: blood to a centrifuge tube con-
1. Water 18.0 cc. taining 0.1 cc. 50.0% glucose.
2. Blood 12.0 cc. (2) Defibrinate the blood-glucose mix-
Preparation ture with a glass rod.
(1) Rub the skin at the bend of the elbow (3) Centrifuge.
with a pledget of cotton soaked in (4) Observe the development of para-
alcohol. sites at 41 C. under anaerobic
(2) Take the blood with a large sized conditions.
Wassermann needle kept dry steril- (b) Stitt gave the following method of
ized in a test tube. preparation:
(3) Distend the vein by a knotted piece (1) Place 0.1 cc. of a 50.0% glucose
of rubber tubing above, and the solution in a centrifuge tube.
patients clinched fist below. (2) Add from 10.0 to 20.0 cc. of malarial
(4) Plunge the needle into the vein and patients blood to each tube of (1).
allow about 10.0 cc. of blood to run (3) Defibrinate the blood by extending
into a dry sterile test tube. This a piece of glass rod or piece of
washes out the needle and lessens the tubing into the bottom of the cen-
chance of contamination. trifuge tube.
(5) Run about 12.0 cc. of the blood then (4) Centrifuge.
into a tube containing 18.0 cc. of (5) There should be at least one inch of
sterile water. serum above the cell sediment.
Sterilization: Sterilization given under (c) Park, Williams and Krumwiede used
preparation. the following method
Use: Blood culture. Author reported that (1) Defibrinate carefully 10.0 cc. of
this method was especially good in case blood taken from a malarial
of pneumococcic and streptococcic in- patient.
fections. (2) Place in small test tubes in 1.0 cc.
Reference: Wurtz and Sappington (1918 amounts.
p. 373). (3) One per cent of a 50 per cent
solution of dextrose added to each
is
1273. Bass, Foster and Johns' Glucose small test tube before adding the
Blood Solution blood.
Constituents: (4) The red corpuscles settle so that a
1. Blood (malarial) lOO.O cc. 0.5cm. layer of serum is left above
2. Glucose 0.5 g. them.
References: Bass, Foster and Johns (1912 (3) Remove blood under aseptic condi-
p. 570), Harvey (1921-22 p. 72), Stitt tions from the jugular vein of a
(1923 p. 53), Park, Williams and Krum- horse.
wiede (1924 p. 133). (4) Defibrinate (3) and store in the ice
box if not to be used at once.
1274. Besson's Defibrinated Blood (5) Add 9.0 cc. of (4) to each tube of
sterile (2) under strictly aseptic
Constituents:
conditions.
1. Blood.
(6) Mix well and place in an incubator at
Preparation:
25C. for 24 hours. At the end of
(1) Collect blood under aseptic condi-
this time the mixture is clear.
tions in a sterile flask containing glass carefully.
beads.
(Method not given.)
(2) Shake thoroly for 10 minutes. and
Use: Cultivation of Babesia caballi
(3) Remove the liquid from the fibrin by
Nuttallia equi. To inoculate carefully
asparating.
place the end of a sterile fine pointed
(4) Distribute the fluid in sterile test
pipette containing infected blood into
tubes.
the red blood cells in the bottom of the
tube, and allow the infected blood to run
Variants: Harvey did not separate the fluid
into the tube.
from the fibrin and incubated for 48 hours
Reference: Carpano (1914 p. 43).
to test sterility.
References: Besson (1920 p. 34), Harvey 1277. Waksman's Basal Fibrin Solution
(1921-22 p. 73).
Constituents:
1. Water 1000.0 cc.
1275. Row's Defibrinated Blood Solution 30.0
2. Glycerol g.
(Harvey)
3. K2HPO4 10 g.
Constituents: 4. KCl 0.5 g.
2. Blood, defibrinated 100.0 cc. 6. FeS04 0.01 g.
3. NaCl (1.2% solution) 2200.0 cc. 7. Fibrin g. 5.0
(1) Defibrinated human or rabbit blood. (1) Dissolve 2, 3, 4, 5, 6 and one of the
(2) Add 10 times its volume distilled added nutrients in 1.
water. (2) Tube in 10-12 cc. lots.
(3) Add 1 volume of the laked blood thus (3) Add small pieces of fibrin to each
obtained to 2 volumes 1.2% sterile tube.
sodium chloride solution. Sterilization: Sterilize at 15 pounds for 15
Sterilization: Not specified. minutes.
Use: Cultivation of Leishmania. Use: To study metabolism of actino-
References: Harvey (1921-22 p. 72), Stitt mycetes.
(1923 p. 52). Added nutrients: The author added 2.0 g.
1276. Carpano's Defibrinated Blood NaNOa (NH5)2S04
Solution (NHOiCOa
NaN02
Constituents Reference: Waksman (1920 p. 3).
1278. Stoklasa's Glucose Fibrin Solution
2. NaCl (c.p.) 7.0 g.
3. Sodium citrate (c.p.) 7.0 g. Constituents:
4.Defibrinated horse blood 1. Water 1000.0 cc.
Preparation: 2. Sodium phosphate 0.5 g.
(1) Dissolve 2 and 3 in 1. 3. K2SO4 0.2 g.
(2) Distribute in 1.0 cc. lots into test 4. MgCl2 0.05 g.

tubes. 5. Glucose 2.5 g.
6. CaCOs 7.5 g. 1280. Fischer's Blood Meal Solution

7. Fibrin 125.0 g.
Constituents:
Preparation:
(1) Dissolve 0.5 g. sodium phosphate,
2. Blood meal 10.0 g.
0.2 g. K2SO4, and 0.05 g. MgCh in
3. KH2PO4 1.0 g.
500.0 cc. water.
Dissolve 2.5 glucose in 500.0 cc.
4. CaCU 0.1 g.
(2) g.
5. MgS04 0.3 g.
water.
6. NaCl 0.1 g.
(3) Mix 200.0 cc. (1) and 120.0 cc. of (2).
7. Fe.CU 0.01 g.
(4) Dissolve 7.5 g. CaCOs in (3).
Preparation:
(1) Dissolve 3, 4, 5, 6 and 7 in a liter of
(6) Add 25.0 g. of fibrin from blood to
water.
each flask.
(3) Add 2.0 g. of blood meal to each flask
Use: To study utilization of nitrogen by
of (2).
Bacillus megatherium, {Bacillus Ellen-
bachii) or "Alinit." The
author re-
Use: To study decomposition of organic
ported that more nitrogen was found to
nitrogen by soil forms. Each flask was
be in solution after incubation.
inoculated with 25.0 cc. of a soil suspen-
Variants: The author also gave the follow-
sion prepared from 100.0 g. soil in 500.0
ing method of preparation:
cc. of water. The author reported that
(1) Dissolve 0.5 g. sodium phosphate,
the flask containing a trace of acid gave
0.2 g. potassium sulphate and 0.05
the most ammonia while the one with a
g. magnesium chloride in 500.0 cc.
trace of alkali showed least ammonia
water.
production.
(2) Dissolve 2.5 g. glucose in 500.0 cc.
water.
variants:
(3) Mix 400.0 cc. (1) with 240.0 cc. of (2).
(a) Dissolve 2.0 g. of blood meal in 200.0
(4) Dissolve 6 in (3).
Dilute to 4 liters.
cc. of water. Add no salts.
(5)
(b) Dissolve 2.0 g. of blood meal in 200.0
(6) Place 1 liter in a large flask and add
cc. of water, no salts, and add 5.0 cc.
40.0 g. fibrin to each flask.
of a 1 to 10,000 diluted 0.5 normal
H0SO4.
1279. Beijerinck's Sucrose Fibrin Solution (c) Dissolve 2.0 g. of blood meal in 200.0
(Percival) cc. of water, no salts, and add 5.0
cc. of a 1 to 10,000 diluted 0.5 normal
Constituents:
1. Water 1000.0 cc.
NaOH.
Reference: Fischer (1909 p. 65).
3. Fibrin 50.0 g.
4. CaCOs (precipitated) 3.0 g.
1281. Remy and Rosing's Blood Albumin
Solution
6. MgS04 0.5 g. Constituents
7. NaCl 0.5 g. 1. Water 1000.0 cc.
Preparation 2. Blood albumin 50.0 g.
(1) Boil 2, 3, 4, 5, 6 and 7 in 1 briskly for Preparation
2 or 3 minutes. The fibrin is to be (1) Grind dry blood albumin fine.
finely ground. (2) Add 1.0 g. sterile (1) to 20.0 cc. of
Sterilization: Not specified. sterile water.
Use: Isolation of Granulobacillus sac- (3) Distribute into small Erlenmeyer
char obutyricus. The medium was in- flasks.
oculated by the addition of 10.0 to 15.0 Sterilization: Sterilize (1) twice at 100C.
g. of fresh garden soil while still boiling. for 30 minutes. Method of sterilization
Reference: Percival (1920 p. 288). of the water not given. Sterilize the
finished medium in the steamer, using the causes the bile acid to precipitate out
fractional method. in silky crystals so-called crystalline
Use: To study decomposition of organic N bile.
materials by soil forms. Author reported (5) Dry and pulverize the crystals.
little change in total nitrogen content (6) Add one or two knife points of (5)
after 12 days when inoculated with soil. to sterile test tubes.

Reference: Remy and Rosing (1911 p. 39). Sterilization: Medium sterilized during the
preparation.
1282. Klimmer's Hemoglobin Solution Use: Enrichment of typhoid bacilli from
Constituents: patients blood. Diagnosis of typhoid
1. Blood 1000.0 cc. fever. The author added 2.0 to 4.0 cc. of
2. Physiological salt solution . . 1000.0 cc. the patients blood to each tube of (6).
Preparation: Variants: The following authors have pre-
(1) Collect pigeon blood under aseptic pared media in the manner indicated.
conditions and add to an equal (a) Kayser
volume of physiological salt (0.85%) (1) Secure normal beef bile immedi-
solution. ately after the death of an animal in
(2) Mix well and centrifuge, or allow to large sterile flasks.
stand in the cold for 24 hours. (2) Distribute in amounts of at least
(3) Thoroly wash the red residue twice 5.0 cc. in test tubes.
more with sterile NaCl solution. (3) Sterilize at 110.
(4) Dissolve the hemoglobin with a trace (4) Put 2.5 cc. of blood of patient into
of ether. this at bedside.
(5) Evaporate the ether at 30C. (b) Bezangon.
(6) Separate the hemoglobin from the (1) Collect bile at a slaughter house.
stromata by passing thru a bacterial (2) Sterilize at 100C.
filter. (3) Allow to settle.
Sterilization: Method of sterilization of (4) Decant the clear liquid.
salt solution not specified. (5) Tube in 3.0 cc. quantities in sterile
Use: This hemoglobin solution is added to tubes.
other media which is used for the cultiva- (6) Sterilize at 105C. for 20 minutes.
tion of pneumococci, vibrio, etc. One (c) Klimmer.
cubic centimeter of this solution is added (1) Puncture the gall bladder of a beef
to 8.0 cc. of media, or this solution is with a knife, and collect the bile.
streaked on solid media. (2) Boil (1) in a steamer for 15 minutes.
Variants: The author also used commercial (3) Distribute in 5.0 cc. quantities in
hemoglobin. This solution was prepared test tubes.
as follows: (4) Steam on each of 3 successive days
(1) Mix 10.0 g. of commercial hemoglobin for 20 to 30 minutes.
with 90.0 cc. of distilled water. References: Meyerstein (1906 p. 1864),
(2) Add about 10.0 cc. of 10.0% KOH. Kayser (1906 pp. 823-826), Harvey
(3) Sterilize in the steamer. (1921-22 p. 89), Bezangon (1920 p. 122),
Reference: Klimmer (1923 p. 196). Klimmer (1923 p. 202), Stitt (1923 p. 47).
1283. Meyerstein's Bile Medium

1284. Ottolenghi's Nitrate Bile Solution
Constituents:
1. Bile, beef. Constituents:
1. Bile 1000.0 cc.
Preparation:
(1) Evaporate beef with animal char-
bile 2. NasCOa (10.0% soln.)
(3.0%) 30.0 cc.

coal to dryness on a water bath.
(2) Take up the residue in alcohol. 3. KNOj (1.0%) 1.0 g.
(3) Filter. Preparation:

(4) Add quite a lot of ether to the some- (1) Filter fresh ox bile thru a paper.
what concentrated filtrate. This (2) Add 3.0% of a 10.0% watery solution
of crystalline Na2C03 and 0.1% Use: Enrichment of typhoid bacilli from

KNOstoCl). patients blood. Add 1.0 cc. of patients
(3) Distribute in 5.0 cc. lots. blood to each tube of (6).
Sterilization: Sterilize in the autoclave for Reference: Meyerstein (1906 p. 1864).
15 to 20 minutes at 0.5 atmosphere.
1287. Thoinot and Masselin's Aqueous
Use: Enrichment medium for cholera
Humor Mediimi
vibrio.
Constituents:
Reference: Ottolenghi (1911 p. 370).
1. Aqueous humor.
1285. Jackson's Lactose Bile Solution Preparation
Constituents: (1) Obtain the aqueous humor from the
ox
Bile,
eye of an animal by searing the
1. 1000.0 cc.
2. Lactose (1.0%)
cornea with a hot piece of iron and
10.0 g.
Preparation: then puncturing the cornea with a
Draw bile, and sterilize undiluted. pipette.
(1)
It may be kept in stock in this (2) Distribute as desired.
manner. Sterilization: Not specified.
When ready for use, decant or filter. Use: Cultivation of anthrax bacilli.
(2)
Add 1.0% lactose that has previously Reference: Thoinot and Alasselin (1902
(3)
been dissolved in small (amount not p. 28).
given) amount of water. 1288. Ohira and Noguchi's Glucose Ascitic

(4) Mi.x (2) and (3) and tube. Fluid Solution
Sterilization: Sterilize in autoclave for 30 Constituents:
minutes at 15 pounds pressure. 1. Ringer's Solution 1000.0 cc.
Use: Enrichment medium used in water 2. Ascitic fluid 1000.0 cc.
analysis. Preparation
Variants: Stitt reported that a 15 to 20.0%
(1) Mix equal portions of ascitic fluid and
solution of a good quality of inspissated Ringer's solution. (See medium 180).
ox bile may be substituted for fresh ox Do
not adjust the reaction.
(2)
bile.
References: Jackson (1907 p. 31), Stitt Use: Cultivation of Trichomonas of the
(1923 p. 47). human mouth. {Tetratrichoinonas hom-
inis).
1286. Meyerstein's Glycerol Bile Solution
Reference: Ohira and Noguchi (1917 p. 342).
Constituents:
1. Bile, ox. 1289. Sinton's Glucose Body Fluid Solution
2. Glycerol. Constituents:
Preparation 1. Ascitic fluid 100.0 cc.
(1) Evaporate ox gall with animal char- 2. Glucose (50.0% soln.). 1.5 to 2.0 cc.
coal on a water bath. Preparation:
(2) Take up the residue in alcohol. (1) Draw off ascitic fluid with strict
(3) Filter. aseptic precautions into sterile flasks
(4) Add quite a lot of ether to the some- until each contains 100.0 cc.
what concentrated This
filtrate. (2) Add 1.5 to 2.0 cc. of 50.0% sterile
causes the bile acid to precipitate out glucose solution to each flask.
in silky crystals so-called crystalline (3) Heat for 30 minutes at 56C. to kill as
bile. much complement as possible.
(5) Dry and pulverize the crystals. Sterilization: Method of sterilization of
(6) Dissolve about 30.0 or 40.0% of (5) in 50.0% glucose solution not given.
glycerol by heating. Use: Cultivation of Plasmodium falci-
(7) Place several drops of (6) in test parum.
tubes. Variants: The author used hydrocele fluid
Sterilization: Medium sterilized during the instead of ascitic fluid.
preparation. Reference: Sinton (1922-23 p. 205).
1290. Tanner's Milk Powder Solution 1293. Reinsch's Milk Medium
1. Distilled water 1000.0 cc. 1. Milk.
2. Milk Powder, Merrell- Preparation

Soule 100.0 g. (1) Place 500.0 cc. of fresh cow milk in a
Preparation: separatory funnel and add 1.0 g.

(1) Add 2 to 1. NaOH (2.5 cc. solution of 400.0 g.
(2) Beat with an egg beater. NaOH in a liter).
(3) Filter. (2) Shake well and allow to stand at

(4) Distribute in 75.0 cc. quantities. about 18C. for 48 hours.
Sterilization: Sterilize in the steamer. (3) Remove the nearly transparent milk
Use: General culture Author
medium. from the bottom of the funnel and
cultivated green fluorescent bacteria from add it to a second separatory funnel.
water. (4) Add 250.0 cc. ether and shake well.
Reference: Tanner (1918 p. 83), (1919 p. (5) Allow to stand for 48 hours.
71). (6) Place the opalescent liquid now in a
large sterile flask, plug with cotton
1291. Brown and Howe's Milk Solution and heat to 50C.
Constituents: (7) Place under the receiver of a water
1. Distilled water 1000.0 cc. suction pump for 3 or 4 hours until all
2. Milk (skim) 500.0 cc. the ether is evaporated.

3. Sodium citrate 4.0 g. SterilizationGiven under preparation.
:
Preparation: Use: General culture medium. Medium is
(1) Add 500.0 cc. of milk to a liter of fat free.

water. Variants
(2) Add 4.0 g. of sodium citrate and allow (a) Migula gave the following method of
to stand for one hour. preparation:

(3) Filter. (1) Obtain milk under aseptic condi-
(4) Adjust to pH 6.8. tions in a sterile flask.
(5) Tube. (2) Add 2.0 cc. of chloroform to 100.0

(A pre-
Sterilization: Sterilize in Arnold. cc. of (1).
cipitate appears when heated but disap- (3) Seal the flask with a rubber stopper
pears when cool). and shake thoroly.
Use: General culture medium. (4) Allow to stand for several days.
Variants: The author used 4.0 g. sodium (5) Distribute in sterile tubes.
oxalate instead of 4.0 g. sodium citrate. (6) Incubate for 48 hours.
Citrated milk was found to be more (b) Smith prepared the medium as
satisfactory than oxalated milk. follows:
Reference: Brown and Howe (1922 p. 512). (1) Obtain fresh milk and free from fat
by running the milk thru a separa-
1292. Boekhout's Sucrose Milk Solution tor or by allowing the cream to rise
Constituents: spontaneously.
1. Milk 1000.0 cc. (2) Test the reaction. It should not
2. Sucrose (8.0%) 80.0 g. be more than +2.
Preparation: (3) Tube.
(1) Dissolve 8.0% sucrose in milk. (4) Sterilize at 100C. by the fractional
Sterilization: Not specified. method.
Use: Cultivation of dextran formers. A large number of investigators have
{Streptococcus hornensis). Organism prepared milk in a manner very
may be isolated from flowers, honey and similar to that of Smith. Some
bees. sterilized the milk in the autoclave.
Reference: Boekhout (1900 p. 162). The differences in preparation are not
outstanding enough to warrant an (3) Add sufficient Kahlbaum's azolitmus

individual discussion of each method, to give a color.
(c) Tanner prepared "Dialyzed Milk" as (4) Filter.
follows: (5) Distribute in 75.0 cc. quantities.
(1) Add 0.1 cc. of hydrogen peroxide to Sterilization: Method not given.
500.0 CO. of milk. Use: General culture medium. Green flu-
Allow to stand for 2 hours.
(2) orescent bacteria of water, B. coli, B.
Add hydrogen peroxide again
(3) welchii and Bad. acidi lactici were used.
(amount not specified). Variants: Hamilton prepared a similar
(4) Allow to stand 4 or 5 hours. medium by mixing 1 part purified litmus
(5) Dialyze (method not given). or azolitmin with 52.7 parts milk powder
(6) Dilute the milk one fifth with water. or 1 part purified azolitmin with 49.6
References: Reinsch (1892 p. 31), Migula parts milk flour. One part of this mix-
(1901 p. 19), Smith (1902 p. 104), Thoinot ture was dissolved in 9.5 parts water, and
and Masselin (1902 p. 27), Committee the medium was sterilized in the
A.P.H.A. (1905 p. 109), Smith (1905 p. 46), autoclave.
(1905-06 p. 202), Abel (1912 p. 29), Lohnis References: Tanner (1918 p. 83), Hamilton
(1913 p. 20), Tanner (1914 p. 72), Ball (1921 pp. 43, 44).
(1919 p. 83), Besson (1920 p. 32), Percival
1296. Bacto Litmus Milk (Dehydrated)
(1920 p. 57), Abbott (1921 p. 138), Dopter
and Sacquepee (1921 p. 122), Harvey Constituents:
(1921-22 p. 94), Cunningham (1924 p. 16), 1. Distilled water 1000.0 cc.
Park, Williams and Krumwiede (1924 2. Litmus milk, Bacto 105.0 g.
p. 120). Preparation:
(1) Dissolve 105.0 g. of Bacto litmus milk
1294. Hiss' Basal Litmus Milk Solution
(dehydrated) in 1000.0 cc. distilled
Constituents: water.
1. Milk 1000.0 cc. Sterilization Sterilize in the usual manner.
:
2. Litmus solution Use: Cultivation of organisms causing the

Preparation: souring of milk and dairy products.
(1) Tinge milk with litmus solution. Reference: Digestive Ferments Co. (1925
(2) Dissolve 1.0% of one of the added p. 13).
nutrients in (1).
1297. Calandra's Picric Acid Litmus Milk
Solution
Use: To study fermentation by dysentery
group. Constituents:
Added nutrients: The author added 1.0% 1. Litmus solution.
of one of the following: 2. Picric acid (saturated solution).
glucose dextrin Preparation
maltose mannitol (1) Mix 3 parts of a sterile saturated
sucrose solution of picric acid, 1 part sterile
Reference: Hiss (1904-05 p. 31). 20.0% NaOH solution and 3 parts
sterilelitmus tincture solution. Do
1295. Tanner's Azolitmin Milk Powder not heat this mixture.
Solution
(2) Add the above sterile indicator mix-
Constituents: ture to sterile milk.
1. Distilled water 1000.0 cc. Sterilization: Sterilize the NaOH, litmus
2. Milk powder, Merrell- and picric acid separately in the auto-
Soule 100.0 g. clave. Method of sterilization of milk
3. Azolitmus, Kahlbaum's not given.
Preparation: Use: Differentiation between typhoid
(1) Add 100.0 g. of Merrell-Soule's milk bacilli and colon bacilli. Author re-
powder to 1000.0 cc. distilled water. ported that typhoid bacilli did not cause a
(2) Beat with an egg beater. change in color nor cause coagulation.
379
Colon gave a pea yellow after 24

bacilli
warrant a separate discussion for each
a few more drops of picric acid investigator.
hours. If
References: Smith (1902 p. 106), Frost
be added to the medium typhoid will not
(1903 p. 66), Heinemann (1905 p. 28),
grow while B. coli will cause a slight
Smith (1905 48), Committee A.F.H.A.
change in color. This indicator may be
p.
(1905 p. 109), Roddy (1917 p. 41), Ball
added to media other than milk.
(1919 p. 83), Tanner (1919 p. 71), Percival
Reference: Calandra (1910 p. 570).
(1920 p. 57), Besson (1920 p. 59), Bezan-
1298. Park and Krumwiede's Litmus gon (1920 p. 116), Abbott (1921 p. 139),
Glycerol Milk Solution Levine (1921 p. 109), Dopter and Sacque-
pee (1921 p. 122), Giltner (1921 p. 25),
Constituents:
1000.0 cc. Harvey (1921-22 p. 94), Pitfield (1922 p.
1. Milk
117), Stitt (1923 p. 39), Park, Williams and
3. Litmus, 5.0% watery solu- Krumwiede (1924 p. 120).
tion Merck's 50.0 cc. Indicator Milk

1300. Sherman and Albus'
Preparation Solution
(1) Free milk from cream.
glycerol and 5.0% of a 5.0% Constituents:
(2) Add 5.0%
watery solution of Merck's purified 1.
2. Milk 1000.0 cc.

litmus.
Method not given. 3. Methylene blue (medicinal) . 0.5 g.
Sterilization:
of tubercle bacilli. After Preparation:
Use: Cultivation
(1) Dissolve 0.5 g. medicinal methylene
inoculation seal either with cork alone or
blue in 1000.0 water.
cc. distilled
preferably dip the cotton stopper also in
(2) Add 1.0 cc. of sterile (1) to each 10.0
paraffin.
cc. of sterile milk.
Reference: Park and Krumwiede (1910 p.
Sterilization: Sterilize the dye and milk
215).
separately. Method not given.
1299. Smith's Litmus Milk Solution Use: To demonstrate the reduction by
streptococci of the Streptococcus lacticus
Constituents:
and Streptococcus pyogenes types. The
1. Milk.
2. Litmus.
medium was decolorized if reduction
occurred.
Preparation:
(1) Obtain fresh milk and free from fat
variants:
by running the milk thru a separator
(a) Dissolved 1.0 g. of Kahlbaum's indigo
or by allowing the cream to rise
carmine in 1000.0 water
cc. distilled
spontaneously.
should not be and used instead of methylene blue
Test the reaction. It
(2)
solution.
more than +2.
(b) Dissolved 1.0 g. of GriJbler's neutral
(3) Tube.
red in 1000.0 cc. of distilled water and
(4) Add sufficient sterile litmus solution
used instead of the methylene blue
to give a distinct pale blue tinge to
solution.
the milk, to sterile (3).
the Reference: Sherman and Albus (1918 p.
Sterilization: Sterilize at 100C. using
167).
fractional method.
Use: General culture medium. 1301. Harvey's Brom Cresol Purple Milk
Variants: Various investigators have pre- Solution
pared media in a number of similar ways.
Constituents:
Some investigators used azolitmin instead 1000.0 cc.
of litmus, and some sterilized the
medium I ^^li\\^
the 2. Brom cresol purple

in the autoclave. Others sterilized
Preparation:
medium after the litmus was added. brom cresol purple in a
(1) Dissolve
The differences in preparation did not and
importance to minimum amount of alcohol,
seem to be of sufficient
make up to 0.04% brom cresol purple Preparation

by the addition of water. (1) Dissolve 105.0 g. of Bacto Purple
(2) Add sufficient of (1) to milk to give a Milk (dehydrated) in 1000.0 cc. of
distinct color. distilled water.
Sterilization: Method not specified. Sterilization: Sterilize in the usual manner.
Use: General culture medium. Kahn cul- The dye stand heating to a total
will
tivated spore forming anaerobes. not over 70 minutes.
Variants: Kahn cultivated spore forming Use: General culture medium. Acid pro-
anaerobes in a medium prepared as ducers change the color to yellow, alkali
follows producers to purple.
(1) Add 40.0 cc. of brom cresol purple Reference: Digestive Ferments Co. (1925
indicator solution to 1 liter partially p. 13).
skimmed milk (strength of indicator
solution not specified). 1304. Bacto Whey Broth (Dehydrated)
(2) Tube in 10.0 cc. lots. Constituents:
(3) Autoclave at 15 pounds for 20 1. Water 1000.0 cc.
minutes. 2. Whey, Broth, Bacto 30.0 g.
(4) The tubes are sealed with a vaseline Preparation
cap. (1) Dissolve 30.0 g. of Bacto Whey Broth
References: Harvey (1921-22, p. 88), Kahn (dehydrated) in 1000.0 cc. of distilled
(1922, p. 175). water.
(2) If sterilized at 15 pounds pressure for
1302. Harvey's China Blue Rosolic Acid 20 minutes pH = 6.5 .
Milk Solution Sterilization: Sterilize in the usual manner.
Constituents Use: Cultivation of Lactobacillus bul-

garicus.
1. Water 100.0 cc.
Milk, skimmed Reference: Digestive Ferments Co. (1925
2. 300.0 cc.
3. China blue (1.0% solution) P-14).
4. Rosolic acid (0.5% solution) 1305. Stutzer and Hartleb's Whey Solution
Preparation
Mix Constituents:
(1) 1 part creamed milk with 3 parts
water. 1. Whey.
Preparation
(2) Mix equal parts of a 1.0% watery
china blue solution and a 0.5% al- (1) Separate the cream from milk.
coholic rosolic acid solution. (2) Heat to 40C. and add a little rennet.
To each (3) After the casein has coagulated heat

(3) 100.0 cc. of (1) add 2.5 cc. of
(2).
to 75C. and filter.
Raise to boiling point and boil 5 (4) Neutralize by the addition of soda
(4)
(indicator not specified).
minutes.
Adjust the reaction by bringing the Sterilization: Not specified.
(5)
color, with the addition of alkali,
Use: Cultivation of the causitive agent of
the foot and mouth disease. Other in-
to a pale grey.
vestigators employed the medium as a
(6) Distribute into test tubes containing
general culture medium.
gas tubes which should project above
the surface. Variants
(a) The author added Na2C03 to obtain
Use: General culture medium. an alkaline reaction, or lactic acid to

obtain an acid reaction.
(b) Percival prepared the medium in the
1303. Bacto
following manner:
Purple Milk, (Dehydrated)
(1) Add 10.0 cc. of distilled water
Constituents: containing 8 to 10 drops of com-
1. Distilled water 1000.0 cc. mercial rennet extract to 1000.0 cc.
2. Purple milk Bacto 105.0 g. of skimmed or separated milk.
(2) Stir briskly for a few seconds. (14) Distribute in test tubes.
(3) Allow to coagulate. (15) Incubate two days in the incubator
(4) When the curd has formed cut with and 8 days at room temperature to
a knife, and leave it for an hour so test sterility.
that the whey may separate. Sterilization: Sterilization is effected in
(5) Filter thru muslin. step (13) above.

(6) Add the white of an egg whipped up Use: Cultivation of Bacillus leprae. May
with 100.0 g. of water. be used as a general culture medium.
(7) Heat in a steam sterilizer for an Variants The author used the medium with
:
hour. a neutral, alkaline or acid reaction.

(8) Filter into test tubes. References: Emile-Weil (1917 p. 380), Bes-
on 3 successive days for 20
(9) Sterilize
son(1920p.59).
minutes each day. Whey Solution
1307, Durham's Litmus
(c) Klimmer gave the following method
of preparation: Constituents:
(1) Add a little rennet to milk obtained 1. Whey
under the cleanest possible condi- 2. Litmus
tions and heat to 40C. until the Preparation:
casein is coagulated. (1) Fresh milk free from antiseptic
(2) Separate the casein from the whey adulteration is slightly warmed and
by straining thru a straining cloth. clotted by means of rennet.
(3) Filter the whey thru filter paper (2) Drain the whey and hang the clot
off
and then thru an asbestos filter. to drain in a piece of muslin.
(4) Distribute in sterile flasks. (3) Neutralize the turbid or yellow whey
(5) Sterilize by the addition of 1.5% with 4.0% citric acid solution, using
chloroform. litmus as an indicator.
References: Stutzer and Hartleb (1897 p. (4) Heat upon the water bath for 30
403), Percival (1920 p. 58), Klimmer (1923 minutes to coagulate the proteins.
p. 203). (5) Filter and add litmus to obtain a
suitable color.
1306. Emile-Weil's Litmus "Whey Solution Distribute in 10.0 cc. quantities if
(6)
Constituents desired.
1. Whey 1000.0 cc. Sterilization: Sterilize 100C. If not
at
2. Litmus perfectly clearthru a Berkefeld

filter
3. CaCh 2.0 g. filter after allowing to settle a few days.
Preparation Use: Cultivation of the colon-typhoid

(1) Skim milk. group.
(2) Heat to 40 C. Variants
(3) Add an excess of rennet tablets (6 (a) Heinemann prepared the medium as
tablets per liter). follows:
(4) Allow to coagulate for 30 minutes. (1) Precipitate casein from milk with
(5) Cut the curd into large pieces. rennet extract.
(6) Filter thru a thin cloth. (2) Neutralize the whey with 4.0%
(7) Make the filtrate alkaline to phenol-
citric acid solution.
phthalein by the addition of soda. (3) Heat on the water bath for 30
(8) Add 2.0 g. CaCh per liter.
minutes.
(9) Heat at 110 for 15 minutes. (4) Filter.
(10) Filter thru paper until the filtrate is (5) Add litmus solution until a decided
clear. blue color is obtained.
(11) The reaction should be a deep violet (6) Sterilization not specified.
to litmus. (b) Harvey prepared a similar medium
(12) Add 2.0% litmus solution to obtain as follows:
the desired shade. (1) Add rennet to fresh milk.
(13) Filter thru a candle. (2) Keepat60C.
(3) Strain the separated whey thru a 1309. Bronfenbrenner, Davis and Mori-
thick, clean cloth. shima's China Blue Rosolic
(4) Make the reaction of the fluid Acid Whey Solution
neutral to litmus by means of an
Constituents:
organic acid, such as citric acid.
1. Water 200.0 cc.
while hot thru well-wetted,
2. Whey 100.0 cc.
(6) Filter
thick filter paper.
3. MnCU (10.0% soln.) 2.5 cc.
4. China blue, rosolic acid
(7) Add
litmus solution to give a deep
indicator 3.0 cc.
purple red color.
Preparation
(8) Distribute into flasks or test tubes,
(1) Allow fresh milk to stand and syphon
(c) Klimmer gave the following method
the milk from under the cream.
of preparation.
(2) Bring it to boiling and add 2.5 cubic
(1) Coagulate the casein in milk by
the addition of rennet by heating
centimeters of 10% MnCU solution to
each 100 cubic centimeters of milk.
at 40C.
(3) Cool the mixture as soon as a clot is
(2) Filter.
Boil the filtrate for 2 hours.
formed and filter thru a single layer
(3)
of cloth.
(4) Neutralize.
(4) Titrate an aliquot portion hot and
(5) Filter.
The whey should be water clear adjust the bulk of medium to neutral
(6)
and slightly yellow. reaction (1 X 10"').
(7) Add 5.0 cc. of sterile litmus solu- (5) Bring quickly to boiling, cool and
tion to each 100.0 cc. of whey. filter thru paper.
(8) Add acid or alkali until the me- (6) Dilute the filtrate with double its
dium is a violet color. volume of water and add 1 cubic
(9) Filter.
centimeter of china blue rosolic acid
(10) Sterilize (method not given). indicator, see medium 535, under
References: Durham (1900-01 p. 379), variants, for each 100 cubic centi-
Heinemann (1905 p. 128), Abbott (1921 meters of medium. (At this point,
p. 140), Harvey (1921-22 p. 95), Klimmer the medium will have an intensely
(1923 p. 207). blue color).
(7) Distribute into sterile tubes con-
1308. Giltner's Sour Whey taining inverted fermentation tubes.
Constituents: Sterilization: Autoclave at 15 pounds pres-
1. Whey. sure for 10 minutes. Immediately after
Preparation autoclaving the medium will have a pink
(1) Inoculate sweet milk with a pure color. On cooling it will become colorless
active culture of Bad. lactis acidi or properly neutralized.
if it is
Bad. bulgaricum as desired and in- Use: Cultivation of colon-typhoid group.

cubate at 30C. Author reported that the medium gave
(2) Allow the maximum acidity to form. as good results as lactose-peptone water
(3) Cut the curd and heat in flowing as a medium for colon-typhoid bacteria
steam for 20 to 30 minutes. and was cheaper and more easily
(4) Strain thru cheese cloth and allow to prepared.
drain. Reference: Bronfenbrenner, Davis and
(5) Filter thru filter paper. Morishima (1918-19 p. 347).
(6) If clear whey is desired it is necessary
to clear the medium by the addition 1310. Jouan's Litmus Whey Solution
of egg albumin.
Use: To determine the acid destroying 1. Water 2000.0 or 3000.0 cc.
ability of bacteria. 2. Whey 1000.0 cc.
Reference: Giltner (1921 p. 365). 3. CaCli (25.0% soln.) 10.0 cc.

Preparation Variants
To 1 liter of milk add 10.0 cc. of 15 (a) Migula prepared a similar medium as
(1)
follows:
Baume (equivalent to 25.0 g. crystal-
water) Mix equal volumes of fresh milk
lineCaCh per 100.0 cc. (1)
CaCh solution. and water.

Heat in the autoclave at 115 for 4 or (2) Add HCl until the casein is
(2)
more or less, depending precipitated.
5 minutes,
on the coagulation. (3) Filter.
(4) Carefully neutralize the whey.
(3) Cool, without shaking.
(5) Boil for 1 to 2 hours.
(4) Filter thru linen.
soda solution until (6) Filter and allow to stand until
(5) Add a dilute
the whey is completely clear.
litmus is colored.
Boil for one minute and allow to cool. (7) The whey may be clarified by the
(6)
The filtrate addition of egg white.
(7) Decant and filter. is
(8) Tube, or distribute in flasks and
clear.
volumes of distilled store until ready for use. Steri-
(8) Mix with 2 or 3
steam.
lize in
water.
Add a tincture of litmus solution (9) When ready for use add sufficient
(9)
litmus tincture (About 5.0 cc. per
(amount not given) to give the de-
100.0 cc. whey) to give a violet
sired color.
color.
Sterilization: Sterilize at 110 to 112C. (10) Steam again.
(b) Bezanfon gave the following method
of preparation for the medium:
Variants: Besson and Harvey did not
dilute the whey with 2 or 3 volumes of (1) Add drop by drop from a burette
above. 10.0% HCl to 100.0 cc. of boiling

water as in step (8)
Besson milk until the casein is completely
Reference: Jouan (1916 p. 520),
coagulated. Stir constantly.
(1920 p. 60), Harvey (1921-22 p. 96).
(2) Add a sufficient quantity of HCl,
1311. Petruchsky's Litmus Whey Solution (calculated from (1)), to cause
(Grimbert and Legros) complete coagulation of casein in
1000.0 cc. of boiling milk. Add
Constituents:
the HCl drop by drop, stirring
1. Whey.
constantly.
2. Litmus.
(3) Allow to stand for several hours.
Preparation:
to a gentle heat (tem- (4) Decant the liquid whey.
(1) Heat cold milk
(5) Filter several times until clear.
perature not given).
HCl to precipitate the (6) Neutralize.
(2) Add sufficient
casein. (7) Add sufficient litmus solution to
give a violet tint.
(3) Filter.
solution until the reaction (8) Tube.
(4) Add soda
(9) Sterilize by heating at 100C. by
isbut only slightly acid.
the intermittent method.
(5) Heat in a Koch steamer for several
(c) Besson prepared the medium as
hours.
follows:
(6) Filter.
(1) Dilute 1.0 cc. of HCl in 20.0 cc. of
(7) Neutralize exactly to litmus.
water.
(8) Color with sensitive litmus.
Sterilization: Not specified. (2) Add (1) to 1 liter of fresh milk.
of colon-typhoid (3) Heat at 110 for 15 minutes.

Use: Differentiation
Allow to cool.
group. Author reported that colon bacilli (4)
(5) Filter thru Chardin paper.
turned medium strongly red. Typhoid
bacilli produced only slight acidity. B. (6) Make slightly alkaline and heat.
faecalis alcaligenes gave an alkaline (7) Add litmus until a light violet
color is obtained.
reaction.
(8) Filter thru a candle. (b) Hetsch prepared the medium as

(9) Distribute. follows:
(d) Harvey prepared the medium as (1) Boil 10.0 g. nutrose, and 5.0 g.
follows: NaCl in 1000.0 cc. of distilled water
(1) Add 1.5 cc. strong HCl acid or for 2 hours.
glacial acetic acid to 1000.0 cc. (2) Boil 20.0 g. mannitol or 25.0 g.
fresh, slightly warm milk. maltose in 50.0 cc. of Kahlbaum's
(2) Boil.
litmus solution for 10 minutes.
(3) Filter thru well-wetted, thick (3) Prepare solutions (strength not
paper.
filter given) of the sugars and alcohols
(4) Make the reaction of the filtrate listed.
neutral to litmus by the addition (4) Tube in about 10.0 cc. lots and
of dilute sodium carbonate sterilize in flowing steam for J hour.
Bolution. (5) Add 1 of (4) to (2).
(5) Filter, while hot thru well-wetted, (c) Hiss dissolved 10.0 g. nutrose, 4.0
thick filter paper. cc. of normal NaOH 10.0 cc. of 5.0%
(6) Add litmus solution to give a deep litmus solution and one of the follow-
purple red color. ing in 1000.0 cc. distilled water.
(7) Distribute into test tubes or flasks. Glucose Dextrin
(8) Sterilize. Maltose Mannitol
References: Grimbert and Legros (1901, p. Sucrose
913), Migula (1901 p. 21), Abel (1912 p. (d) Elser and Huntoon prepared the
49), Tanner (1919 p. 71), Bezanôn (1920 medium as follows:

Besson (1920 p. 59), Dopter and (1) Dissolve 10.0 g. nutrose and 5.0 g.
p. 116),
Sacqu6p6e (1921 p. 123), Harvey (1921-22 NaCl in 1000.0 cc. distilled water.
p. 95). (2) Add 5.0 to 7.5 cc. of a watery solu-

tion of Merk's highly sensitized
Nutrose Solution litmus.
1312. Barsiekow's Basal
(Segin) (3) Sterilize, in usual manner (exact
method not specified).
Constituents (4) Prepare a 10.0% solution of one of
1. Water 1000.0 cc. the following in distilled water.
2. Nutrose 10.0 g. Glucose Dulcitol
3. NaCl 5.0 g. Galactose Inulin
4. Litmus tincture 100.0 cc. Levulose Dextrin
Preparation Lactose Maltose
(1) Dissolve 2 and 3 in 1. Sucrose Mannitol
(2) Tube. (5) Sterilize (4) at 100C. for 10
(3) Prepare solutions of one of two added minutes.
nutrients in the litmus tincture. (6) Mix (5) and (3).
(4) Add quantity of sterile (3)
sufficient (7) Tube in sterile tubes.
to each sterile tube of (2) to give the (8) Incubate for 3 days to detect acci-
desired concentration of fermentable dental contamination.
material. (e) Tanner prepared the medium as
Sterilization: Sterilize (2) for an hour in follows:
the steamer. Sterilize (3) for 15 minutes (1) Dissolve 10.0 g. nutrose and 5.0 g.
in the steamer NaCl in 750 cc. distilled water.
Use To determine fermentation reactions.
: (2) Dissolve 10.0 g. of any desired
Added nutrients and variants carbohydrates, alcohol, etc. in
(a) Segin added one of the following 250.0 cc. of water.
materials: (3) Add litmus solution to (2) to give
Lactose Erythritol an amethyst color.
Glucose Dulcitol (4) Cool (1) and (3).
Maltose Mannitol (5) Mix (1) and (3).

(6) Tube. Added nutrients: The authors added any

not specified.
(7) Sterilization desired carbohydrates.
(f) Klimmer gave the following method Reference: Purwin and McNutt (1924 p.
of preparation: 297).
(1) Mix 10.0 g. of nutrose and 4.0 g.

NaCl in a mortar and add water, 1314. Th5ni and Allemann's Nutrose
drop by drop until a thick paste is Solution
obtained. Constituents:
(2) Steam for 1 to 2 hours. 1. Water.., 3000.0 cc.
(3) Allow to settle.
2. Nutrose...,, 30.0 g.
(4) Carefully decant and filter thru a Preparation
folded filter paper. sodium caseinate
(1) Dissolve 30.0 g. of
(5) Dissolve one of the following in 50.0 (nutrose) in 3 liters of water.
cc. of Kubel and Tiemann's litmus Sterilization: Not specified.
solution. Use: Cultivation of Bacillus putrificus.
lactose 10.0 g.
Inoculate and incubate under anaerobic
glucose 10.0 g.
conditions.
maltose 20.0 g.
Reference : Thoni and AUemann (1916
sucrose 20.0 g.
p. 101).
(6) Add (5) to (4).
(7) Add 0.1 normal NaOH until the 1315. Ficker and Hoffmann's Caffeine
medium is neutral to the litmus. Nutrose Solution
tubes. Constituents:
(9) Sterilize for 25 to 30 minutes in the 1. Distilled water. .
, 200.0 cc.
steamer. 2. Nutrose 10.0 g.
References: Segin (1903, p. 203), Hetsch 3. Caffeine 5.0 g.
(1903, p. 580), Hiss (1904-05, p. 31), 4. Crystal violet (Hochst) 0.1 g.
Elser and Huntoon (1909, p. 404), Tanner Preparation

(1919, p. 59), Besson (1920, p. 59), Harvey (1) Dissolve 10.0 g. nutrose in 200.0 cc.
(1921-22, p. 97), Klimmer (1923, p. 211). distilled water by boiling on the

water bath for several hours.
1313. Purwin and McNutt's Basal Nutrose (2) Make up the loss of water after
Solution cooling.
(3) Dissolve 5.0 g. caffeine in 20.0 cc.
Constituents distilled water at about 80C. Cool
1. Water 990.0 cc. to 55 to 60C.
2. Nutrose 10.0 g.
(4) Dissolve exactly 0.1 g. Hochst's
3. NaCl 0.5 g. crystal violet in 100.0 cc. distilled
4. Andrade indicator (1.0%) water.
(1) Mix 2 and 3 with 1 and allow to stand Use: Enrichment of typhoid and cholera
over night. bacilli from water and other materials.
(2) Adjust the reaction to pH 7.7. The author added 900.0 cc. of the water
(3) Steam in the Arnold for 2 hours. under investigation in a flask. Pour
(4) Filter thru cotton. sterile (2) into the flask containing (3).
(5) Add 1.0% Andrade indicator. (Do not reverse the order). Mix thoroly.
(6) Adjust to a very slight pink if Add mixture to the water under
this
necessary. investigation and add 10.0 cc. of (4).
(7) Add one of the added nutrients. Shake thoroly. Incubate for 12 to 13
Sterilization: Sterilize in the Arnold on hours at 37C. and then plate on medium.
each of 3 successive days. Reitz used the medium for the bacteri-
Use: Sugar free broth to study fer- ological examination of butter. He
mentation. added 200.0 g. of butter under investiga-
tion,melted at 20 or 30C., to the medium 4. Nutrose 10.0 g.

instead of 900.0 cc. of water as above. 5. Litmus solution, Kahlbaum 30.0 cc.
References: Ficker and Hoffmann (1904 p. Preparation
268), Reitz (1906 p. 722). (1) Add 2, 3 and 4 to 1.
(2) Heat in the water bath until solution

1316. Loeffler's Lactose Nutrose Solution is complete.
1. Distilled water 1000.0 cc. (4) Add 30.0 cc. of Kahlbaum's Litmus
2. Nutrose., 10.0 g. Solution.
3. Lactose 20.0 g. (5) Distribute into flasks or fermentation
4. Malachite green (2.0% soln). 50.0 cc. tubes.
Preparation Sterilization: Sterilize in streaming steam
for 15minutes on two successive days.
(2) Add 5.0% of a 2.0% malachite green Use: Differentiation of colon typhoid and
solution to (1). dysentery group. The author reported
Sterilization: Not specified. that Flexner's strains gave red coloration
Use: Enrichment of typhoid bacilli from after 24 hours. Shiga group caused no
faeces. change. Typhoid and coli forms gave
Variants: Klimmer prepared the medium the same reaction as in dextrose-nutrose
as follows: medium, coli causing a precipitation of
(1) Dissolve 10.0 g. nutrose in 1 liter hot the nutrose in 24 hours while the typhoid
distilled water. organisms required a longer period to
(2) Add 20.0 g. lactose to (1). produce precipitation.
(3) Flask in 100.0 cc. quantities. Variants Lehman prepared the medium as
:
(4) Sterilize on each of 3 successive days follows:

for 10 minutes in streaming steam. (1) Dissolve 4.0 g. Nutrose (Kahlbaum)
(5) Add 5.0 cc. of a 2.0% solution of 120 and 2.0 g. NaCl in 400.0 cc. distilled
Hochet malachite green to each 100.0 water.
cc. lot. (2) Sterilize for 45 minutes in the
References: Loeffler (1906, p. 289-295), steamer.
Klimmer (1923, p. 213). (3) Filter off 300.0 cc. immediately after
sterilization.
1317. Hill's Artificial Milk Medium (4) Dissolve 3.0 g. mannitol in 15.0 cc. of
litmus solution by heating slightly.
Constituents:
(5) Mix (4) and (3).
(6) Distribute into 4.0 cc. lots and
2. Nutrose.... 20.4 g.
sterilize for 20 minutes in the steamer.
3. Lactose 10.0 g.
Preparation
Reference: Doerr (1903 p. 395), Lehman
(1916-17 p. 102).
(1) Mix 1, 2 and 3 and allow to stand for
12 hours in the cold.
1319. Whittaker's Lactose Caseinogen
(2) Shake thoroly and filter thru cotton.
Solution
(3) Tube.
Sterilization: Tube and sterilize at 110 for Constituents
20 minutes. 1. Distilled water 1000.0 cc.
Use: Substitute for milk. General culture 2. Caseinogen 15.0 g.
medium. 3. Lactose 10.0 g.
Reference: Hill (1909 p. 379). 4. CaClo 0.1 g.
Preparation
1318. Doerr's Mannitol Nutrose Solution Prepare a 1.0% solution of NaOH in
(1)
Constituents: distilled water.
1. Distilled water 1000.0 cc. (2) Dissolve 15.0 g. of caseinogen in 100.0
2. Mannitol 10.0 g. cc. of (1). (18 to 24 hours may be
3. NaCl 5.0 g. required for this.)
(3) Dilute to 900.0 cc. with distilled Preparation

water. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
(4) Add 10.0 g. lactose and 0.1 g. CaCh (2) Suspend 30.0 g. of casein in (1).
to (3). Sterilization: Method not given.
(5) Make the solution up to 1000.0 cc. Use : Cultivation of oidium.
by the addition of distilled water. Reference: Laxa (1901-02 p. 129).
(6) Neutralize and make 0.3 with + 1323. Baginsky's Casein Solution
normal HCl, using phenolphthalein
as an indicator. Constituents:
Sterilization: Sterilize in the autoclave at 1. Water 1000.0 cc.
107 for 20 minutes. 2. Casein 1000.0 g.
Use: Substitute for milk. General culture Preparation
medium. (1) Add 20.0 g. of casein to 20.0 cc. of
Reference: Whittaker (1912 p. 162). water (or nutrient solution, composi-
tion not given).
1320. Waksman's Basal Casein Solution
Distribute into sterile flasks.
(2)
Constituents: Sterilization: Sterilize in the steamer.
1. Water 1000.0 cc. Use: To study fermentation by B. lactis.
2. Glycerol 30.0 g. Reference: Baginsky (1888 pp. 43'^462).
3. K2HPO4 1.0 g.
1324. Harvey's Alkaline Casein Solution
4. KCl 0.5 g.
6. FeS04 0.01 g. 1. Water 1000.0 cc.
7. Casein 5.0 g. 2. Casein 20.0 g.
Preparation: 3. NaOH (normal) 10.0 cc.
(1) Dissolve 2, 3, 4, 5, 6 and one of the Preparation
added nutrients in 1. (1) Mix 1, 2 and 3.
(2) Dissolve casein in N/10 NaOH and Sterilization: Not specified.
add to (1). Use: Cultivation of anaerobic organisms.
(3) Tube in 10 to 12 cc. lots. Author specified that nutrose or any other
Sterilization: Sterilize at 15 pounds for 15 casein product could be used instead of
minutes. casein.
Use: To study metabolism of actinomy- Reference: Harvey (1921-22 p. 96).
cetes.
1325. Geilinger's Basal Urine Solution
of one of the following: Constituents:
NaXOa (NH4)2S04 1. Urine, cow.
NaXOj (NH 4)200, Preparation:
Reference: Waksman (1920 p. 3). (1) Mix one of the added nutrients with
sterile cow's urine.
1321. Seliber's Basal Casein Solution
Sterilization: Sterilize cow's urine by heat-
(Harvey)
ing for one hour at 0.5 atmosphere pres-
Same as medium 743 but not containing sure or preferably by filtering thru a
peptone. Chamberland clay candle filter.
1322. Laxa's Lactic Acid Casein Solution
splitting urea, Bac. urea.
Constituents: Added nutrients and variants
1. Water 1000.0 cc. (a) The author added 1.0 cc. of one of the
2. NaCl 5.0 g. following to each 20.0 cc. of sterile
3. CaClj 0.1 g. urine.
4. MgCIa 0.2 g. The added nutrients were prepared as
5. Potassium phosphate follows:
(neutral) 2.5 g. (a) Straw infusion.
6. Lactic acid 3.0 g. (1) Mix 9 parts by weight of
7. Casein 30.0 g. water with 1 part by weight
of finely chopped straw and (g) Neutralize urine with K2HPO4 and
heat in steamer for one dilute 5 times with water.
hour. Reference: Bokorny (1920 p. 27).
(2) Filter.
(3) Sterilize for 45 minutes at 1327, Besson's Urine Medium (Tanner)
0.5 atmosphere pressure. Constituents
(b) Feces infusion. 1. Urine.
(1) Mix one part cow feces Preparation

with 4 parts water. (1) Boil some recently passed urine.
(2) Heat for one hour in the (2) If the reaction is markedly alkaline
steamer. after boiling, add a little tartaric acid
(3) Filter. solution. Use litmus paper as an
(4) Sterilize for 45 minutes indicator.
under 0.5 atmosphere pres- (3) Filter.
sure. (4) Tube.
(c) Peptone solution. Sterilization: Sterilize at 155C. It is
(1) Prepare and filter a 10.0% better to filter thru a Chamberland
peptone solution. bougie.
(2) Sterilize (method not Use: General culture medium.
given). Variants The following variants have been
:
(b) The author used cow's urine without given by the following authors:
any additions. (a) Besson
Reference: Geilinger (1917 p. 246). (1) Collect urine under aseptic condi-
tions in sterile flasks.
1326, Bokorny's Sucrose Urine Solution (2) Distribute in tubes.
(3) Incubate for 24 hours at 37C. to
Constituents
test sterility.
1. Urine 1000.0 cc.
(b) Besson filtered fresh urine thru a
2. Sucrose SO.O g.
Chamberland filter.
Preparation
(c) Besson
(1) Add 80.0 g. sucrose to 1000.0 cc. of
(1) Boil fresh urine.
undiluted and not neutralized urine.
(2) Neutralize to litmus by the addition
of tartaric acid if necessary.
Use: To study the growth of yeast. The
(3) Filter.
author reported that neutralized urine Tube.
(4)
showed better growth than non-neutral- 115C.
(5) Sterilize at
ized. The effect of diluting the urine was (d) Harvey
that of diluting the food materials.
(1) Collect urine fresh.
Growth best if diluted urine be neutral-
(2) Boil.
ized.
(3) Filter.
Variants : The author added 80.0 g. of sucrose (4) Sterilize in the steamer or auto-
to 1000.0 cc. of urine prepared in the clave.
following manner: (e) Harvey
(a) Undiluted urine neutralized with Distribute into test tubes freshly
(1)
K5HPO4. passed urine diluted to specific
(b) Undiluted urine partially neutral- gravity 1.010.
ized. (2) Sterilize in the steamer or auto-
(c) Unneutralized urine mixed with an clave.
equal volume of water. References: Tanner (1919 p. 58), Besson
(d) Neutralize urine with K2HPO4 and (1920 p. 33), Harvey (1921-22 p. 84).
then add an equal amount of water.
1328. Burri and Stutzer's Nitrate Feces
(e) Neutralize urine with K2HPO4 and
Solution
dilute with 3 volumes of water.
(f) Dilute unneutralized urine with 5 Constituents:
times water. 1. Water 1000.0 cc.
2. Feces (horse) 50.0 g. beef with one liter of water, and

3. NaNOa 3.2 g. allow to stand in the ice box for 12
Preparation: to 24 hours.
(1) Mix 1, 2 and 3. (2) Press the liquid thru a towel and
Sterilization: Do not sterilize. make up the volume to one liter.
Use: Enrichment of nitrate reducers, and (3) Boil in the steam cooker for 30
to stud)' denitrification. minutes.
Variants (4) The infusion may be boiled for an
(a) Ampola and Garino specified cow hour before removing the meat,
feces instead of horse. and then filtered thru paper. If
(b) Jensen mixed 10.0 g. NaNO,, 100.0 g. the liquid is still red, boil again for
cow or horse feces and 0.0 or 20.0 cc. 15 minutes.
of He reported that the
glycerol. (5) Distribute in flasks.
presence of glycerol aided denitri- (6) Boil for an hour to sterilize.
fication. (7) Store until ready for use. Seal the
References: Burri and Stutzer (1895 p. 261), flask with paraffin, gum arable or a
Ampola and Garino (1896 p. 671), Jensen beaker if it is to be stored for a
(1897 p. 693). long time.
(b) Abel-
1329. Dimitroff's Fecal Infusion
(1) Chop 500.0 g. of fat-free meat and
Constituents: add to a water at 50C.
liter of
1. Tap water 1000.0 cc. (2) Keep at 50C. for 30 minutes and
2. Feces (human) (1.0 to then boil for 30 to 45 minutes.
2.0%) 10.0 to 20.0 g. (3) Filter or strain the fluid from the
Preparation meat.
(1) Prepare an emulsion of 1.0 to 2.0% (4) Make up the fluid to one liter.
human feces in tap water. (5) Sterilize on each of 3 successive
Sterilization: Method not given. days in the steamer, or autoclave,
Use Cultivation of Leptospira biflexa.
: for 15 minutes at 120C.
Reference: Dimitroff (1927 p. 511). (c) Linde
(1) Chop 500.0 g. of beef into small

1330. Dunham's Meat Infusion Solution
pieces.
Constituents: (2) Boil with one liter of water.
1. Water 1000.0 cc. (3) Filter.
2. Beef 500.0 g. (4) Use filtrate without any additions
Preparation or add a concentrated Na2C03
(1) Boil finely chopped beef with a solution to give alkaline reaction.
double weight of water for 2 hours. (5) Sterilization not specified.
(2) Filter. (d) Davis and Ferry used beef infusion
(3) Make slightly alkaline; 0.5% NaCl (or a 1.0% meat extract solution) as a
may be added if desired. basic solution and added one of the
Sterilization: Not specified. following to 1000.0 cc. of the basic
Use: Dunham
used the solution for the solution:
detection of cholera bacilli. He reported Cystine 0.5 g
that the cholera vibrio gave a red ring Tryptophane 0.6 g
where the H2SO4 and medium met when Tyrosine., 1-25 g
concentrated H2SO4 was poured down the Glutaminic acid hydrochloride. 2.5 g
side of the tube containing a culture. Histidine dichloride 0.5 g
Similar media were used by different Leucine 3.0 g
investigators for a variety of purposes. Glycocoll 0.75 g
Variants: The following investigators pre- Sodium asparaginate 1.5 g
pared media as follows: Glucoseamine hydrochloride... . 2.0 g
(a) Migula fCreatine 0.2 g
(1) Mix 500.0 g. of finely chopped lean [Creatinine 0.15 g
fXanthin 0.05 g. a cool place (refrigerator) for not

\Hypoxanthia 0.05 g. more than 16 to 24 hours.
(3) Strain the infusion thru a cheese
NaCl 4.0 g.
cloth, thoroly pressing out all the
K2HPO4 3.0 g.
juice.
lMgS04 0.4 g.
[KNO3 0.2 g.
(4) Make up to 500.0 cc. by the addi-
for the culti- tion of tap water if necessary.
These media were used
(5) Place (4) in a sterile liter Erlen-
vation of Bact. diphtheriae and toxin
meyer flask.
production by it.
(e) Waksman studied the metabolism of
for 30 minutes.
actinomyces in media prepared as
(h) Harvey
follows:
water, filter and (1) Mince finely fat-free beef.
(1) Boil beef in
Note: Veal, chicken, ox or
sterilize.
horse heart, horse flesh, rabbit
(2) Inoculate with B. coli communor,
flesh, fish, blood, placenta, liver,
incubate at 37 for 24 hours.
spleen, kidneys, brain and vege-
(3) Boil for 10 minutes, adjust to pH
table materials, such as yeast,
= 7.6 to 7.7. Boil again, filter and
wheat, etc., may serve to furnish
sterilize.
any desired carbo- the extract used as basis for the
(4) Add 1.0% of
medium.
hydrate, alcohol, etc., and 1.0%
of Andrade indicator.
water or clear tap water.
(5) Distribute in fermentation
tubes.
minutes to (3) Heat the mixture 20 minutes over
(6) Steam in Arnold for 30
a free flame, at a temperature not
sterilize.
exceeding 50C.
The author reported that the actin-
Note: Or simply keep in a cool
omycetes developed in the infusion
place over night.
alone without the addition of other
(4) Skim off fat floating on the surface.
nutrients.
(f) Besson
point.
(1) Pour 1000.0 cc. of water on 500.0 g.
of finely chopped beef, and place
(7) Pour the mixture on to a wet,
in the ice box for 12 hours.
thick, clean cloth.
(2) Stir the mixture well.
(8) Add sodium chloride 5.0 g. to the
(3) Filter thru a cloth and press
the
filtrate.
meat free from juice. Steam 45 minutes.
(9)
(4) Filter the juice thru paper. Bring the volume up to 1000.0 cc.
(10)
(5) Add 5.0 g. NaCl. by the addition of water.
(6) Boil. (11) Estimate and adjust the reaction.
(7) Make slightly alkaline or neutral- (12) Steam 30 minutes.
ize to litmus by the addition of
(13) Filter, while hot, thru well-wetted,
soda. thick filter paper.
(8) Heat at 115 to 117C. for 5 minutes. References: Dunham
(1887 p. 338), Migula
(9) Filter until clear. (1901 p. Abel (1912 p. 13), Linde
13),
(10) Make up to 1000.0 cc. by the addi- (1913 p. 386), Davis and Ferry (1919 p.
tion of distilled water. 235), Waksman (1919 p. 316), Besson
(11) Distribute as desired. (1920 p. 28), Dopter and Sacquepee
110 to 115C. for 20 (1921 p. 120), Giltner (1921 p. 27), Harvey
(12) Sterilize at
minutes. (1921-22 p. 94).
(g) Giltner
1331. Stutzer and Hartleb's Nitrite Infusion
(1) Add 500.0 g. of tap water to 500.0
Solution
g. finely chopped fresh lean beef in
ware pail.
3.5 liter agate Constituents
1. Water... 2000.0 cc
(2) Mix thoroly and allow to stand in
2. Meat 1000.0 g. 3. NaCl 5.0 g.

3. NaN02 (2.0%) 40.0 g. 4. Starch 10.0 g.
(1) Add 2 liters of water to 1000.0 g. of (1) Prepare meat infusion solution from
finely chopped lean meat. 1, 2 and 3 as indicated in medium 850.
(2) Heat in thesteamer at 100C. for 2 (2) Add 10.0 g. starch to 1000.0 cc. of (1).
hours. (3) Adjust to 0.4% to phenolphthalein.
(3) Pour off the liquid. Sterilization: Not specified.
(4) Make up to 2 liters.
(3) Use: Cultivation of gonococci.
(5) Neutralize by the addition of soda, Reference: Harvey (1921-22 p. 112).
indicator not specified.
1334. Beijerinck's Urea Meat Infusion
(6) Add 2.0% NaNOj.
(7) Filter. Constituents
Sterilization: Method not given. 1. Meat infu.sion 1000.0 cc.
Use: Cultivation of bacteria from case of 2. Urea (4.0%) 40.0 g.
foot and mouth disease. Preparation
Reference: Stutzer and Hartleb (1897 p. (1) Add 4.0% urea to meat infusion.
404). Sterilization : Not specified.
Use: Cultivation of Urococcus ureae, Uro-
1332. Besson's Glucose Infusion Solution
bacillus pasteurii, Urobacillus leubei,
Constituents Planosarcina ureae and other bacteria.
1. Beef infusion solution 1000.0 cc. Variants
2. Glucose 4.0 g. (a) The author used 5.0, 6.0, or 10.0%
3. Neutral red (1.0% soln.).... 3.0 cc. urea instead of 4.0%.

Preparation: (b) Sohngen added 0.5 to 6.0% urea to
(1) Method of preparation of beef infu- meat infusion or bouillon to cultivate
sion solution not given. Bacillus erythrogenes.
(2) Dissolve 2 in (1). (c) Groenewege cultivated
Phytobacter
(3) Add 3.0 cc. of a 1.0% watery neutral lycopersicum, (causing tomato rot)
red solution. in meat infusion solution containing
(4) Distribute in tubes containing fer- 0.5% urea.
mentation tubes. (d) Wojtkiewicz studied urea decompo-
Sterilization: Sterilize at 110C., time not sition by soil forms in a meat infusion
specified. solution containing 0.5% NaCl and
Use: Differentiation of colon typhoid 10.0% urea.
group. Author reported that Bad. ty- References: Beijerinck (1901 p. 52), Sohn-
phosum and dysentery gave amaranth gen (1909 p. 95), Groenewege (1913 p. 24),
tint, but no gas. Paratyphoid A, ama- Wojtkiewicz (1914 p. 259).
ranth tint with a gas bubble. Para-
1335. Klimmer's Blood Infusion
typhoid B gave orange color in the tube
with yellow in the fermentation tube, Constituents:
gas was produced. B. proteus same as 1. Water 1000.0 cc.
paratyphoid B but much less gas was 2. Blood 500.0 cc.
produced. Colon bacilli, fluorescence Preparation
with a yellow tint, gas was produced. (1) Mix blood with a double amount of
B. fecal alcaligenes, no fluorescence. water. The clot of the blood is
Culture salmon colored in the tube, gas previously run thru a meat chopping
was not produced. machine.
Reference: Besson (1918 p. 929). (2) Boil for 10 minutes, stirring con-
tinuously to prevent burning.
1333. Harvey's Starch Beef Infusion
(3) Acidify slightly by the addition of
Constituents acetic acid.
1. Water 1000.0 cc. (4) Boil for 5 more minutes.
2. Beef 500.0 g. (5) Allow to and cool.
settle
(6) Decant carefully and strain thru a (5) Make up to the original volume,
straining cloth. adjust the reaction as desired in
(7) Filter thru paper. the same manner as for meat
Sterilization: Not specified. extract.
Use : Use as meat water in the preparation (6) The bouillon may be filtered until
of media. clear.
Reference: Klimmer (1923 p. 172). Usually 0.5% glycerol is added.
(7)
not specified.
(8) Sterilization
1336. Szasz's Blood Clot Infusion
(b) Stefanopoulo cultivated Spirochaeta
Constituents: icterohemorragiae on a medium pre-
1. Distilled water 2000.0 cc. pared as follows:
2. Blood clot , 1000.0 g. (1) Separate the serum from the clot
Preparation of coagulated horse blood.
(1) Take 1000.0 g. of blood from which the (2) Press the clot thru a fine wire gauze.
serum has been separated, coagulated (3) Dilute with two times its volume
blood or blood clots and add 2000.0 with physiological salt solution
cc. of distilled water. prepared by dissolving 8.0 g. of
(2) Boil for a short time. (Time not NaCl in 1000.0 cc. of water.
specified.) (4) Heat for 15 minutes at 80C.
(3) Separate the clot into pieces the size (5) Filter thru paper, and then on a
of a nut and boil longer. Do not cut Chamberland filter.
the clot before heating. Take care (6) Distribute in sterile tubes and cover
that the blood clot does not sink to with a layer of sterile vaseline.
the bottom of the kettle and burn. References: Szasz (1914-15 p. 491), (1915-
Place the kettle or container in which 16 p. Ill), Stefanopoulo (1921 p. 813).
the boiling is taking place over a free
flame. It may be stirred with a 1337. Wellman's Placenta Infusion
wooden spoon. A linen towel may Constituents
be placed in the bottom of the con- 1. Distilled water 1000.0 cc.
tainer and extend part way up the 2. Placenta, human 1000.0 g.
side walls or all the way up and be Preparation
fastened with a cord. This is the (1) Grind fresh human placenta thoroly
simplest way to prevent burning. in a meat chopping machine after
(4) Filter thru a large linen towel. first washing out the blood by running
(5) The bouillon may be clarified with sterile salt solution thru the attached
egg white. vessel.
Sterilization: Not specified. (2) To each kilogram of macerated
Use: Inexpensive medium. Meat infusion placental tissue add 1 liter of distilled
or extract substitute. water.
Variants (3) Infuse for 48 hours in the ice box.
(a) The author prepared a similar me- (4) Tube sterile (3).
dium as follows: (5) Store the medium at 40-41 C. for 2
(1) Divide the blood clots into pieces days before use to inactivate the
the size of a walnut or hazel nut by complement.
means of a piece of wood or hands. Sterilization: Pass thru a No. N Berkefeld
(2) To the clot obtained from every filter that will hold back ordinary bac-
kilogram of blood, add 1.5 liters teria. To facilitate this fill the cylinder
of water. of the filter with a clean fine sterile sand
(3) Mix well and allow to stand in the until the cylinder is completely covered.
cold for 20 to 24 hours, stirring Use: Cultivation of parasitic bacteria.
several times. Author used Bacillus leprae and tubercle
(4) Filter thru a coarse linen cloth and bacilli.
boil the filtrate until brown clumps Variants
are formed and the liquid is yellow. (a) Groer and Srnka studied the produc-
tion of toxin by diphtheria bacilli, (2) Mix equal parts of (1) and distilled
and cultivated them on a medium water.
prepared as follows: (3) Shake vigorously.
(1) Mix 4 liters of medium fine placenta (4) Filter thru Buchner filters and then
with 4 liters of water. thru Berkefeld filters (finest grade).
(2) Boil for about 3 hours with the (5) Distribute to fermentation tubes.
addition of water. (6) Incubate for 24 hours to determine
(3) Filter. sterility.
(4) Concentrate the filtrate to 3 liters. Sterilization: Sterilization given under
(5) Add N/1 10.0% NaOH until phenol- preparation.
phthalein is turned weakly red. Use: Cultivation of a spore bearing an-
(Hot titration). aerobe from a dog's liver. The medium
Distribute into flasks and sterilize.
(6) obtained at first is rather viscous, red and
(Method not given). slightly acid in reaction. After 48 hours
(b) Park, Williams and Krumwiede pre- incubation proteins are precipitated.
pared a similar medium as follows: Reference: Wolbach and Saiki (1909 p.
(1) Add 500.0 g. of ground up tissue 270).
(placenta) to a liter of water.
Soak for 90 minutes.
1340. Brieger's Thymus Gland Infusion
(2)
(Besson)
(3) Strain thru cheese cloth and
squeeze by twisting the cloth or Constituents:
use a meat press. 1. Distiled water 1000.0 cc.
(4) Filter thru paper or sand. 2. Thymus gland (beef) 1000.0 g.
(5) Sterilize by filtration thru a filter Preparation
candle. (1) Remove the thymus glands from dead
References: Wellman (1912 p. 143), Groer beef.
and Srnka (1918-19 p. 334), Park, Williams (2) Chop into a pulp and add an equal
and Krumwiede (1924 p. 125). weight of distilled water.
(3) Allow to soak for 12 hours.
1338. Moon's Brain Infusion
(4) Filter thru a gauze, pressing out the
Constituents: liquid.
1. Physiological salt solution.. . 900.0 cc. (5) Add anequal weight of water to the
2. Brain (dog) 100.0 g. turbid viscous liquid.
Preparation (6) Make slightly alkaline by the addition
(1) Remove brain matter from normal of a 1.0% solution of sodium bicar-
dog under the most careful aseptic bonate.
precautions. (7) Heat at 100C. for 15 minutes in the
(2) Add 900.0 cc. of physiological salt autoclave or salt bath.
solution to 100.0 g. of (1). (8) Filter thru a fine linen cloth.
(3) Emulsify by shaking with glass beads. (9) Distribute in sterile tubes.
Sterilization: Filter thru a coarse Berke- Sterilization: Sterilize at 100 for 15
feld filter. minutes on two days.
Use: Cultivation of Negri bodies. Author Use: General culture medium. Besson re-
reported that growth was poor, both ported that the cholera vibrio did not
anaerobically and aerobically. develop in this medium unless it was
Reference: Moon (1913 p. 233). diluted with 5 or 6 volumes of sterile
water before use.
1339. Wolbach and Saiki's Liver Infusion
Constituents:
1341. Nencki, Sieber and Wyznikiewicz's
2. Liver (dog) Salivary Gland Infusion
1000.0 g.
(1) Pass fresh dog's liver thru the meat 1. Distilled water 1000.0 cc.
grinder. 2. Salivary glands 200.0 g.
Preparation Variants: Robertson and Davis prepared a

(1) Obtain beef salivary glands from the similar medium in the following manner:
slaughter house. (1) Grind lean beef heart finely and just
(2) Cut in small pieces with a meat cover with sterile distilled water.
chopper. (2) Place in ice box 18 hours.
(3) Add 5 volumes of water. (3) Pass thru Berkefeld filter.
(4) Filter thru blotting paper. (4) Mix (3) with sterile (method of
(5) Distribute sterile (4) into sterile sterilization not given) physiological
tubes. salt solution.
Sterilization: Filter thru a Chamberland Reference: Graham-Smith (1920-21 p. 136),
filter to sterilize. Robertson and Davis (1923 p. 154).
Use Medium when inoculated with plague
:
containing material contained round cells 1343, Robertson and Davis' Heart Infusion
after two days. Medium
Variants
Constituents:
(a) The author added 3.0% NaCl or 2.0
1. Water, sterile distilled 1000.0 cc.
to3.0% NaCl plus 0.2 to 0.5 g. NaOH
2. Asparagin (Merck) 3.4 g.
or KOH per liter.
(b) Mayer prepared a similar medium as

3. CaCU 0.1 g.
4. Glucose 20.0 g.
follows
Chop fresh salivary glands in a 5. MgS04 0.2 g.
(1)
meat chopping machine. 6. K2HPO4 1.0 g,
7. NaCl 5.0 g.
(2) Mix with an equal weight of water,
8. Heart, beef.
(3) Infuse on ice for 24 hours after
Preparation
stirring strongly.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1 by
(4) Press the mass in a meat press.
boiling 3 minutes.
(5) Sterilize the thick, slimy liquid in
(2) Restore original volume with sterile
streaming steam for 30 minutes.
distilled water.
Mayer reported that a meat extract
(3) Adjust reaction to pH = 7.4 and tube.
medium generally gave better
(4) Grind lean beef heart finely and just
growth of parasitic and saprophytic
cover with sterile distilled water.
bacteria.
(5) Place in ice box 18 hours.
References: Nencki, Sieber and Wyzni-
(6) Pass thru Berkefeled filter.
kiewicz (1898 p. 530), Mayer (1899 p.
(7) Add various amounts of (6) to each
747).
tube of sterile (3).
1342. Graham-Smith's Heart Infusion Sterilization: Autoclave (3) at 20 pounds
pressure for 30 minutes. Heart infusion
Constituents
is sterilized by filtration thru Berkefeld
filter.
2. Heart, bullock 100.0 g.
Use: To study influence of vitamins on
Preparation
bacterial growth. Author reported a
(1) Remove the fat and vessels from
luxuriant growth of yeast in this medium.
fresh bullock's heart muscle.
Reference: Robertson and Davis (1923 p.
(2) Pass thru a mincing machine.
154).
(3) To each 100.0 g. of (2) add 250.0 cc.
of water.
1344, Mereshkowsky's Egg White Infusion
(4) Boil gently for 99 minutes.
(5) Filter. Constituents:
Sterilization: Sterilize for 20 minutes on 3 1. Water 1000.0 cc.
successive days in a steam sterilizer. 2. Egg albumin 100.0 g.
Use: General culture medium. Robertson Preparation
and Davis used a similar medium to study (1) Remove the shell from hard boiled
the influence of vitamines on bacterial hen eggs.
growth. They reported that the medium (2) Cut the egg in 4 lengthwise strips
did not give continued growth of yeast. and remove the egg yolk.
(3) Pass the egg white thru a meat grind- Preparation

ing machine. (1) Remove mussels from their shells.
(4) To each 100.0 g. of finely chopped egg (2) Work (1) into a pulp and mi.x with a
white add 1000.0 cc. of water. double weight of water.
(5) Place in a flask and plug. (3) Place in the autoclave for 15 to 20
(6) Autoclave for 15 minutes under one minutes under 1 atmosphere pressure.
atmosphere of pressure. (4) Filter thru filter paper until a not
(7) Filter thru cotton, and then thru exccessive turbid, at the most a
paper. slightly opalescent, liquid is obtained.
(8) Distribute into test tubes. (5) Place in the autoclave for 30 minutes
Sterilization: Method not given. again under 1 atmosphere pressure.
Use: Cultivation of Bacillus Danysz. The (6) Filter. The filtrate is a clear amber
author reported that Bacillus Danysz did yellow.
not lose its virulence on repeated cultiva- (7) Distribute into sterile tubes.
tion on this medium. Sterilization: The medium may be steri-
Reference: Mershkowsky (1912 p. 394). lized in the autoclave if necessary.
1345. Proca's Spleen Infusion Solution Use: Cultivation of typhoid, cholera and
other intestinal forms. Author reported
Constituents:
that the cholera vibrio lost its character-
1. Ringer solution 1000.0 cc.
istic appearance on this medium.
2. Spleen 400.0 g.
Preparation: Variants
(1) Add 400.0 g. of finely chopped fresh (a) The author used oysters instead of
beef spleen to 1000.0 cc. of Ringer's mussels.
solution (see medium ^180). (b) The medium was prepared as follows:
(2) Heat at 115C. for 30 minutes. (1)Remove oysters or mussels from
(3) Filter while hot thru paper. their shell.
(4) Distribute in tubes or flasks. (The (2) Work (1) into a pulp and mix with a
filtrate may be solidfied with 1.4% double weight of sterile phyiologi-
agar or 10.0% gelatin). cal salt solution.
Sterilization: Sterilize in the autoclave. (3) Allow the mixture to stand for 24 to
Use: General culture medium. 48 hours at 15C.
Reference: Proca (1924 p. 1164). (4) Pour off the liquid, filter thru cotton
then thru paper and finally thru a
1346. Harde and Hauser's Fish Infusion
Berkefeld candle.
Constituents: (5) This gives a yellowish amber fluid.
1. Water 1000.0 cc. The author reported that the organ-
2. Fish (Whiting) 500.0 g. isms failed to develop on this
Preparation: medium.
(1) Boil 500.0 g. of chopped whiting fish
Reference: Pergola (1912 p. 171).
with 1 liter of water for 20 minutes.
1343. Besson's Chicken Infusion (Tanner)
(3) Reaction is neutral to litmus.
Sterilization: Sterilize at 120^ for 20 Constituents:
minutes. 1. Water 1000.0 cc.
Use: Substitute for beef media and as a 2. Chicken 500.0 g.
general culture medium.
Preparation:
Reference: Harde and Hauser (1919 p. lean chicken meat in
(1) Soak 500.0 g. of
1259).
1000.0 cc. water.
1347. Pergola's Mussel Infusion (2) Filter.
Constituents
1. Water 1000.0 cc. Use: Substitute for beef infusion.

2. Mussels 500.0 g. Reference: Tanner (1917 p. 46).
1349. Biisgen and Hoflich's Meat Extract Reference: Stutzer (1901 p. 83), Tanner
Solution (Linde) (1919 p. 45).
Constituents:
1. Water 1000.0 cc. 1351. Zikes' Basal Glucose Meat Extract
2. Meat
extract (0.5%) ........ 5.0 g. Solution
(1) Dissolve 2 in 1. 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Meat extract (0.5%) 5.0 g.
Use : Enrichment of Cladothrix, Cladothrix 3. Glucose (0.25%) 2.5 g.
dichotoma. Cultivation of other Preparation
organisms. (1) Dissolve 2, 3 and one of the added
Variants nutrients in 1.
(a) The author added a concentrated Distribute as desired.
(2)
Na2C03 solution to obtain an alkaline Sterilization: Not specified.
reaction. Use: Author used the medium to study the
(b) Zikes cultivated cladothrix and nitrogen sources suited to the development
Sphaerotilus natans in a medium of Cladothrix dichotoma and Cladothrix
containing 0.5, 0.25, 0.125, 0.063 or natans. He reported that Cladothrix
0.031% meat extract. dichotoma grew well on any nitrogen
(c) Besson prepared the medium as source but Cladothrix natans grew only
follows: when peptone or asparagin was added.
(1) Dissolve 0.5% Liebig's meat ex- Bloch studied the nitrogen sources suited
tract (or 20.0 g. Cibils meat extract) for the development of Zoogloea rami-
in 1000.0 cc. of water. gera. He reported that asparagin pep-
(2) Make alkaline if necessary. tone, ammonium sulphate and then
(3) Autoclave at 115 to 117 for 5 nitrate was the order of the materials as a
minutes. nitrogen source for Zoogloea ramigera.
(4) Filter while hot thru a wet filter. Added nutrients: Zikes as well as Bloch
(5) Tube. added 0.25% of one of the following:
(6) Sterilize at 110 to 115C. (NH 4)280 4 asparagin
Reference: Linde (1913 p. 372), Zikes KNO3 peptone
(1915 p. 530), Besson (1920 p. 30). Reference: Zikes (1915 p. 542), Bloch (1918
p. 51).
1350. Stutzer's Nitrate Meat Extract
Solution
1352. Stutzer's Glucose Meat Extract
Constituents:
Solution
1. Water
2. KNO3 Constituents
3. Liebig's Meat Extract 1. Water.
Preparation 2. Glucose.
(1) Dissolve 2 and 3 in 1. 3. KNO3
(2) Adjust the reaction to a slight 4. Liebig's ]\Ieat Extract.
alkalinity. Preparation
(3) Distribute in 10.0 cc. lots. (1) Dissolve 2, 3 and 4 in 1. (Peptone
Sterilization: Method not given. may be added).
Use: To study denitrification by B. agilis, (2) Reaction to be slightly alkaline.
B. niirovorus, B. Stuizeri, B. Hartlebi. (3) Distribute in 10.0 cc. lots.
Variants Sterilization: Method not given.
(a) The author used Schiilke and Meyer Use: To study denitrification by B. agilis,
or Cibils meat extract instead of B. niirovorus, B. Stutzeri, B. Hartlebi.
Liebig's. Variants
(b) Tanner dissolved 10.0 g. c.p. NaNOa (a) The author substituted Schiilke and
and 3.0 g. of Liebig's meat extract in Meyer's meat extract or Cibils meat
1000.0 cc. of water. extract for Liebig's.
(b) Kappen studied nitrate decomposi- d-galactose

tion by cyanamide decomposing
bacteria, in a medium composed of
5.0 g. meat extract, 2.0 g. glucose and
2.0 g. KNO3 per liter.
Reference: Stutzer (1901 p. 83), Kappen

(1909 p. 395).
1353. Hurler's Succinate Meat Extract

Solution
Constituents
1. Water 1000.0 cc.
2. Meat e.xtract, Cibils 100.0 to 200.0 cc.
3. Ammonium succi-
nate 10.0 g.
Preparation
(1) Thicken 100 to 200.0 cc. of Cibils
meat extract on the water bath.
(2) Transfer the viscous mass to a cru-
cible and carbonize and ash under the
hood.
(3) Take 3.0 g. of the grey white ash thus
obtained and dissolve in 1 liter of
water by boiling an hour.
(4) Filter repeatedly thru cotton.
(5) Add 10.0 g. of ammonium succinate
and neutralize with dilute ammonia.
(6) Distribute the slightly opalescent
medium in tubes.
Sterilization: Sterilize (method not given).
The medium becomes clear.
Use: Cultivation of typhoid and inter-
mediate group.
Reference: Hurler (1912 p. 356).
1354. Bloch's Basal Asparagin Meat Extract

Solution
Constituents:
1. Water 1000.0 cc.
2. Meat extract (1.0%) 10.0 g.
3. Asparagin (0.25%) 2.5 g.
Preparation:
(1) Dissolve 2, 3 and 0.25% of one of the
added nutrients in 1.
(2) Tube or flask.
Use: Cultivation of Zoogloea ramigera.
Author reported that growth was gener-
ally better without carbohydrates.
d-glucose lactose
d-levulose starch
d-mannose raffinose
Add 0.5% Cyanamide to (2). (4) To obtain best results it is well to add
(3)
(4) Distribute in Erlenmeyer flasks. some albuminous material. Peptone
Sterilization: Method not given. may be added but a material from the
Use: To study cyanamide decomposition bone jelly gives equally good results.
by bacteria. Mix equal parts of water, bone jelly
Reference: Kappen (1909 p. 392). and HCl (strength not specified) and
heat in a water bath for 24 hours.
1358. Homer's Tryptophane Gelatin Neutralize with soda, clarify and
Solution filter.
Constituents (5) Add 1.0% of (4) to (3).
2. Nutrient salts Use: Ine.xpensive culture medium.

3. Tryptophane (0.15%) 1.5 g. Reference Standfuss and Kallert (1920-21
:
4. Gelatin (1.0%) 10.0 g. p. 223).
Preparation :
1361. Berman and Rettger's Gelatin
(1) Add 1.0% gelatin and 0.15%, tryopto-
Solution
phane to a solution containing neces-
Constituents:
sary nutrient salts.
2. Gelatin 2.5 g.
Use: Indol production.
Variants: The author added 1.0% glucose.
3. NaCl 5.0 g.
Preparation
Reference: Homer (1906 p. 402).
(1) Dissolve 2 and 3 in 1. (2.5 g. Liebig's
1359. Remy and Rosing's Gelatin Solution meat extract may be added.)
Constituents
1. Water 1000.0 cc.
Use: To study bacterial nutrition.
2. Gelatin (0.8%) 8.0 g.
Reference: Berman and Rettger (1918 p.
Preparation
381).
(1) Add 0.8% gelatin to 1000.0 cc. water.
(2) Neutralize with soda. 1362. Fleming's Blood and Minced Meat
(3) Distribute into small Erlenmeyer Medium
flasks. Constituents:
steam
Sterilization: Sterilize in streaming 1. Meat
by means method.
of the fractional 2. Blood.
Use: To study decomposition of organic N Preparation
materials by soil forms. (1) Add a little blood to a minced meat
Reference: Remy and Rosing (1911 p. 39). medium such as is commonly used to
grow anaerobes.
1360. Standfuss and Kallert's Bone Jelly
Solution
Use: Preservation of B. influenzae.
Constituents Author reported that subcultures from
1. Water. this medium after 6 weeks gave growths as
2. Bone jelly. copious as after two days.
Preparation Variants: Harvey added a little whole
(1) Bone jelly is obtained from a factory blood to the meat medium as prepared in
where fresh bones are rendered under medium 1239.
a pressure of 2 to 4 atmospheres in an References: Fleming (1919 p. 139), Harvey
autoclave. The
rendered material (1921-22 p. 72).
is evaporated or condensed to a jelly-
like material which is quite stable.

1363. Dean and Nouat's Blood Egg Medium
(Cutler)
(2) Dissolve some of the jelly in hot
water. The amount depends on the Constituents:
consistency of the jelly. 1. Water 300.0 cc.
(3) Clarify and filter, obtaining a com- 2. Egg 1
pletely clear golden yellow fluid. 3. Blood, human drop

Preparation and spirochaetes. The author added a

(1) Break up the white and yolk of one layer of sterile paraffin oil to each tube
egg in a flask containing beads and and incubated under strict anaerobic
shake thoroly. conditions at 35 to 37C.
(2) Add 300.0 cc. of water and again Variants: Pitfield prepared a similar me-
shake. dium as follows:
(3) Place the flask in a water bath and (1) Obtain dog, sheep or cow blood under
bring to boiling point. aseptic conditions.
(4) Maintain this temperature for 30 (2) Pipette off the serum with a sterile
minutes, shaking from time to time. pipette.
(5) Tube in 5.0 cc. amounts. (3) Mix 3 to 4 parts of distilled water with
(6) Add a few drops of human blood. 1 part (2).
Sterilization: Sterilize in the autoclave. (4) Tube.
Use: Cultivation of Entamoeba his- (5) Add a bit of sterile animal tissue to
tolytica and other intestinal protozoa as each tube.
amoeba, etc. References: Noguchi (1911 p. 101), (1911
References: Cutler (1918 p. 22), Stitt (1923 pp. 1550-1551), Pitfield (1922 p. 119).
p. 51).
1366. Hata's Serum Tissue Medium
1364. Bruschettini's Egg Yolk Blood Constituents:
Infusion Solution (Kolle and
1. Physiological salt solution 80.0 cc.
Wassermann) Horse serum 40.0 cc.
2.
Constituents 3. Kidney
1. Veal infusion solution 100.0 cc. Preparation
2. Blood, defibrinated, rabbit (1) Draw blood from the vein of a normal
or dog 10.0 cc. horse into tall glass cjdinders and
3. Egg yolk 5.0 cc. allow the serum to separate.
Preparation (2) Pipette 4.0 cc. of the serum into tubes
(1) Mix 1, 2 and 3. having a diameter of 1.5 to 1.7 cm.
Sterilization: Not specified. (3) To each tube add 8.0 cc. of physiologi-
Use: Cultivation of tubercle bacilli. cal salt solution and mix well. The
Reference: Kolle and Wassermann (1912 p. contents of the tube are about 6.5 to
413). 7.0cm. high.
(4) Place in a water bath at 58C. raising
1365. Noguchi's Serum Tissue Medium the temperature gradually until it
Constituents: reaches 70 or 71 C. in three hours.
1. Distilled water 300.0 cc. (5) Heat for 30 minutes at 7rC. The
2. Serum 100.0 cc. contents present a translucent milky
3. Tissue, (rabbit) appearance with a semi-solid surface.
Preparation (6) Immerse one or two small pieces of
(1) Tube a mixture of one part serum rabbit kidney in each tube. It may
(sheep, horse or rabbit) and three be necessary to push the kidney to
parts distilled water in test tubes 20 the bottom by means of a sterile glass
cm. high and 1.5 cm. wide to a depth rod.
ofabout 16 cm. Sterilization: Not specified.
(2) Add to each sterile tube a small piece Use: Cultivation of Spirochaeta recurrens.
of freshly removed sterile rabbit Inoculate the medium with infected
tissue kidney or heart
(testicles, blood by means of a sterile capillary tube.
muscle) (liver being unsuitable). Author reported that in the kidney
(3) Incubate at 37 for 2 days to test medium growth appeared after 24 hours
sterility. and reached a maximum on the 4th day.
Sterilization: Sterilize (1) at 100C. for 15 In the buff coagulum medium growth was
minutes on 3 successive days. at a maximum at the end of the 5th to
Use: Cultivation of Treponema pallidum 7th days. The number seemed to be less
than in the kidney medium. In the buff Variants

medium the spirochaetes kept their (a) Noguchi substituted hydrocele fluid
regular forms of life for a longer period for ascitic.
of time and kept their virulence for at (b) Stitt gave the following method of
least two months. preparation, of a medium for the
Variants In place of the kidney 2 or 3 small
: cultivation of treponemata.
pieces of buff coagulum cut in 1 cc. (1) Fit a test tube with a perforated
dimensions may be added. Buff co- rubber stopper which can be
agulum is a part of the blood clot. The pushed down into the tube.
clot separates into two layers, the cruor, (2) Pass a piece of glass tubing thru
consisting of red corpuscles and a buff the stopper to project slightly into
coagulum consisting of white corpuscles the test tube.
and blood platelets. These pieces of (3) Draw out the other end of the glass
buff coagulum must be pushed to the tube into a capillary tube and
bottom of the medium with a sterile bend at an acute angle.
glass rod. (4) Boil the apparatus.
Reference: Hata (1913 p. 109). (5) When cool a piece of sterile tissue is
dropped into the tube.

1367. Noguchi's Ascitic Fluid Tissue
(6) Draw a strip of gauze thru a glass
Medium
bead and soak the gauze in the
Constituents material to be cultured.
1. Ascitic fluid. (7) Drop (6) into the bottom of the
2. Tissue (rabbit). tube.
Preparation (8) Run in ascitic fluid to the point
(1) Place sterile pieces of tissue, usually where it would be reached by the
rabbit kidney, in test tubes 2 x 20 cm. bottom of the rubber stopper.
(2) After inoculation add quickly about (9) Push in the stopper as quickly as
15.0 cc. of sterile unheated unfiltered possible and when fluid appears in
ascitic fluid. the capillary tube seal off quickly
Sterilization : Materials are obtained under with a small flame.
aseptic conditions. References: Noguchi (1912 p. 201), Flexner
Use: Cultivation of spirochaetae, Spiro- and Noguchi (1913 p. 463), Noguchi
chaeta dutioni, Spirochaeta kochi, Spiro- (1916 p. 622), Rosenow and Towne (1917
chaeta ohermeieri, Spirochaeta novyi, p. 177),Loewe and Strauss (1920 p. 252),
treponemata and others. A few centi- Olitsky and Gates (1921 p. 715), Abbott
meters of citrated blood from the heart (1921 p. 635), Stitt (1923 p. 53).
of an infected rat or mouse was added to
the rabbit tissue before the addition of 1368. Kligler and Robertson's Ascitic Fluid
ascitic fluid.Incubate under anaerobic Egg Medium. (Stitt)
as well as aerobic conditions. Flexner
and Noguchi inoculated the medium Constituents
with a piece of cerebrum or other part of 1. Egg albumin 300.0 cc.
the brain or spinal cord of an infected 2. Ascitic fluid 100.0 cc.
animal to cultivate the organism causing Preparation
epidemic poliomyelitis. Noguchi iso- (1) ]\Iix three parts egg albumin with one
lated and cultivated Spirochaeta galli- part ascitic fluid (horse or rabbit
narum, inoculating the medium with a serum may be used instead).
blood emulsion containing the spiro- (2) Tube in tall tubes.
chaeta. Loewe and Strauss cultivated Sterilization: Not specified.

the organism causing epidemic en- Use: Cultivation of Borrelia recurrentis.
cephalitis. Olitsky and Gates isolated The medium was covered with a layer
and cultivated anaerobic organisms found of oil not greater than 1.5 cm. high.
in influenza, in the medium. Reference: Stitt (1923 p. 54).
401
References: Rettger (1907 p. 75), Tanner

1369. Bacto EggMeat Medium
(Dehydrated) (1919 p. 58).
Constituents 1371. Besredka's Egg Meat Infusion

1. Water 1000-0 cc. Medium (Bezangon)
2. Egg albumin
Constituents:
3. Beef 1-0 lb.
1. Meat infusion solution 1000.0 cc.
4. CaCOa :
5.0 g.
2. Egg.
Preparation:
Preparation:
(1) Suspend 150.0 g. of Bacto Egg-Meat
(1) Prepare a meat infusion solution (no
Medium (Dehydrated) in 1000.0 cc.
peptone or NaCl added).
of distilled water. (Each liter con-
1 pound of (2) To each 100.0 cc. of sterile (1) con-
tains the whites of 6 eggs, 20.0 cc. of a
tained in Roux flasks add
lean beef and 5.0 g. CaCOs).
sterile 10.0% egg white solution and
Distribute an even mixture in tubes.
(2)
from 5.0 to 20.0 cc. of a sterile 10.0%,
egg yolk solution under aseptic
20 minutes, pH = 7.2 .
conditions.
Sterilization: Sterilize 20 minutes at 15
Sterilization: Method not given. The in-
pounds, allowing autoclave to lose pres-
fusion, egg white and egg yolk solutions
sure slowly.
are sterilized separately.
Use: Cultivation of anaerobes.
Reference: Bezangon (1920 p. 546).
p. 11).
Egg-Meat Medium 1372. Picker's Sputum Serum Solution

1370. Rettger's
1000.0 cc. 1. Serum (beef) 200.0 cc.
1. Water
2. Sputum 100.0 cc.
2. Beef 1.0 lb.
3. Glycerol (2.0%) 6.0 g.
3. Egg white
CaCOa (0.5%) 5.0 Preparation:
4.
(1) Mix two parts sterile
beef serum with
Preparation:
Thoroly mix 1 pound of lean chopped one part sterile tuberculous or bron-
(1)
beef with 500.0 cc. of water, chitus sputa.
(2) Neutralize (1) with NasCOs. (2) Add 2.0% glycerolto (1).
Heat in the Arnold sterilizer for 30 Sterilization: Sterilize tuberculous or bron-

(3)
minutes, stirring occasionally. chitus sputa for an hour on 3 successive
Allow to stand for several hours and days in streaming steam. Method of
(4)
remove the fatty scum. sterilization of serum or glycerol not
Mix the whites of 6 eggs with 500.0 given.
(5)
cc. of water.
Neutralize (5). (Method not given). Reference: Ficker (1900 p. 508).
(6)
(7) Heat in the Arnold for 30 minutes 1373. Kayser's Blood Bile Solution
with occasional stirring to coagulate.
Mix (4) and (7) and place into a liter Constituents
(8)
1. Bile, beef 5.0 cc.
flask.
Blood.., 2.5 cc.
(9) Add 0.5% of powdered calcium car- 2.
bonate to (8).
Preparation:
to 112C. (1) Place 5.0 cc. of sterile beef bile into a
Sterilization: Sterilize at 110
sterile test tube.
in the autoclave for 30 minutes.
Add about 2.5 cc. of the patient's
Use: To study putrefaction by B. coli (2)
communis, B. lactic aerogenes, B. putri- blood that has been removed from a
bacillus of malignant oedema, finger tip or an ear under aseptic
ficus,
conditions.
bacillus of symptomatic anthrax. The
author replaced the air in the flask with
hydrogen gas. bile not specified.
Use: Enrichment medium for typhoid and Reference: Berman and Rettger (1918 p.
paratyphoid used in diagnosis by blood 382).
culture method,
Reference: Kayser (1906 p. 186), Klimmer 1376. Adams' Coke Milk Medium
(1923 p. 214). Constituents:
Milk.
1.
1374. Noguchi's Blood Serum Solution
Coke.
2.
1. Serum...., 100.0 cc. (1) Add sterile pieces of coke to skimmed
2. Ringer's Solution 300.0 cc. milk.
3. Plasma, citrated, rabbit 150.0 cc. Sterilization: Method not given in the
Preparation abstract.
(1) Mix 1 part rabbit serum with 3 parts Use: Cultivation of butyric acid bacilli.
Ringer's solution (see medium 180) Before use the oxygen was removed by
and 0.5 part of citrated rabbit plasma. heating and after inoculation with the
Sterilization: Not specified. fecal material the tubes were again
Use: Cultivation of Spirochaeta ictero- heated for 2 to 3 minutes at 70C. to
haemorrhagiae, Leptospira icterohaemor- eliminate the less resistant organisms.
rhagiae and other spirochaeteceae. The Reference: Adams (1921 p. 59). Taken
medium was covered with a layer of from (1921 p. 319).
paraffin oil following
inoculation with
suspected blood material. SUBGROUP I-C. SECTION 17
Variants Liquid media or basal solutions not

(a) The author used 0.9% NaCl solution containing digests, but containing extracts
instead of Ringer's solution. of ashes, soil or similar materials.
(b) The author cultivated Leptospira Ai. Ashed material employed.
icterohaemorrhagiae on a mixture of 1.5 Harrison and Barlow's Basal Wood
parts rabbit serum, 4.5 parts Ringer's Ash Medium . .
, 1377
solution and 1.0 part citrated plasma. Pasteur's Sucrose Yeast Ash Solu-
(c) Kaneko cultivated Spirochaeta ic- tion (Smith) 1378
terohaemorrhagiae and Spirochaeta A2. Soil or soil constituents employed.
hebdomadis on medium prepared as Bi. Humic acid or its salts used.
follows: Krzemieniewska's Glucose Humate
(1) Introduce a drop of dog blood into Solution (Vogel) 1379
2.0 to 3.0 cc. of dog serum diluted Sohngen's Salt Humate Solution... . 1380
with 1 to 5 parts Ringer solution. Bj. Soil infusions used.
Heat for 30 minutes at 56 to 58C.
(2) Ci. Containing no additional organic
(3) Cover with liquid sterile paraffin. nutrients.
References: Noguchi (1917 p. 761), (1918 p. Di. Inorganic nitrogen added.
606), Kaneko (1921-22 p. 354), Abbott Gutzeit's Ammonium Sulphate Soil
(1921 p. 635). Infusion 1381
Stoklasa's Nitrate Soil Infusion I 1382 . .
1375. Berman and Rettger's Casein Meat Stoklasa's Nitrate Soil Infusion II.. 1383
Extract Medium
.
D2. Inorganic nitrogen not added.

Constituents Lohnis and Pillai's Basal Soil In-
1. Water 1000.0 cc. fusion 1384
2. Casein 1.25 g. Buchanan's Basal Soil Infusion 1385
3. Liebig's meat extract 5.0 g. Saltet's Sulphite Mud Solution 1386
4. NaCl... 5.0 g. Lohnis' Soil Infusion Medium 1387
Preparation Zikes' Soil Infusion Medium 1388
(1) Dissolve 2, 3 and 4 in 1. Harvey's Soil Infusion Medium
1389
(2) Tube in 10.0 cc. quantities. C2. Containing additional organic nutrients
Sterilization: Not specified. Di. Organic nitrogen added.
Use: To study bacterial nutrition. Lohnis Urea Soil Infusion Medium.. 1390
L6hni3 and Moll's Asparagin Soil 20 parts of maltose, sucrose or

Infusion Medium 1391 dextrose (see table), maltose being
Perotti's Dicyandiamid Soil In- best.
fusion Medium 1392 (4) Heat steam and boil a

in flowing
Di. Organic nitrogen not added. moment over the flame or heat in
Gutzeit's Mannitol Soil Infusion the autoclave for 20 minutes or
Medium, 1393 more at 10 pounds steam pressure.
Jensen's Nitrate Soil Infusion This medium will vary faintly
Medium 1394 alkaline to phenolphthalein to
alkaline -0.6 according to the
1377. Harrison and Barlow's Basal Wood amount of ash used.
Ash Medium (5) If desired acid potassium phosphate
Constituents: may be added 2 to 5 parts per 1000

1. Distilled water 1000.0 cc. (see table) at any stage in the
2. Wood ashes 15.0 g. preparation but best perhaps after

Preparation: adding the sugar.
Add 15.0 g. wood ashes (maple wood, (6) Sterilize in flowing steam or in the
(1)
mixed beech and maple, elm and autoclave at 10 pounds steam pres-
tamarack give good results) to 1000.0 sure for 10 to 20 minutes.
cc. distilled water. The accompanying table shows the
(2) Heat in flowing steam, boil a minute percentages of constituents used and
and filter. the reaction.
Water
References: Harrison and Barlow (1907 Use: To study nitrogen assimilation by

pp. 267, 269), Percival (1920 p. 204), azotobacter. Vogel reported little differ-
Giltner (1921p. 377). ence in amount of nitrogen assimilated
whether K2SO4 be present or not. Pos-
1378. Pasteur's Sucrose Yeast Ash Solution
sibly there was slightly more nitrogen
(Smith)
assimilated per mg. of glucose utilized if
Constituents: the potassium salt was not added.
1. Distilled water 1000.0 cc. Variants: The author added 0.25 g. K.SO^
2. Ammonium tartrate 10.0 g. to the medium.
3. Yeast ash 10.0 g. Reference: Vogel (1911-12 p. 419).
4. Sucrose 100.0 g.
Preparation 1380. Sohngen's Salt Humate Solution
(1) Dissolve 2, 3 and 4 in 1 in the cold.
Constituents:
2. MgS04 0.5 g.
Variants
3. Bipotassium phosphate 0.5 g.
(a) Roux and Rochaix, and Bezangon,
4. NH4CI 0.5 g.
used 0.75 g. yeast ash and omitted
5. Sodium humate 1-0 g.
the ammonium tartrate.
Preparation
(b) Kolle and Wassermann used the ashes
from 10.0 g. yeast.
(c) Tanner, Besson, Bezangon, Dopter
Use: Cultivation of paraffin and petroleum
and Sacquepee, used 1.0 g. ammo-
oxidizers, Mycobacterium, B. fluorescens
nium tartrate and used 0.75 g. yeast
liquefaciens and Micrococcus paraffinae.
ashes.
Author reported that sodium humate was
References: Smith (1905 p. 197), Roux and
utilized by these organisms.
Rochaix (1911 p. 103), Kolle and Wasser-
Reference: Sohngen (1913 p. 603).
mann (1912 p. 393), Tanner (1919 p. 66),
Besson (1920 p. 36), Bezangon (1920 p.
and Sacquepee (1921 p.
1381. Gutzeit's Ammonium Sulphate Soil
115) Dopter
Infusion
121), Harvey (1921-22 p. 120).
Constituents
1379. Krzemieniewska's Glucose Humate
1. Water.. 1000.0 cc.
Solution (Vogel)
2. Soil
Constituents 3. (NH4)2S04 (1.0%) 10.0 g.
1. Water 1000.0 cc. 4. CaCOs (0.5%) 5.0 g.
2. MgS04-7H20 0.125 g. Preparation
3. CaHP04-2H20, 0.25 g. (1) Prepare soil extract.
4. Sodium humate 10.0 cc. (2) Add 1.0% (NH4)2S04 and 0.5%
5. Glucose 9.0 g. CaCOsto (1).
Preparation: Sterilization: Not specified.
(1) Prepare a sodium humate solution Use: To study denitrification.

by shaking 1000.0 g. of humus soil Variants: Lohnis prepared a similar me-
in a dilute solution of NaOH. dium as follows:
(2) Filter thru glass wool. (1) Heat 1000.0 g. of good rich garden
(3) Acidify the filtrate with HCl. soilwith a liter of tap water for 30
(4) Wash the precipitate until the wash minutes in the autoclave under pres-
water shows a very weak acid sure of 1 atmosphere or boil with 2
reaction. liters of water over a free flame.
(5) Dissolve the filtrate in weak NaOH (2) Pour the turbid liquid.
off
solution. (3) Mix with the liquid.

talc
(6) Dissolve 2, 3 and 5 in 1. (4) Filter thru a double filter paper.
(7) Add 10.0 cc. of (5) to (6). (5) Make up the volume to 800.0 cc. if
Sterilization: Not specified. necessary.

Dissolve 1.0% (NH 4)2804 and 0.5% 1384. Lohnis and Pillai's Basal Soil Infusion
(6)
K2HPO4 in (5). Constituents
(7) Add chalk after inoculation with soil.
1. Water 1000.0 cc.
not specified.
(8) Sterilization 2. Soil 1000.0 g.
Reference: Gutzeit (1906 p. 370), Lohnis 3. K2HPO4 (0.5%) 5.0 g.
(1913 p. 110). 4. CaCOs (1.0 to 2.0%). 10.0 to 20.0 g.
Preparation
1382. Stoklasa's Nitrate Soil Infusion I
(1) Add 1000.0 g. soil to 1 liter of water.
Constituents: (2) Place in a covered pot and autoclave
1. Distilled water for 30 minutes at 1 atmosphere pres-
2. Soil 9000.0 g. sure.
3. NaNO, 1-5 g.
(3) Filter. One obtains about 600.0 cc.
4. K2HPO4 10 g-
(4) To add 0.5% K2HPO4 and 1.0%
(3)
5. MgSOi 10 g- of the added nutrients.
Preparation (5) Add 1.0 to 2.0% CaCO,.
(1) Extract 9000.0 g. of soil with distilled Sterilization: Not specified.
water at 60 C. (Amount of water or Use: To study N assimilation. Author
time not specified.) reported that more nitrogen was assim-
(2) Concentrate the extract (19 liters) ilated with mannitol than glucose,
to 1 liter. This liter contains 2.2 g. of CaCOa favored the nitrogen assimilation
carbon in the form of soluble organic with sugar but not with the acids.
compounds. Variants: The author omitted the CaCOs.
(3) Add 1.5 g. NaNOa, 1.0 g. KjHPO* Added nutrients: The author added 1.0%
and 1.0 g. MgS04
to 600.0 cc. of (2). of one of the following:
Sterilization: Sterilize four times in the mannitol
steamer. glucose
Use: To study denitrification by Bad. tartaric acid (neutralized with soda)
Hartlebi. Author reported no deni-
Reference: Lohnis and Pillai (1907 p. 88).
trification.
1385. Buchanan's Basal Soil Infusion
Constituents
1383. Stoklasa's Nitrate Soil Infusion II 1000.0 cc.
1. Water
Constituents 2. Soil 100.0 g.
1. Distilled water Preparation
2. Soil 2500.0 g. (1) Boil 100.0 g. of rich garden soil
with 1
3. NaNOs 0.5 g. liter of water.

4. CaCOs 2.5 g. (2) Filter until clear.
Preparation (3) Dissolve 2.0% of one of the added
(1) Extract 2500.0 g. soil with distilled nutrients in (2).
water three times (15 liters of water in minutes on
Sterilization: Sterilize for 20
all). each of 3 successive days in streaming
(2) Evaporate to 160.0 cc. This extract steam.
contains 2.55 g. organic carbon per Use: To study gum production.
liter (determined by oxidation with Added nutrients and variants: The author
chromic acid). added 2.0% of one of the following:
(3) Add 0.5 g. NaNOs, containing 0.08 glucose sucrose
g. N and 2.5 g. CaCOa to 150.0 cc. levulose lactose
of (2). galactose mannitol
Sterilization: Sterilize four times in /the maltose
steamer. Reference: Buchanan (1909 p. 388).
Use: To study denitrification by Bad.
Author reported no deni- 1386. Saltet's Sulphite Mud Solution
Hartlebi.
trification. Constituents
Reference: Stoklasa (1907 p. 33). 1. Water (ditch).
2. Mud. (2) Filter.

3. NasSO,. (3) Add 0.3% KH2PO4 and 0.2% MgSOi-
(1) Mix ditch water and mud. Use: Cultivation of Apiculatus yeast, To-
(2) Distribute in test tubes. rula alba, Torvla Molischiana, Myco-
(3) To each tube of sterile (2) add two derma cerevisiae Blasioderma salmoni-
drops of a 10.0% NajSOs solution. color.
Sterilization: Sterilize (2) at 110C. Reference: Zikes (1911 p. 147).
Use: To study sulphite reduction by B.
desulfuricans 1389. Harvey's Soil Infusion Medium
Variants The author added two drops of a
:
10.0% Na2S20j solution to each tube Constituents:

instead of NajSOa- 1. Water (tap) 1000.0 cc.
Reference: Saltet (1900 p. 697). 2. Soil lOC.O g.
3. K2HPO4 0.5 g.
1387. Lohnis' Soil Infusion Me4ium Preparation
(1) Boil 100.0 g. of rich garden soil in
Constituents
200.0 cc. of water.
2. Soil
(2) Pour ofT the turbid liquid and add a
1000.0 g.
little talc.
Preparation
Heat 1000.0 g. of good rich garden (3) Filter until clear thru thick filter
(1)
paper.
soil with a liter of tap water for 30
minutes in the autoclave under pres- (4) Add 900.0 cc. of tap water and 0.5 g.
sure of 1 atmosphere or boil with 2

K2HPO4 to 100.0 cc. of (3).
liters of water over a free flame.
Pour Use: Cultivation of flagellates and ciliates

(2) off the turbid liquid.
Mix talc with the liquid.
from the soil.
(3)
(4) Filter thru a double filter paper.
(5) Make up the volume to 800.0 cc. if
necessary. 1390. Lohnis Urea Soil Infusion Medium

Constituents
Use: Cultivation of soil microorganisms.
1. Water 1000.0 cc.
Variants
2. Soil 1000.0 g.
(a) Sherman boiled one part soil with 3
3. K2HPO4 (0.05%) 0.5 g.
parts water, filtered until clear and
4. Urea (5.0%,) 40.0 g.
then added a small excess of CaCOs.
Preparation
Hereported that this medium was
Prepare soil infusion as indicated in
(1)
especially adapted for the cultivation
medium 1387.
of flagellates, ciliates and amoebae.
(2) Dissolve 0.05% K2HPO4 and 5.0%
(b) Giltner used 2 liters of water to 1000.0
urea in (1).
g. soil.
References: Lohnis (1913 p. 101), Sherman
(1916 p. 42), Giltner (1921 p. 370), Harvey
utilizing urea.
(1921-22 p. 105), Cunningham (1924 p.
Reference: Lohnis (1913 p. 108), Giltner
136).
(1921 p. 374).
1388. Zikes' Soil Infusion Medium
1391. Lohnis and Moll's Asparagln Soil
Constituents
Infusion Medium
1. Water 1000.0 cc.
2. Soil 100.0 g. Constituents:
3. KH2PO4 (0.3%) 3.0 g. 1. Water 1000.0 cc.
4. MgS04 (0.2%) 2.0 g. 2. Soil 500.0 g.
Preparation 3. K2HPO4 (0.5%) 5.0 g.
(1) Extract 100.0 g. of soil with 1000.0 4. Glucose (0.1%) '.
1.0 g.
cc. water by boiling. 5. Asparagin (0.1%) 1.0 g.
Preparation: 1393. Gutzeit's Mannitol Soil Infusion
Allow 50.0% soil to stand in water for Medium

(1)
20 hours and then filter. Constituents
(2) Dissolve 2, 3 and 4 in (1). 1. Water 1000.0 cc.
Sterilization: Medium may or may not be 2. Soil
sterilized. 3. Mannitol (1.0%) 10.0 g.
Use: Cultivation of organisms capable of Preparation

utilizing calcium cyanamide. (1) Dissolve 1.0% mannitol in a soil
Variants infusion.
(a) The authors added 0.014 g. of calcium Sterilization: Not specified.
cyanamide. Use: Gutzeit used the medium to study

(b) Lohnis prepared the medium by denitrification. Lohnis and Cunning-
dissolving 0.2% calcium cyanamide, ham used similar media for the enrich-
0.01% glucose, 0.05% K2HPO4 and ment of nodule bacteria and azotobacter.
0.01% asparagin in soil infusion pre- Variants: Lohnis added 1.0% mannitol and
pared as in medium 1387. 0.05% K2HPO4 to soil infusion (see
medium 1387) and sterilized in the auto-
References: Lohnis and Moll (1907 p. 265),
clave.
Lohnis (1913 p. 108).
References: Gutzeit (1906 p. 370), Lohnis
(1913 p. 112), Cunningham (1924 p. 155).
1392. Perotti's Dicyandiamid Soil Infusion
Medium 1394. Jensen's Nitrate Soil Infusion
Medium
Constituents:
2000.0 cc. Constituents
1. Water
1000.0 g. 1. Water 1000.0 cc.
2. Garden soil
2. Soil 500.0 g.
3. Dicyandiamid 2.0 g.
4. K2HPO4 0.5 g.
3. NaNOa 20.0 g.
4. Glycerol
5. Glucose 0.1 g.
Preparation:
Preparation
(1) Add 1000.0 cc. of 2.0% solution of
(1) Boil 1000.0 g. garden soil with 200.0
NaNOs to 500.0 g. of soil that has
water for 2 hours.
cc. of
been passed thru a 0.5 mm. sieve.
(2) Concentrate the liquid a little and
(2) Add 0.0, 5.0, 20.0 or 50.0 g. of glycerol
filter.
to(l).
(3) Dilute until the filtrate contains
0.4% mineral substances.
Use: To study denitrification by soil forms.
(4) Adds, 4 and 5 to (3).
Author reported that the addition of
Sterilization: Not specified. glycerol aided denitrification. No deni-
Use: Cultivation of organisms utilizing trification occurred without glycerol at
dicyandiamid from the soil. the end of 14 days.
Reference: Perotti (1908 p. 207). Reference: Jensen (1897 p. 690).
GROUP II. LIQUEFIABLE AGAR MEDIA
SUBGROUPS OF LIQUEFIABLE AGAR under one atmosphere pressure for
MEDIA 15 minutes.
Ai. Water and agar only. ^^^- Cultivation amoebae, algae, para-
of
Subgroup II A. (Med. 1395-1401) ^^*^^ fiagellata ciliata. and

Frost used a
similar medium to study the antagonistic
Aj. Water and agar with other constituents.
Bi. All other constituents inorganic. ^^^'^ ^ bacteria.
Variants:
Subgroup II B. (Med. 1402-1430)
B2. Containing one or more organic con- ^^^ ^^^* studied the antagonistic action
stituent (other than agar). between saprophytic organisms and
Subgroup II C. (Med. 1431-2198) ^- ^yPôsus in medium prepared by
the following method:
SUBGROUP II-A (1) Dissolve 2.0% agar in water.
(2) Sterilize.
Media Consisting of Water and Agar (3) Pour in deep Petri dish to cool.
Only (^) When solid, cut in little squares
with sterile knife.
Basal or complete media containing agar gtab with needle seeded with organ-
(5)
and water only. Here may be included jsm to be studied.
methods used for purifying agar for use in (g) Seal top of block with red hot nail
media. It is rarely used without the addi- head
tion of other constituents.
(7) pi^ce by means of sterile forceps
Ai. Agar and water only. (Agar not into broth culture of other
P""fied.) organism.
Tischutkin's Agar Solution 1395 Frost reported that if there was antag-
Pringsheim's Basal Agar 1396 onistic action, no growth occurred in
A2. Agar purified by washing with water. the block.
Beijerinck's Basal Agar Solution (b) Walker cultivated parasitic fiagellata
(Si*h) 1397 and ciliata in a 2.0% agar solution
Ayers, Mudge and Rupp's Washed adjusted to an alkalinity of 1.0% to
^g^^ 1398 phenolphthalein.
As. Agar purified by washing with alco- References: Tischutkin (1897 p. 185), Frost
hoi, acids, etc.
(19Q4 p go4). Walker (1908 p. 490).
Dominikiewicz's Purified Agar 1399
Cunningham's Purified Agar 1400 ^,r^< r, , u , -c a
TT 7 T. -c J A , .^, 1^96. Pringsheim's Basal
, 1
Agar
Harvey's Purified Agar 1401
1395. Tischutkin's Agar Solution

^TwZT. '. 1000.0 cc.
Constituents: 2. Agar 2.5 g.
1. Water 1000.0 cc. Preparation: Dissolve 2 and one of the
(1)
2. Agar (1.0%) 10.0 g. added nutrients in 1.
Preparation: Sterilization: Not specified.
(1) Dissolve 1.0% agar (best quality) in Use: To study nitrogen assimilation by
water by steaming in the autoclave Clostridium americanum and Azotobac-
under 2 atmospheres pressure for ter chroococcum with Bacillus gelaticus.
10 minutes. Author reported that Bacillus gelaticus
(2) Filter thru paper. with Clostridium americanum assimilated
(3) Tube. more nitrogen than did Bacillus gelaticus
Sterilization: Sterilize in the autoclave with Azotobacter chroococcum.
408
Added nutrients and variants: The author (3) Pour off as much water as possible by
prepared one of the following media, placing a piece of cheese cloth over
using water and varying amounts of agar the top of the flask.
as a base: (4) Add make up
fresh distilled water to
(a) 2.5 agar and 0.5 g. glucose

g. per for water poured off.
1000.0 cc. water. (5) Allow to soak for another 24 hours.
(b) 2.0 g. agar and 0.05 g. (NHOaPOj per (6) Filter thru a cotton flannel cloth and
1000.0 cc. water. wash once with a liter of distilled

(c) 5.0 g. agar + 1.0 g. mannitol per water.
1000.0 cc. water. (7) Allow the agar to drain as much as
(d) 5.0 g. agar +
0.4 g. mannitol per possible. Press out the remaining
1000.0 cc. water. water by squeezing the filter cloth
Author used Bacillus gelaticus with with the hands.
Clostridium americanum with (a) and (8) The washed agar may be dissolved
(b), and Bacillus gelaticus with Azoto- in water for the preparation of media
bacter chroococcum in solution (c) and (d). or dried in air and used as ordinary
Reference: Pringsheim (1910 p. 230). agar. In this case use 1.0% of the dry
material to obtain a medium having
Basal Agar Solution the same jelly strength as a 1.5%

1397. Beijerinck's
shred agar medium.
(Smith)
Constituents: Use : Purified agar. Giltner used a similar
1. Distilled water. medium the preparation of a solid
in
2. Agar. synthetic medium and for testing food
Preparation: requirements and selective power of
(1) Add ordinary agar to distilled water bacteria.
and heat until solution is complete. Variants: Giltner used the agar, prepared
(2) Pour into Erlenmeyer flasks and in the following manner:
allow to solidify.
(1) Place 15 parts agar in 1000.0 parts
(3) When cold, fill the flasks with dis- distilled water.
tilledwater and set away.
(2) Cover the mouth of the bottle with
(4) Change the water several times, parchment paper, or several layers
during a lapse of one or two weeks. of clean cheese cloth.
(5) Add any desired inorganic nutrients
(3) Allow to ferment spontaneously.
and some pure precipitated CaCOa. (4) Change the water in the bottle occa-
Sterilization: Method not given. sionally, replacing the amount re-
Use: Cultivation of nitrite bacteria. moved with the same amount of
Added nutrients: Smith specified the addi- clean distilled water.
tion of any solution of nutrient salts
(5) When active fermentation has ceased
containing ammonium salts and states as noted by the evolution of gas
that Beijerinck recommended NH4, place the agar in an agate ware pail.
NaHP04'H20 as the best ammonium (6) Weigh.
salt to be used. Boil over a free flame to dissolve the
(7)
Reference: Smith (1905 p. 199). agar.
(8) Make up any loss in weight by the
1398. Ayers, Mudge and Rupp's Washed
addition of distilled water.
Agar
Constituents: (10) Autoclave.
1. Distilled water 2000.0 cc. References: Ayers, Mudge and Rupp (1923
2. Agar 30.0 g.
p. 591), Giltner (1921 p. 365).
Preparation
(1) Place 30.0 g. of shredded agar in a
1399. Dominikiewicz's Purified Agar
flask with 2000.0 cc. distilled water. Constituents
(2) Allow to stand for 24 hours at room 1. Water 200.0 to 400.0 cc.
temperature. 2. Agar 30.0 to 40.0 g.
Preparation :
(9) Cool by placing the tins in cold
(1) Dissolve 30.0 to 40.0 g. of agar in water. The agar sets into a very
200 to 400.0 cc. of water. firm jelly which is cut into slices
(2) Add slowly, 700-800 cc. of alcohol which are broken up by a meat
that has been acidified with mincing machine with a very fine
CH3COOH. bore.
(3) Shake the mixture thoroly and allow (10) Place the small vermicular threads
the precipitated agar to settle. obtained from (9) in trays, and
(4) Pour off the alcohol and place the agar desiccate in a drying oven at a
precipitate on a Biicher funnel. moderate heat. When the threads
(5) Wash the precipitate until the wash are dry they have a light brown
water is neutral. color.
(6) The agar may be washed on a piece of Use: A purified agar. A 1.0% solution of
strain cloth instead of a Biicher the agar gives a clear firm medium.
funnel. Variants: The threads may be milled into
(7) After the alcohol has been removed a fine powder.
from the agar, the agar is dried at Reference: Cunningham (1918-19 p. 561).
100C.
(8) The dried agar is a snow white
1401. Harvey's Purified Agar.
powder.
Use: A purified form of agar. When this Constituents
powdered agar is used in the preparation 1- Water 200.0 g.
of various media, it requires no filtration. 2. Agar loO.O g.
Reference: Dominikiewicz (1908 p. 668). Preparation
(1) Mix 1.0 part fibre agar, 0.05 part
1400. Cunningham's Purified Agar
glacial acetic acid and 2.0 parts water.
Constituents (2) Allow the agar to soak in the acidified
1. Water. water 15 minutes.
2. China grass. (3) Remove the fibre agar and wash
Preparation thoroly with water until quite free
(1) Weigh a quantity of china grass from any trace of acid reaction to
and place in a vessel containing suffi- litmus paper.
cient dilute acid (0.01% H2SO4 gives (4) Squeeze in a cloth to get rid of excess
the best results) to completely cover of water.
the fibre. Use: Used in the preparation of media, to
(2) Soak in the acid for 10 minutes. give a 3% solution of agar, calculating
(3) Remove and wash in running water from the weight of the agar in the original
until all traces of acid have been dry condition.
removed. Both the fibre and wash- Variants Harvey treated the agar obtained
:
ings are to be alkaline to litmus. above as follows:

(4) Express the superfluous water by (1) Place the fibre on a filter funnel fur-
placing the fibre in a stout cloth and nished with moistened cotton wool
subjecting it to a pressure by twist- for filtration.
ing the ends in opposite directions. Note: The filtering wool is sup-
(5) Place a layer of cotton wool on a ported on butter muslin and the
layer of butter muslin. Moisten free edges of the latter are turned
and remove the superfluous water over the rim of the funnel and tied
by careful wringing. there. This prevents the wool from
(6) Fit (5) to the filter funnel with the sinking down into the narrow part of
cotton up. the funnel and blocking the fil-
(7) Pile the agar (4) on the funnels, and tration.
then the funnels are placed in a posi- (2) Place the filter funnel with agar in
tion over a series of tiltering tins. an autoclave over a collecting re-
(8) Place in the autoclave, and autoclave ceptacle.
at 120C. for 1 hour. (3) Keep one hour at 120C.
411
now 1402. Davis' Sodium Chloride Agar

(4) Cool rapidly the receptacle,
containing the cleaned filtered agar. Constituents;
(5) Remove the agar when set. 1. Water 1000.0 cc.
(6) Mince finely by passing the agar 2. Agar 15.0 g.
thru a mincing machine furnished 3. NaCl 5.0 g.
with a disc having small holes. Preparation: Dissolve 2 and 3 in
(1) 1.
(7) Spread the vermicular threads ob- (Pure or impure sugars may be added.)
tained in shallow layers on trays. Sterilization: Not specified.
(8) Desiccate in a drying oven at Use: To produce chlamydospores by Sporo-
moderate temperature. thrix schenckii. Smith and Smillie used
(9) Reduce the desiccated material to a similar medium to obtain coccidia
powder. spores from sparrow feces. Teague and
(10) Preserve in the dry state. Deibert studied the constituents neces-
(11) Use for the preparation of media in sary for the growth of Unna-Ducrey's
a strength of one per cent. bacillus and reported no growth on the
Reference: Harvey (1921-22 p. 66, 67). medium used.
Variants: Smith and Smillie, and Teague
SUBGROUP II-B and Deibert dissolved 2.0% agar in a
0.5% aqueous NaCl solution.
Agar Media with All Other Constit- References: Davis (1914 p. 485), Smith and
Smillie (1917 p. 415), Teague and Deibert
uents Inorganic
(1922 p. 70).
Basal or complete media containing agar; Agar
1403. Beijerinck's Thiosulphate
all other constituents inorganic.
Constituents
Key to the Sections of Subgroup II B 1. Water 1000.0 cc.
2. NaoSaOs, 5H2O 5.0 g.
Ai. Nitrogen present as free or elementary
3. K2HPO4 0.1 g.
nitrogen.... Section 1 (Med. 1402-1410)
4. NaHC03 0-2 g.
A2. Nitrogen supplied as ammonium salts
5. Agar 20.0 g.
Section 2 (Med. 1411-1424)
A3. Nitrogen supplied as nitrites or ni-
trates Section 3 (Med. 1425-1430)
Use: Isolation of Thiobacillus denitrificans
and T. thioparus.
SUBGROUP II-B. SECTION 1
Reference: Beijerinck (1903-04 p. 599).
Basal or complete media containing agar
1404. Gordon's Basal Salt Agar
with inorganic salts. Nitrogen present as
free or atmospheric nitrogen only. Constituents:
Ai. Containing salts of monovalent cations 1.
only. 2. K2HPO4 (0.1%) 1.0 g.
Davis' Sodium Chloride Agar 1402 3. MgS04 (0.2%) 2.0 g.
Beijerinck's Thiosulphate Agar 1403 4. CaCl. (0.01%) 0.1 g.
As. Containing salts of monovalent and 5. NaCl (0.5%) 5.0 g.
other cations. 6. Agar (3.0%) 30.0 g.
Bi. Calcium salts added. Preparation

Gordon's Basal Salt Agar 1404 (1) Dissolve 2, 3, 4, 5 and 3.0% of a
Carrel and Burrows' Locke's Solu- thoroughly washed and then dried
tion Agar 1405 agar in 1.
Omeliansky's Basal Carbonate Agar. 1406 (2) Dissolve one of the added nutrients
Ashby's Salt Agar (Murray) 1407 in (1).
Winogradski's Salt Agar (Murray) . . 1408 Sterilization: Not specified in the abstract.
B2. Calcium salts not added. Use: To determine nitrogen requirement of

Liot's Basal Salt Agar 1409 some of the commoner pathogenic bac-
Dox's Salt Agar (Tanner) 1410 teria.
412 CULTURE MEDIA FOR CULTIVATION OF MICROROGANISMS
Added nutrients: Gordon added various Use: To study nitrogen fixation and to
nitrogen compounds. The compounds determine number of organisms in soil.
used or concentration not specified. Used both anaerobically and aerobically.
Reference: Gordon (1917 p. 371) taken from Reference: Murray (1916 p. 608).
(1917 p. 299).
1408. Winogradski's Salt Agar (Murray)
1405. Carrel and Burrows' Locke's Solution
Constituents:
Agar
1. Water 1000.0 cc.
Constituents: 2. K.HPO4 1.0 g.
1, Locke's solution 1000.0 cc. 3. MgS04 3.0 g.
'
2. Agar (2.0%) 20.0 g. 4. NaCl 0.01 g.
Preparation: (1) Dissolve 2.0% agar in 5. MnS04 0.01 g.
Locke's solution (see medium 6 for 6. CaCO, 10.0 g.
Locke's solution). 7. FeCIa (10% soln.) 2 drops
Sterilization: Not specified. 8. Agar 15.0 g.
Use: Cultivation of tissue in vitrio. Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7
Author reported that the medium did not and 8 in 1.
support growth nearly as well as did Sterilization: Not specified.
plasma. Use: lo study nitrogen fixation and to
Reference: Carrel and Burrows (1911 determine the number of bacteria in the
p. 245). soil. Used anaerobically and aerobi-
cally.
1406. Omeliansky's Basal Carbonate Agar Reference: Murray (1916 p. 608).
Constituents
Water 1409. Liot's Basal Salt Agar
1. 1000.0 cc.
2. Agar 15.0 g. Constituents:
3. NaoCOs 1.0 g. 1. Double distilled water 1000.0 cc.
4. K2HPO4 trace 2. Sodium phosphate 5.0 g.
Preparation: Dissolve 2,
(1) 3, 4 and one of 3. MgS04 2.5 g.
the added nutrients in 1. 4. Agar 25.0 g.
Use: To study oxidation of sulphites and (1) Macerate 25.0 g. of commercial
phosphites. The author reported that agar in 750.0 cc. of double distilled
neither the phosphite nor sulphite were water for 48 hours.
oxidized. (2) Dissolve in the autoclave at about
Added nutrients: The author added 0.3 g. 100C.
of one of the following: (3) Filter while hot on a Chardin filter
KNO2, KNO3, Peptone. paper.
Variants: The author added 0.0 or 2.0 g. (4) Dissolve 5.0 g. sodium phosphate in
of NaoSOa or NaaHPOj. 125.0 cc. of double distilled water.
Reference: Omeliansky (1902 p. 64). (5) Dissolve 2.5 g. MgS04 in 125.0 cc.
double distilled water.
1407. Ashby's Salt Agar (Murray)
(6) Add sterile (4) to (3). Mix well.
Constituents (7) Add sterile (5) to (6). Mix well.
1. Water 1000.0 cc. (8) Tube in 10.0 cc. quantities.
2. K2HPO4 0.2 g. (9) When ready for use add 0.5 cc. of a
3. MgS04 0.2 g. 1.0% sterile solution of one of the
4. NaCl 0.2 g. added nutrients to each sterile tube.
5. CaS04 0.2 g. (The sodium phosphate and MgS04
6. CaCOj 5.0 g. may be omitted, leaving a non-
7. Agar 15.0 g. mineral agar.)
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 Sterilization: Sterilize (4) and (5) at 115
in 1. for 15 minutes. Sterilize (8) in the usual
Sterilization: Not specified. manner. (Method not given.) Method of
413
sterilization of the added nutrients not Fremlin's Ammonium Sulphate Agar. 1417
Nencki, Sieber and Wynikiwicz's
given.
Ammonium Sulphate Agar 1418
pyocyanius bacilli. Gowda's Ammonium Sulphate Agar. 1419
Heinemann's Ammonium Sulphate
Added nutrients: The author added 0.5 cc. 1420
1.0% solution of one of the following Agar
of a
Committee S. A. B. Basal Ammo-
to each 10.0 cc. of the medium:
nium Phosphate Agar 1421
Ammonia salts of organic acids
A Ammonia
4. present as salts of phos-
Amides
Monobasic acids phoric acid.
Beijerinck's Sodium Ammonium
Polybasic acids
Phosphate Agar. - 1422
Amines
Ammonia salts of inorganic acids Stutzer's Ammonium Magnesium
Phosphate Agar 1423
Polyatomic alcohols
Monosaccharides Carper's Basal Ammonium Phos-
phate Agar 1424
Polysaccharides
Monoses 1411. Beijerinck's Thiosulphate Ammonium
Reference: Liot (1923 p. 238). Chloride Agar
1410. Dox's Salt Agar (Tanner) Solidify Beijerinck's Thiosulphate Solu-

tion, see medium 76 by the addition of
Constituents
1. Distilled water 3000.0 cc. 2.0% agar.
Waksman's Thiosulphate Ammonium

2. MgS04 1-^ g-
1412.
3. K2HPO4 3.0 g.
Chloride Agar
4. KCl 1-5 S-
0.03g. Constituents:
5. FeS04 cc.
Distilled water 1000.0
solidify 1.
6. Agar to
Preparation 2. NaoS203-5H20 5.0 g.
2, 3, 4 and 5 in 1. 3. KH2PO4 3.0 g.

(1) Dissolve
Solidify by the addition of agar. 4. NH4CI 0.1 g.
(2)
Sterilization: Not specified. 5. MgCh 0.1 g.
CaCU 0-2-5g-
6.
Use: Cultivation of fungi.
Agar 20.0_g.
Reference: Tanner (1919 p. 65). 7.
Preparation: Dissolve 6 and 7 m 1.
2, 3, 4, 5,
SUBGROUP II-B. SECTION 2 Sterilization: Sterilize at 15 pounds pres-
agar sure for 15 minutes.

Basal or complete media containing Th.
supplied Use: Cultivation and isolation of
with inorganic salts. Nitrogen
thiooxidans. Author reported that
this
as ammonium salts.
organism can grow at pH = 1.0.
Maxi-
Ai. Ammonia present as ammonium chlo-
ride.
mum pH was between 3.0 and 4.0.
Thiosulphate Ammo- Reference: Waksman (1922 p. 607).
Beijerinck's
nium Chloride Agar 1411
1413. Beijerinck and van Delden's Ammo-
Waksman's Thiosulphate Ammo- nium Chloride Agar
1412
nium Chloride Agar
Constituents:
Beijerinck and van Delden's Ammo- 1000.0 cc.
1413 1. Distilled water
nium Chloride Agar 15.0 g.
as ammonium 2. Agar
A2. Ammonia present 0.1 g.
3. K2HPO4
nitrate. 0.1 g.
Ammonium Agar. 1414 4. NH4CI
Munter's Basal .
.
Dissolve 2, 3 and 4 in 1.
Ammonium Nitrate Preparation: (1)
Beijerinck's
.--"IS Sterilization: Not specified.
Agar oligocarbophilus.
present as ammonium Use: Isolation of Bacillus
Ammonia
Author reported that colonies were
A3. dry,
sulphate. agar
Ammonium vSulphate snowv white or rose colored. If the
Pesch's Basal with straw or an
1416 plate" be inoculated
Agar
impure culture of the organism, a pure

culture may be obtained.
(1903 p. 37).
1414. Mijnter's Basal Ammonium Nitrate

Agar
Constituents:
1. Water 1000.0 cc.
2. MgS04 0.5 g.
3. NaCl 0.5 g.
4. K2HPO4 1.5 g.
5. NH4NO3 1.5g.
6. CaCU 0.2 g.
7. FeClj trace
8. Agar 1.25% 12.5 g.
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and one of
(2) Make by the addi-
slightly alkaline
Na2C03.
tion of
Use: Cultivation of actinomycetes. The
author reported that oxalic acid, tartaric
acid and hippuric acid were not suitable
carbon sources for the actinomycetes
studied. The remaining acids were gen-
erally fair carbon sources for most of the
actinomycetes studied.
the following materials:
acetic acid aspartic acid
lactic acid citric acid
oxalic acid hippuric acid
succinic acid uric acid
malic acid humus acid
Reference: Miinter (1913 p. 371).
1415. Beijerinck's Ammonium Nitrate Agar
Constituents:
2. Agar
3. NH4NO3
4. Potassium phosphate..,
5. MgS04
6. CaCh
Preparation
(1) Wash agar with distilled water for
some time to separate the soluble
material.
(3) Dissolve 2.0% (dry) (1) in (2).
(4) Pour into plates.
415
lOg. Reference: Nencki, Sieber and Wyzni-

4. MgCO3(1.0%).
Agar 15.0 g. kiewicz (1898 p. 530).
5.
Preparation:
1419. Gowda's Ammonium Sulphate Agar
(2) Add 5 and boil.

Constituents:
(3) Tube. 1. Conductivity water.
(4) Add 1.0% sterile MgCOa to sterile
2. K2HPO4.
(3). 3. (NH4)2S04.
Sterilization: Method of sterilization of (3)
4. MgS04.
or MgCOa not given. 5. FeS04.
Use: Isolation of nitroso bacteria and to 6. NaCl.
study nitrogen oxidation by them.
7. NajCOa.
Author reported that colonies appeared, 8. Agar.
when eye was nearly on the level with the Preparation
colony, as a colorless, ground glass mass Soak agar in water for 8 to 10 days.
(1)
1.0 mm. or more in diameter. Colonies
(2) Dissolve agar in water.
in the depth of medium appeared as Pour in plates, and while still liquid
(3)
points to the naked eye. add the salts as indicated in the table.
Variants: Gage prepared the medium as (4) The table at top of page 416 gives the
follows: amounts of salts used, sterilization,
Wash agar agar ten times in water added to each
(1) and amount of solution
to remove all nitrogenous material.
agar plate.
Dissolve (NH4)2S04, 1.0 g.
1.0 g.
(2) Sterilization: Indicated in table.
potassium phosphate and 15.0 g. of Use: To study nitrification.
(1) in 1000.0 cc. of nitrite
free water.
Reference: Gowda (1924 p. 253).
(3) Dissolve 1.0 g. MgCOs in 100.0 g.
nitrite free water. 1420. Helnemann's Ammonium Sulphate
and (3) separately.
(4) Sterilize (2) Agar
(5) Mix (2) and (3) under aseptic con-
Constituents:
ditions.
References: Fremlin (1903 p. 375), Gage 1.
2. FeS04 0.4 g.
(1910 p. 15).
3. MgSOi 0.5 g.
1418. Nencki, Sieber and Wyzniklewlcz's 4. K2HPO4 10 g.
Ammonium Sulphate Agar 5. NaCl 2.0 g.
6. (NH4)2S04 10 g.
Constituents 20.0 g.
1000.0 cc.
7. Agar
1. Water 10.0 g.
10.0 to 15.0 g.
8. CaCOa,
2. Agar
Preparation
3. K2HPO4. 0.5 g.
(1) Dissolve 2, 3, 4, 5 and 6
in 1.
NajCOa 1.0 g.
4.
(2) Dissolve 20.0 g. of agar
in (1).
(NH4)2S04 2.5 g.
5.
(3) Add 10.0 g. precipitated
CaCOs to (2).
6. NaCl 5.0 to 10.0 g.
(4) Shake well.
Preparation
(5) Tube.
Soak 10.0 to 15.0 g. agar with 2 or 3
(1)
times the volume of distilled water.
Use: To study nitrification.
(2) Dissolve the agar in 1000.0 cc. of hot
water.
(3) Adds, 4, 5 and 6 to (2). Committee S. A. B. Basal Ammonium
1421.
(4) Filter. Phosphate Agar
Constituents:
Use Study cause of cattle plague. Author
:
Water 1000.0 cc.

reported that medium when inoculated
1.
^
KCl 0.2 g.
with cattle plague containing material
2.
two days showed 1-3m round cells. 3. NH4H2PO4 10 g.

after
(3) Pour distilled water over the solidified 3. Ammonium phosphate 3.0 g.
agar and allow to stand for several 4. Agar 30.0 g.
days. Pour off this water and add Preparation
fresh distilled water from time to (1) Dissolve 2, 3 and 4 in 1 by boiling.
time. This process removes all solu- (2) Neutralize to litmus by the addition
ble material from the agar. of NaOH.
(4) Add 0.2 to 0.5% NH4XaHP04 and (3) Add one of the added nutrients listed
0.05% KCl to the agar, also pre- below.
cipitated CaCOs (amount not given). Sterilization: Sterilize in the autoclave.
(5) Boil. (This serves to sterilize.) Use: Cultivation of tubercle bacilli.
(6) Distribute into sterile dishes. Added nutrients: The author added one of
Sterilization: Sterilization is accomplished the following:
in step (5) above. (a) Glycerol 5.0%
Use: To show nitrite formation by Amoeba (b) Glycerol 5.0%
nitrophil. Author reported that the Beef extract (Liebig's) 0.3%
colonies producing nitrites formed a (c) Glycerol 5.0%
clear ring around the colonies due to the Peptone (Witte) 1.0%
action of nitrites on CaCOa. Killer culti- (d) Glycerol 5.0%
vated protozoa and other soil forms on a Beef extract (Liebig's) 0.3%
similar medium. Peptone (Witte) 1.0%
Variants: Killer specified the use of 0.2% (e) Glycerol 5.0%
NH,NaHP04 4H2O and omitted the
Add 1.0% defibrinated rabbit blood
CaCOs. at 43C. and inspissate on each of
References: Beijerinck (1896 p. 258), Killer 3 successive days for 90 minutes.
(1913 p. 523). (f) Same as (e) but using laked blood.
(g) Same as (e) but using cow's milk,
1423. Stutzer's Ammonium Magnesium Same
(h) as (e) but using 10.0% casein.
Phosphate Agar
(i) Same as (e) but using whole egg.
Constituents: (j) Same as (e) but using egg white.
1. Water ^,
1000.0 cc. (k) Glycerol 5.0%
2. Agar
'.
10.0 g. Add 1.0% sodium nucleinate, pre-
3. K2HPO4 1.0 g. pared by neutralizing yeast nu-
4. NaCl 5.0 g. cleic acid in warm water by means
5. FeS04 0.5 g. of Na2C03 solution. Inspissate as
6. Ammonium magnesium in (e).
phosphate 20.0 g. (1) Glycerol 5.0%
7. MgCOa 20.0 g. Adda suspension of yeast nuclein
Preparation: make a 1.0% suspen-
sufficient to
(1) Dissolve 2, 3, 4 and 5 in 1. sion. Inspissate as in (e).
(2) Mix equal amounts of 6 and 7 in a (m) Glycerol 5.0%
mortar. Add 1.0% of a mush of testes from
(3) Add about a half teaspoon of (2) to rabbits. Inspissate as in (e).
test tubes. (n) Same as (m) but use liver from rab-
(4) Add 10.0 to 15.0 cc. of melted (1) to bits instead of testes.
each tube. (o) Same as (m) but use brain from rab-
Sterilization: Method not specified. bits instead of testes.
Use: To study nitrification by soil forms. Reference: Corper (1919 p. 463).
1424. Corper's Basal Ammonium Phosphate SUBGROUP II-B. SECTION 3
Agar
Constituents with inorganic constituents. Nitrogen sup-
1. Water, tap 1000.0 cc. plied as nitrites or nitrates.
2. Salt (sea salt) 5.0 g. Ai. Nitrogen supplied as nitrites.
Winogradsky's Sodium Nitrite Agar

(Fraenkel) 1425
Sodium Nitrite Agar
Stutzer's 1426
Heinemann's Sodium Nitrite Agar. . 1427
A2. Nitrogen supplied as nitrates.

Trautwein's Thiosulphate Nitrate
Agar..., 1428
Beijerinck and van Delden's Nitrate
Agar 1429
Noyes' Ammonium Nitrate agar... 1430
1425. Winogradsky's Sodium Nitrite Agar

Constituents:
1. Water. 1000.0 cc.
NaNOj (Merck) 2.0 g. 1000.0 cc.
2.
3. Soda (water free) 1.0 g.
5. Agar agar 15.0 g.
Preparation:
(2) Dissolve a knife point of potassium

phosphate in (1).
Use: To study nitrification by nitrifying

organisms. Stutzer and Hartleb used a
similar medium for the cultivation of the
supposedly foot and mouth disease
bacteria.
Variants
(a) Stutzer and Hartleb used a medium
of the following composition:
(1) Water 1000.0 cc.
(2) Agar 20.0 g.
(3) Potassium phosphate. . . 1.0 g.
(4) NasCOs..., 1-0 g-
(5) NaN02 2.0 g.

(b) Fraenkel specified the use of 1.0 g.
potassium phosphate and used 12.5 g.
of agar instead of 15.0 g.

(c) Omeliansky added a trace of potas-
sium phosphate instead of a knife
point.
(d) Wimmer specified the use of 0.5 g.
KH2PO4 instead of a knife point of
potassium phosphate.
(e) Smith added 0.05 g. of K2HPO4 in-
stead of a knife point of potassium

phosphate.
(f) Lohnis added 0.5 g. K2HPO4 instead
of a knife point of potassium phos-
phate.
(g) Percival and Harvey used 0.2 g. of
K2HPO4 instead of a knife point of
potassium phosphate.
lactose sodium tartrate B2. Not containing digests.

raffinose sodium malate Ci. Containing unknown constituents of
sodium acetate sodium lactate plant origin. .Section 8 (Med. 2079-2141)
He reported that lactose, sodium acetate, C2. Containing unknown constituents of
propionate, formate, oxalate or butyrate
animal origin. .Section 9 (Med. 2142-2192)
were not suitable carbon sources and
C3. Containing unknown constituents of
showed no growth. Remaining materials
ash or soil origin
supported growth. Best growth was ob-
Section 10 (Med. 2193-2198)
tained with sucrose, glucose and levulose.
References: Beijerinck and van Delden SUBGROUP II-C. SECTION 1
(1903 p. 37), Groenewege (1913 p. 24),
Basal or complete media containing agar,
Lohnis (1913 p. 106).
with all other constituents of known chem-
1430. Noyes' Ammonium Nitrate Agar ical composition; nitrogen inorganic, car-
bon organic.
Constituents:
Water Ai.* Nitrogen present as free or atmospheric
1. 1000.0 cc.
2. Agar (best) nitrogen only.
15.0 g.
3. NH4NO3 1.0 g. Bi. Carbon supplied as carbohydrates.
Preparation: (1) Dissolve 2 and 3 in 1. Ci. Monosaccharides employed.
Sterilization: Not specified. Di. Inorganic salts not added.
Use: Cultivation of soil organisms. The Milburn's Basal Glucose Agar 1431
medium was inoculated with soil. Zipfel's Basal Glucose Agar 1432
Reference: Noyes (1916 p. 93). Lantzsch's Basal Nitrate Agar 1433
D2. Inorganic salts added.
SUBGROUP II-C
El. Containing salts of monovalent cations,
Agar Media with One or More only.
Constituents Organic Davis' Glucose Agar 1434
Key to the Sections of Subgroup II-C Gerlach and Vogel's Glucose Phos-
phate Agar 1435
Ai. Chemical composition of all constit-
Sullivan's Glucose Agar 1436
uents known.
Gage's Glucose Agar 1437
Bi. Nitrogen inorganic, carbon organic
Section 1 (Med. 1431-1495) E2. Containing salts of mono and di-valent
cations.
B2. Nitrogen organic
Section 2 (Med. 1496-1529) Kisch's Basal Glucose Agar 1438
A2. Chemical composition of at least one
Lipman and Brown's Basal Glucose
constituent not definitely known. Agar 1439
Bi. Containing digests.
Beijerinck's Glucose Agar 1440
Ci. Containing a commercial digest.
Heinemann's Glucose Agar 1441
Di. All additional constituents of known Miinter's Basal Salt Agar 1442
chemical composition C2. Disaccharides employed.
Section 3 (Med. 1530-1596) Groenewege's Basal Sucrose Agar. . 1443
D2. At least one of the additional constit- Owen's Sucrose Agar 1444
uents of unknown chemical composition. Buchanan's Sucrose Agar 1445
El. Additional unknown constituent of Lohnis' Congo Red Sucrose Agar. . . 1446
plant or soil origin C3. Polysaccharides employed.
Section 4 (Med. 1597-1627) Krainsky's Basal Starch Agar 1447
E2. Additional unknown constituent of Wyant and Tweed's Starch Agar. . . 1448
animal origin. Welch's Dextrin Agar 1449
Fi. Not containing extracts or infusions of B2.t Carbon supplied as alcohols.
animal origin. .Sections (Med. 1628-1660)
Ci. Inorganic salts not added.
F2. Containing extracts or infusion of ani-
Hesse's Glycerol Agar 1450
mal origin. . . .Section 6 (Med. 1661-2028)
C2. Inorganic salts added.
C2. Notcontaining commercial digests;
containing non-commercial digests. * See page 420 for A2 and A3.
Section 7 (Med. 2029-2078) t See page 420 for B3.
Di. Containing salts of monovalent cations, Bs. Ammonia supplied as salts of phos-
only. phoric acid.
Beijerinck's Mannitol Agar 1451 Ci. Basal media, employed with the addi-
Dj. Containing salts of monovalent and tion of other nutrients.
other cations. C2. Complete media.
Lohnis' Basal Glj'cerol Agar 1452
Di. Only one source of organic carbon
Sackett's Mannitol Salt Agar 1453
added.
Ashby's Mannitol Agar (Jones) .... 1454
El. Carbon supplied as carbohydrates.
Beijerinck's Mannitol Agar (Omeli-
Bengis' Glucose Ammonium
Phos-
ansky and Ssewerowa) 1455
phate Agar 1475
Shunk's Mannitol Agar 1456
Ayers and Rupp's Fuchsin Sulphite
Krainsky's Mannitol Agar 1457
Agar 1476
Heinemann's Mannitol Agar 1458
Cunningham's Cellulose Ammonium
B3.* Carbon supplied other than alcohols or
Phosphate Agar 1477
carbohydrates.
E2. Carbon supplied as organic acids or
Lieske's Acetate Agar 1459
their salts.
Sohngen's Petroleum Agar 1460
Bengis' Lactate Ammonium Phos-
A2. Nitrogen present as ammonium salts.
phate Agar 1478
Bi. Ammonium chloride employed.
Tausz and Peter's Napthenate Am-
Ci. Carbon supplied as carbohydrates.
monium Phosphate Agar 1479
Sohngen's Manganese Oxide Agar. 1461 .
Simmons' Citrate Agar 1480

Conn and Breed's Glucose Ammo-
nium Chloride Agar 1462 D2. More than one source of organic car-
Fulmer and Grimes' Sucrose Ammo- bon added.

nium Chloride Agar. 1463 Dolt's Lactose Ammonium Phos-
Groenewege's Cellulose Agar 1464 phate Agar 1481
Harrison and van der Leek's Aescu-
C2. Carbon supplied as organic acids or
lin Ammonium Phosphate Agar 1482 . .
their salts.
Sohngen's Organic Acid Agar 1465 Be. Ammonium salts of organic acids em-
Krainsky's Malate Agar 1466 ployed.
B2. Ammonium nitrate employed. Bengis'Ammonium Lactate Agar.. 1483
Noyes' Starch Ammonium Nitrate Cohn's Ammonium Tartrate Agar
Agar 1467 (Klimmer) 1484
B3. Ammonium
sulphate employed. Nelson's Ammonium Succinate Agar 1485
Carbon supplied as carbohydrate. Dolt's Lactose Lactate Agar 1486
Ci. Containing monosaccharides. Harrison and van der Leek's Aescu-

Kellerman and McBeth's Glucose culin Lactate Agar 1487
Ammonium
Sulphate Agar 1468 Fischer's Nitrate Tartrate Agar. . . . 1488
Higgins' Glucose Nitrate Agar 1469 As. Nitrogen supplied as nitrates (e.xclusive
C2. Containing disaccharides. of (NH3NO3).
Mortensen's Sucrose Ammonium Bi. Only one source of organic carbon
Sulphate Agar 1470 added.
C3. Containing polysaccharides. Ci. Organic carbon supplied as carbo-
Kellerman and McBeth's Polysac- hydrate.
charide Ammonium Sulphate Sackett's Glucose Nitrate Agar 1489
Agar 1471
Conn and Breed's Carbohydrate Ni-
Sanborn's Ammonium Sulphate Cel- trate Agar 1490
lulose Agar 1472 Groenewege's Sucrose Nitrate Agar. 1491
Vierling's Cellulose Ammonium Sul- Czapek's Sucrose Nitrate Agar
phate Agar 1473 (Conn) 1492
B4. Ammonium
carbonate employed. Pinoy's Dextrin Nitrate Agar 1493
Proskauer and Beck's Glycerol Am- Giltay's Glucose Nitrate Agar
monium Carbonate Agar (Klim- (Giltner) 1494
mer) 1474
C2. Organic carbon supplied as organic
* See also B4, Bs and Be. acids or their salts.
Bj. More than one source of organic car- Added nutrients: The author added 2.0%
bon added. of one of the following:
Conn and Breed's Double Sugar Ni- plasmon sanatogen
trate Agar 1495 tropon nutrose
roborat
1431. Milburn's Basal Glucose Agar Reference: Zipfel (1911 p. 128).
Constituents: 1433. Lantzsch's Basal Nitrate Agar

1. Water 1000.0 cc.
Constituents:
2. Agar
1. Water 1000.0 cc.
3. Glucose (5.0%) 50.0 g.
2. Agar (2.0%) 20.0 g.
Preparation:
3. K2HPO4 (0.01%) 0.1 g.
(1) Dissolve 3 and one of the added nu-
4. NaN03 (0.01%) 0.1 g.
trients in 1.
5. CaCls (0.005%o) 0.05 g.
(2) Solidify by the addition of agar.
6. MgS04 traces
7. FeCla trace
Use: To study color formation. The
Preparation
author reported that tyrosine and aspara-
(1) Wash agar shreds in running water
gin gave concentric circles. Leucin and
for a week.
albumin gave poorly defined green and
(2) Soak (1) in distilled water for one day.
yellowish white rings. Remaining ma-
(3) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
terials gave no rings. Krainsky used a
(4) Pour thick plates.
similar medium for the cultivation of
(5) Incubate the inoculated medium in
actinomyces.
an atmosphere of one of the added
nutrients.
tyrosine alcumin
Use: Cultivation of Actinomyces oligo-
asparagin nuclean
carbophilus (Bacillus oligocarbophilus)
leucine peptone
Added nutrients: The author incubated the
albumin
medium an atmosphere of acetic acid,
in
Variants: Krainsky used 1.0% glucose and
butyric or acetone. The atmos-
acid
added one of the following materials:
phere should contain 0.005 g. acetic acid,
KNO3 0.05%
0.005 g. acetone or 0.004 g. butyric acid
NH4CI 0.05%
per 100.0 cc. atmosphere.
Calcium malate 1 .0%
Reference: Lantzsch (1922 p. 312).
Peptone 0.05%
Asparagin 0.05% 1434. Davis' Glucose Agar
References: Milburn (1904 p. 136),
Constituents:
Krainsky's (1914 p. 662).
1. Water 1000.0 cc.
Glucose Agar
2. Agar 15.0 g.
1432. Zipfel's Basal
3. NaCl 5.0 g.
Constituents: 4. Glucose 20.0 g.
1. Water 1000.0 cc. Preparation: (1) Dissolve 2, 3 and 4 in 1.
2. Agar (3.0%) 30.0 g. Sterilization: Not specified.
3. Glucose (1.0%,) 10.0 g. Use: To study chromogenesis by sporo-
4. Malic acid tricha. Author reported that if maltose
Preparation be substituted for glucose, growth and
(1) Dissolve 2, 3 and one of the added pigment production was more luxuriant.
nutrients in 1. Using glucose, pigment formation was
(2) Acidify slightly by the addition of slight. Growth without glucose about
malic acid. the same as when glucose (or impure glu-
Sterilization: Not specified. cose) was employed.
Use : Cultivation of nodule bacteria. Reference: Davis (1915 p. 178).
1435. Gerlach and Vogel's Glucose Preparation:

Phosphate Agar (1) Wash agar thoroly in water until it is
free from nitrogen.

Constituents:
(2) Dissolve 2, 3, 15.0 g. of (1) and 1.0 g.
1. Water 1000.0 cc.
of glucose in 1000.0 cc. of nitrite free
2. Agar 20.0 g.
water.
3. Potassium bi-phosphate. .. 2.0 g.
(3) Distribute into large Erlenmeyer
4. Glucose 2.0 g.
flasks.
Use: To study nitrogen fixation by nitroso
Use: Cultivation of Azotobacter (nitrogen
bacteria. The author also substituted
assimilators from the soil). Other in-
lactose for glucose.
vestigators used different nitrogen fixing
organisms.
Variants 1438. Kisch's Basal Glucose Agar
(a) Beijerinck and van Delden used 0.5 g.
Medium the same as 129, but solidified by
K2HPO4 instead of 2.0 g. of potassium
the addition of 2.0% agar.
bi-phosphate, and solidified the me-
dium with agar, not specifying the 1439. Lipman and Brown's Basal Glucose
exact amount used. These investi- Agar
gators cultivated chroococcum.
Constituents:
(b) Stoklasa isolated radiobacteria, azo-
1. Water 1000.0 cc.
tobacter and chroococcum in a me-
2. Glucose 10.0 g.
dium composed of 1000.0 cc. of IMol-
3. K2HPO4 0.5 g.
dau river water, 20.0 g. agar, 20.0 g.
4. MgS04 0.2 g.
glucose and 5.0 g. potassium di-
5. Agar 20.0 g.
phosphate.
Preparation
(c) Percival used 2.0 g. K2HPO4 per liter.
(1) Dissolve 2, 3, 4, 5 and one of the added
References: Gerlach and Vogel (1902
nutrients in 1.
p. 670), Beijerinck and van Delden
(2) Add NaOH to neutralize one-half
(1902 p. 8), Stoklasa (1908 p. 489), Per-
or one-fourth of the acid or add no
cival (1920 p. 182).
NaOH.
Agar
1436. Sullivan's Glucose
Use: To determine bacterial count of soils.
Constituents: Other investigators used the basic me-
1. Water 1000.0 cc. dium without additions for the cultiva-
2. Glucose 20.0 g. tion of yeast.
3. NasPOi 5.0 g. Added nutrients The authors added 0.05 g.
:
4. K2HPO4 5.0 g. KNO3, 0.04 g. (NH4)2S04 or 0.05 g. pep-

5. Agar 10.0 g. tone to the basic solution.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Variants
Sterilization: Not specified. (a) Dombrowski cultivated milk yeast
Use: General culture medium. Author re- in a medium of the following com-
ported that few organisms were capable position:
of growing in this medium. 1. Water 1000.0 cc.
Reference: Sullivan (1905-06 p. 117). 2. MgS04 (0.1%) 1.0 g.
3. K2HPO4 (0.2%) 2.0 g.
1437. Gage's Glucose Agar
4. Dextrose (5.0%) 50.0 g.
Constituents: 0. Agar (1.5%) 15.0 g.
1. Water (nitrate free) 1000.0 cc. (b) Beijerinck cultivated Odium lactis
2. K2HPO4 1.0 g. in a medium composed of the follow-

3. NaCl 1.0 g. ing constituents:
4. Agar 15.0 g. 1. Water 1000.0 cc.
5. Glucose 1.0 g. 2. Agar 20.0 g.
3. Glucose 50.0 g. tract neutralized with N/1 HCI

4. KH2PO4 0.5 g. for peptone (Brown Artificial
5. MgS04 0.2 g. humus agar G).
(c) Beijerinck also used the following (13) Substitute 0.05 g. casein for pep-
medium: tone (Brown casein agar B).
1. Water 1000.0 g. (14) Substitute 0.05 g. albumin for pep-
2. Glucose 100.0 g. tone (Brown albumin agar C).
3. KH2PO4 1.0 g. (15) Substitute 0.5 g. albumin for pep-
4. MgS04 1.0 g. tone (Brown albumin agar D).
5. Agar 20.0 g. (16) Substitute 1.0 g. albumin for pep-
(d) Brown added a trace of FeaSOi to the tone (Brown albumin agar E).
basic medium and added 0.05 g. pep- (17) Substitute 0.5 g. casein for pep-
tone. This medium was modified as tone (Brown casein agar C).
follows: (e) Greaves studied the bacterial count
(1) Substitute 0.05 g. urea for peptone on a medium composed of the
of soil
(Brown urea agar). following constituents:
(2) Substitute 0.05 g. asparagin for 1. Distilled water 1000.0 cc.
peptone (Brown asparagin agar). 2. Dextrose 10 g
(3) Substitute 0.1 g. casein for peptone 3. K2HPO4 g'
0^5
(Brown casein agar A). 4. MgS04 0.2 g.
(4) Substitute 0.1 g. albumin for pep- 5. Agar (powdered) 20.0 g.
tone (Brown albumin agar A). References: Lipman and Brown (1910
(5) Omit the peptone from (1) and p. 447), Dombrowski (1910 p. 380), Beij-
substitute 10.0 g. of albumin for erinck (1911 pp. 164, 165), Brown (1913
the peptone (Brown albumin p. 498), Greaves (1914 p. 447).
agar B).
(6) Substitute 20.0 cc. of extract of 1440. Beijerinck's Glucose Agar
humus (method of preparation not
given) for peptone (Brown Arti-
Same as medium 185, but solidified by the
addition of 2.0% agar.
ficialhumus agar A).
(7) Substitute 10.0 cc. of extract of
1441. Heinemann's Glucose Aâr
humus (method of preparation not
given) for peptone (Brown Arti- Constituents:
humus agar B).
ficial 1. Distilled water 1000.0 cc.
(8) Substitute 20.0 cc. of humus ex- 2. KH2PO4 1.0 g
tract (method of preparation not 3. MgSOi. 0.5 g
given) for peptone and add 6.0 g. 4. NaCl .
0.01 g
K2HPO4 (Brown Artificial humus 5. FeS04. 0.01 g
agar C). Reaction is 0.5% acid. 6. MnS04. 0.01 g
(9) Substitute 10.0 cc. of humus ex- 7. Glucose 20.0
tract (method of preparation not 8. Agar 2O.O g
given) for peptone and add 3.0 g. Preparation
K2HPO4 (Brown Artificial humus (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
agar D).
Reaction is 0.5% acid. (2) Dissolve 20.0 g. of agar in (1).
(10) Same as (9) but omit the dextrose Sterilization: Sterilize in the autoclave.
from the agar. Reaction is 0.5% Use: To study the assimilation of atmos-
acid (Brown Artificial humus pheric nitrogen.
agar E). Reference: Heinemann (1922 p. 39).
(11) Substitute 10.0 cc.humus ex-
of
tract neutralized with HCl for 1442. Miinter's Basal Salt Agar
peptone (Brown Artificial humus Same as medium
but solidified by the
164,
agar F). addition of 1.2% agar. Also solidify me-
(12) Substitute 25.0 cc. of humus ex- dium 140 by the addition of 1.2% agar.
1443. Groenewege's Basal Sucrose Agar hosts. Various authors used the same
or similar media for a variety of purposes.
Constituents:
Variants
(a) Peklo cultivated plant actinomyces
2. Agar to solidify
on a medium solidified with agar con-
3. Sucrose 20.0 g.
taining 0.5% KH,P04, 0.2% MgS04
4. K2HPO4 0.5 g.
and did not specify the use of 1.0%
Preparation: (1) Dissolve 3, 4 and one of
agar as did Buchanan.
the added nutrients in tap water agar
(b) Tanner, citing Moore, cultivated Ps.
(preparation not given).
radicicola on a medium containing
0.1% K2HPO4, 0.05% MgS04, 1.0%
Use: Cultivation of Phytobacter lycoper-
sucrose and 1.0% agar.
sicum n. sp. causing tomato rot. Author
(c) Shunk dissolved 1.0 g. KH2PO4, 0.5 g.
reported growth with all nitrogen sources
MgS04, 10.0 g. sucrose and 10.0 g. or
employed.
15.0 g. agar in tap water. This me-
dium was employed for the isolation
the following nitrogen compounds as a
of nodule bacteria. He reported that
nitrogen source:
if mannitol be substituted for sucrose,
KNO3 the medium could be used to main-
(NH4)2S04
tain cultures.
sodium ammonium tartrate
References: Buchanan (1909 p. 381), Peklo
ammonium citrate
(1910 p. 470), Tanner (1919 p. 50), Shunk
ammonium succinate
(1921 p. 241).
ammonium acetate
ammonium lactate 1446. Lohnis' Congo Red Sucrose Agar
ammonium malate
asparagin Bonstituents:
Reference: Groenewege (1913 p. 25). 1. Water 00.0
2. Sucrose
1444. Owen's Sucrose Agar 3. K2HPO4
Constituents: 4. MgS04
1. Water 1000.0 cc. 5. Agar
2. Agar (2.0%) 20.0 g. 6. Congo red
3. Sucrose (Second 80)
(10.0%) 100.0 g.
Preparation: Dissolve 2 and 3 in 1.
(1)
Use: To determine bacterial count in cane
sugar products.
1445. Buchanan's Sucrose Agar

Constituents
1. Water 1000.0 cc.
2. Agar 10.0 g.
3. Sucrose 20.0 g.
4. KH2PO4 0.2 g.
5. MgS04 0.01 g.
Preparation
(2) Pour sterile (1) into sterile plates.

Use: To study the production of gum by
B. radicicola, isolated from a variety of
(2) Solidify (1) by the addition of agar.

Use: Cultivation of Actinomyces.
KNO2 Peptone
KXO3 Asparagin
NH4CI
Variants: Noyes dissolved 1.0 or 2.0 g. of
starch and 15.0 g. agar in 1000.0 cc. of
water, and used the medium for soil
organisms.
References: Krainsky (1914 p. 662), Noyes
(1916 pp. 93, 94).
1448. Wyant and Tweed's Starch Agar

Constituents:
1. Water..... 1000.0 cc.
2. Starch (potato) 10.0
3. Agar 10.0 g.
4. K2HPO4 0.2 g.
5. MgS04 0.2 g.
6. K2CO3. 0.4
7. CaCls 0.02
8. Fe2(S04)3 0.02 _
9. NaCl 0.02 g.
Preparation:
(1) Prepare a starch solution bj^ suspend-
ing 10.0 g. potato starch in a little
cold water. Then add 500.0 cc. boil-
ing water. Concentrate to 500.0 cc.
(2) Dissolve 3, 4, 5, 6, 7, 8 and 9 in
500.0 cc. water by boiling.
(3) Replace amount of water lost by addi-
tion of distilled water.
(4) Add (1) to (3).
Use: Cultivation of organisms causing flat
sour in peas.
Reference: Wyant and Tweed (1923 p. 12).
1449. Welch's Dextrin Agar
Constituents
Sterilization 1454. Ashby's Mannitol Agar (Jones)

(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Same as medium 170, but solidified by
(2) Dissolve 1.0% glycerol and 0.1% of
one of the added nutrients to (1).
(3) Distribute in 10.0 cc. quantities in Mannitol Agar
1455. Beijerinck's
50.0 cc. Erlenmeyer flasks. (Omeliansky and Ssewerowa)
Sterilization: Not specified. Same as medium 148, but solidified by the
Use: To study assimilation of amide, am- addition of 1.5% agar.
monia and nitrate. The author suggested Murray gave a modification of the same
that any desired carbohydrate, alcohol, medium by solidifying medium 149 by the
etc., might replace the glycerol. addition of 1.5% agar.
Added nutrients: The author suggested the
addition of 0.1% of one of the following: 1456. Shunk's Mannitol Agar
Urea Ammonium acetate Variant medium 1445 solidified
(c) of
Uric acid Ammonium butyrate
with agar.
Sodium hippurate Ammonium lactate
Asparagin NaNOj 1457. Krainsky's Mannitol Agar
(NH 4)280 4 Same as medium 188, but solidified by
Reference: L5hnis (1913 p. 99). the addition of 2.0% agar.
1453. Sackett's Mannitol Salt Agar
1458. Heinemann's Mannitol Agar
2. Na2S04 9.0668 g. 2. K2HPO4 0.2 g.
3. NaCl 20.2621 g. 3. CaCl2 0.02 g.
4. NazCOs 2.0118 g. 4. MgS04 0.2 g.
5. MgS04.... 1.7475 g. 5. FejCl, 0.01 g.
6. Mannitol 15.0 g. 6. Mannitol 15.0 g.
7. CaS04 9.4457 g. 7. Agar 20,0 g.
8. NaNOs 13.5319 g. Preparation
9. Agar 15.0 g.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Preparation Add NaOH solution until the reac-
(2)
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
tion is neutral to phenolphthalein.
(2) Dissolve 15.0 g. of agar in 1000.0 cc. Dissolve 20.0 g. of agar in (2).
(3)
of (1). Sterilization: Sterilize in the autoclave.
(3) Tube. Use: Cultivation of cultures capable of
Sterilization: Autoclave at 120C. for 5 assimilating atmospheric nitrogen.
minutes. Reference: Heinemann (1922 p. 39).
Azotobacter chroococcum. The author 1459. Lleske's Acetate Agar
reported generally a brownish pigment Constituents
was produced. No pigment was pro- 1. Distilled water 1000.0 cc.
duced if mannitol be omitted. 2. Agar 10.0 g.
Variants: The author gave the following 3. Manganese acetate 0.1 g.
variants Preparation: (1) Dissolve 2 and 3 in 1.
(a) Omitted the CaS04 and NaNO, and Sterilization: Not specified.
added 2.8589 g. NajCOs. Use: Isolation of Leptothrix ochracea.
(b) Omitted the NaCl. Inoculate the agar plates when the sur-
(c) Omitted the NaNO 3. face is still moist.
(d) Omitted the Na2S04. Reference: Lieske (1919 p. 418).
(e) Omitted the MgSOi.
1460. Sohngen's Petroleum Agar
(f) Omitted the K2SO4.
(g) Diluted any of the above media with Constituents:
2 or 3 volumes of water. 1. Distilled water 1000.0 cc.
Reference: Sackett (1912 p. 109). 2. Bipotassium phosphate 0.5 g.
3. MgSOi 0.5 g. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7

4. Agar 20.0 g. in 1.
5. Petroleum Sterilization: Not specified.

Preparation Use: Test for reduction of nitrate.
(1) Dissolve 2, 3 and 20.0 g. of washed Reference: Conn and Breed (1919 p. 278).
agar in 1.
1463. Fulmer and Grimes' Sucrose Ammo-
nium Chloride Agar
(3) Pour in Petri dishes.
(4) Invert the Petri dishes, and place a Constituents:
little petroleum in a flat container, 1. Distilled water 1000.0 cc.
on the lid inside the petri dish. This 2. NH4CI 1.88 g.
places the surface of the medium 3. K.,HP04 1.0 g.
directly opposite the surface of the 4. CaCh 1.0 g.
petroleum. 5. Sucrose 50.0 g.
Sterilization: Method not given. 6. Agar... 15.0 g.
Use: Cultivation of petroleum oxidizers, Preparation
soil forms Mycobacterium hyalinum (1) Dissolve 2, 3, 4 and 5 in 1.
Mycobacterium phlei, Mycobacterium lac- (2) Wash agar several times in distilled
ticola, Mycobacterium rubrum, Mycobac- water.
terium album. (3) Add agar to (1), heat in autoclave at
Reference: Sohngen (1913 p. 59S). 15 pounds pressure for 30 minutes.
(4) Filter thru absorbent cotton and tube.
1461. Sohngen's Manganese Oxide Agar Sterilization: Sterilize in live steam for
Constituents: 30 minutes on two successive days.
1. Water agar 1000.0 cc. Use: Cultivation of yeast, Saccharomyces
2. Glucose 5.0 g. cerevisiae and Torula sphaerica.
3. K5HPO4 0.05 g. Variants: The authors omitted the CaCU-
4. NH4CI 0.05 g. Other experiments were reported omitting
5. Manganese oxide the sucrose.
Preparation Reference: Fulmer and Grimes (1923
(1) Dissolve 2, 3 and 4 in water agar p. 586).
(composition not given).
1464. Groenewege's Cellulose Agar
(2) Add manganese oxide until a black
color is formed. Constituents:
Sterilization: Method not given. 1. Water agar 100.0 cc.
Use: Cultivation of manganese bacteria. 2. Cellulose 2.0 g.
Author reported that a clearing of the 3. NH4CI 0.1 g.
medium near the colony indicated the 4. K2HPO4 0.05 g.
decomposition of the manganese oxide. Preparation: Dissolve 2, 3 and 4 in tap
(1)
Acetobacter melanogenum, B. lactis water agar (composition not given).
aerogenes, B. herbicola, B. aceti, B. ran- Sterilization: Not specified.
cens and others decomposed manganese Use: Cultivation of Phytobacter lycoper-
oxide. sicum causing tomato rot. Author re-
Reference: Sohngen (1914 p. 554). ported that organism did not utilize
cellulose. A slight growth appeared
1462. Conn and Breed's Glucose Ammonium after several weeks, however, due possibly
Chloride Agar
to a trace of starch in the filter paper used
Constituents: as cellulose source.
1. Water 1000.0 cc. Reference: Groenewege (1913 p. 25).
2. Agar 15.0 g.
1465. Sohngen's Organic Acid Agar
3. Glucose 10.0 g.
4. KNO3 1.0 g. Constituents
5. CaCl. 0.5 g. 1. Distilled water 1000.0 cc.
6. K2HPO4 0.5 g. 2. Agar 20.0 g.
7. NH4CI 2.0 g. 3. NH4CI 0.5 g.
4. MgS04... 0.5 g. Use: Cultivation of soil forms.

5. Bipotassium phosphate 0.5 g. Reference: Noyes (1916 pp. 93, 94).
6. Sodium butyrate 10.0 g.
7. Chemical elements 1468. Kellerman and McBeth's Glucose
Preparation Ammonium Sulphate Agar
Constituents
(2) Add several elements. (Elements or
1. Water 1000.0 cc.
compounds not given.) 2. Agar 10.0 g.
3. Glucose 10.0 g.
Use: Cultivation of Mycobacteria. Au- 4. K2HPO4 1.0 g.
thor reported that sulphur, potassium,
5. MgS04 1.0 g.
phosphorous nitrogen and carbon were NaCl 1.0 g.
6.
necessary for growth of mycobacteria. (NH4)2S04 2.0 g.
7.
The author also studied the production of 8. CaCOa 2.0 g.
manganese oxide by bacteria on a similar Preparation
medium. and
(1) Dissolve 4, 5, 6, 7 8 in 1000.0 cc.
Variants: The author reported that when of water.
manganese oxide was produced on the Mix 500.0 cc. of (1) and 500.0 cc. of
(2)
following medium a dark brown or black
water.
colony was formed: agar and 10.0
(3) Dissolve 10.0 g. g. glu-
1. Water agar 1000.0 cc.
cose in (2).
2. NH4CI 0.5 g.
3. K2HPO4 0.5 g.
Use: Cultivation of Bacillus flavigena,
4. Calcium malate or calcium Bacillus amylolyticus, Bacillus rossica
gluconate 20.0 g.
n. sp.
5. Manganese sulphate 10.0 g.
Reference: Kellerman and McBeth (1912
Reference: Sohngen (1913 p. 605), (1914
p. 487).
p. 546).
1469. Higgins' Glucose Nitrate Agar
1466. Krainsky's Malate Agar
Constituents
addition of agar.
1. Water 1000.0 cc.
2. Agar to solidify
1470. Mortensen's Sucrose Ammonium
3. NH4CI (0.05%). 0.5 g.
Sulphate Agar
4. Calcium malate (1.0%) 10.0 g.
Preparation Same as medium 207, but solidified by
0.05% NH4CI and 1.0% the addition of 2.0% agar.
(1) Dissolve
calcium malate (or the author sub-
1471. Kellerman and McBeth's Polysac-
stituted 1.0% calcium nitrate) in
charide Ammonium Sulphate Agar
water.
(2) Solidify (1) by the addition of agar. Constituents
Sterilization: Method not given. 1. Water 1000.0 cc.
Use: Cultivation of actinomyces. 2. Cellulose 15.0 g.
Reference: Krainsky (1914 p. 662). 3. Agar 10.0 g.

4. K2HPO4 1.0 g.
1467. Noyes' Starch Ammonium Nitrate
5. MgS04 1.0 g.
Agar 6. NaCl 1.0 g.
Constituents 7 (NH4)2S04 2.0 g.

1. Water 1000.0 cc. 8. CaCOs 2.0 g.
2. Agar (best) 15.0 g. Preparation
3. NH4NO3 1.0 g. (1) Prepare a liter of dilute ammonium
4. Starch 1.0 g. hydroxide solution by adding three
Preparation: Dissolve 2, 3 and 4 in
(1) 1. parts water to ten parts ammonium
Sterilization: Method not given. hydroxide, sp. gr. 0.90.
(2) Add a slight excess of copper car- of water instead of 1000.0 cc. as indi-
bonate. cated by Kellerman and McBeth.
(3) Shake vigorously. Tanner then mixed equal parts salt
(4) Allow to stand over night and then solution on cellulose solution.
siphon off the supernatant solution. (c) Tanner prepared starch agar by dis-
(5) Add 15.0 g. of unwashed sheet filter solving one-half the quantity of salts
paper. given in variant (a), step (3), in
(6) Shake occasionally until the paper 500.0 cc. tap water. Then Tanner
is dissolved. mixed equal parts of salt solution
(7) Dilute to 10 liters and add slowly a and starch water and proceeded as
one to five solution of hydrochloric in variant (a).
acid with vigorous shaking until the (d) Giltner prepared a starch agar as
precipitation of the cellulose is follows:
complete. (1) Dissolve 1.0 g. K2HPO4, 1.0 g.
(8) Dilute to 20 liters, allow the cellu- MgS04-7H20, 1.0 g. NaCl, 2.0 g.
lose to settle and decant the super- (NH4)2S04, and 2.0 g. CaCOa in
natant liquid. 1000.0 cc. of water.
(9) Wash by repeated changes of water, (2) Add 10.0 g. washed agar.
adding HCl each time until the (3) Weigh.
copper color disappears. Then wash (4) Boil over a free flame until the
with water alone until the solution solution of the agar is complete.
is free from chlorine. (5) Weigh and make up the loss in
(10) Allow to settle several days and weight with boiling water.
decant as much of the clear solution (6) Make a smooth paste of 10.0 g. of
as possible. potato starch or other starch in a
(11) If the cellulose percentage is too little cold water.
low centrifuge a portion of the fluid (7) Add 800.0 cc. of boiling water
to bring the cellulose content up to to (6).
one per cent. (8) Concentrate to 500.0 cc. by boiling.
(12) Dissolve 4, 5, 6, 7 and 8 in 1000.0 cc. Mix (5) and (8) and boil a few
(9)
of tap water. minutes.
(13) Mix 500.0 cc. of (11), 500.0 cc. of (12). (10) Strain thru two thicknesses of
(14) Dissolve 10.0 g. of agar in (13). cheese cloth.
Sterilization: Method not given. (11) Add 1.5% china blue rosolic acid
Use: Cultivation of organisms fermenting for indicator.
cellulose. (12) Tube, taking care to keep the
Variants CaCOs well mixed with the media.
(a) The author prepared a similar me- (13) Sterilize (method not given).
dium using potato starch instead of (e) Khouvine prepared a cellulose in the
cellulose as the polysaccharide. The same manner as Kellerman and
medium was prepared as follows McBeth and dissolved 20.0 g. agar in
(1) To 800.0 cc. of boiling water add 1000.0 cc. of water by heating. The
10.0 g. of potato starch suspended inorganic salts, as used by Keller-
in cold water. man and McBeth, were dissolved in
(2) Evaporate by boiling to 500.0 cc. the agar solution. Five hundred
(3) Dissolve 1.0 g. K2HPO4, 1.0 g. cubic centimeters of the cellulose
MgS04, 1.0 g. NaCl, 2.0 g. solution was then added to the agar
(NH4)2S04, and 2.0 g. CaCOj in solution, mi.xed well, tubed and steri-
1000.0 cc. of water. lized at 110 for 15 minutes.
(4) Mix 500.0 cc. (2) with 500.0 cc. (3). (f) Khouvine prepared a starch agar in
(5) Dissolve 10.0 g. of agar in (4). the following manner:
(6) Sterilization not specified. (1) Prepare a paste with 10.0 g. of
(b) Tanner used one-half the amounts of potato starch in a little cold water.
salts and dissolved them in 500.0 cc. (2) Add (1) to 800.0 cc. of boiling water.
(3) Evaporate to 500.0 cc. Cellulomonas folia in the presence of Act.

Dissolve 20.0 g. agar in 1000.0 cc. colorata, Azotobacter, B. subtilis, B.
(4)
water. mycoids and B. cereus.
(5) Dissolve 1.0 g. K2HPO4, 1.0 g. Reference: Sanborn (1926 p. 353).
MgSOi, 1.0 g. NaCl, 2.0g. (NH^z Ammonium
1473. Vierling's Cellulose
SO4 and 2.0 g. CaCO, in (4). Sulphate Agar
(6) Mix (3) and (5).
Constituents:
(7) If deep agar tubes are to be pre-
1. Water 1000.0 cc
pared it is well to filter until
clear. Filtering is carried out in
2. K2HPO4 10 g.
3. CaCl. 0.1 g.
the autoclave.
Distribute in tubes and sterilize
4. MgS04 0.1 g.
(8)
5. FeCls trace
in the autoclave at 100C. for
15 minutes.
6. NaCl trace
Medium may be poured into sterile 7. Agar 20.0 g.

(9)
8. Cellulose (Merck) 10.0 g.
petri dishes.
9. Potassium stearate 1.0 g.
References: Kellerman and McBeth (1912
10. (NH4)2S04 5.0 g.
p. 487), Tanner (1919 pp. 61, 62), Giltner
(1921 p. 371), Khouvine (1923 pp. 714,
11. CaCOs 10 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8,

715).
9, 10 and 11 in 1.
1472. Sanborn's Ammonium Sulphate Sterilization: Method not given.

Cellulose Agar Use: Decomposition of cellulose by Myco-
Constituents: bacteria. Incubate under a glass bell
1. Distilled water 1000.0 cc. jar. Author reported that the filter paper
2. K2HPO4 10 g. showed no signs of being attacked.
3. MgS04 10 g. Growth occurred, however. The agar
4. NasCO, 1.0 g. became turbid. After incubation no
5. (NH4)2S04 2.0 g. clearing occurred around the colonies.
6. Cotton 30.0 g. Cellulose was not decomposed.
7. Agar 2.5 g. Reference: Vierling (1920 p. 206).
8. CR indicator (1.0%)
1474. Proskauer and Beck's Glycerol Am-
Preparation
monium Carbonate Agar (Klimmer)
distilled water. Constituents:
Prepare a 0.5% agar solution with 1. Water 1000.0 cc.
(2)
the remaining 500.0 cc. distilled 2. Glycerol 15.0 g.
water. 3. (NH4)2C03 3.5 g.
(3) Mix (1) and (2). 4. MgS04 (crystalline) . . 2.5 g.
(4) Cut 30.0 g. of cotton (chemically un- 5. KH2PO4 1-3 g.
treated) into small fragments. 6. Agar 15.0 to 30.0 g.
Add 1.0% CR
indicator with constant Preparation: (1) Dissolve 2, 3, 4, 5 and 6
(5)
stirring. (CR indicator prepared by in 1.
mixing equal parts of 0.5% aqueous Sterilization: Method not given.
solution of china blue with 1.0% Use: Cultivation of tubercle bacilli.
solution of rosolic acid in 95.0% Reference: Klimmer (1923 p. 172).
alcohol.) Glucose Ammonium

1475. Bengis'
Mix well to insure an even distribu-
(6) Phosphate Agar
tion of cotton and pour into petri
dishes. Constituents
Sterilization: The medium was autoclaved 1.
after it had been added to the dishes. 2. Agar (powdered) 2.0 g.
The medium is colored red, pH-8.4. 3. Glucose 2.0 g.
Use: To show fermentation of cellulose by 4. (NH4)2HP04 0.1 g.

5. MgS04 0.5 g. (2) Add to this mixture at 90C.: 1%

6. CaCOa 1.0 g. alcoholic basic fuchsin 0.5; freshly
Preparation prepared 5% sodium sulphite 0.5.
(1) Dissolve 2 in 1. References: Ayers and Rupp (1918 pp. 433,
(2) Add 3, 4, 5 and 6 to (1). 434), Harvey (1921-22 p. 103).
(3) Filter to incubating flasks.
Sterilization: Sterilize in autoclave. 1477. Cunningham's Cellulose Ammonium
Use: To cultivate large quantities of Phosphate Agar
B. colt.
Constituents
Reference: Bengis (1916 p. 392). Distilled water
1. 1000.0 cc.
2. Agar (1.5%) 15.0 g.
1476. Ayers and Rupp's Fuchsin Sulphite 3. Cellulose
Agar 4. MgNH4P04 (0.05%) 0.5 g.
Constituents: 5. KjHP04 (0.05%) 0.5 g.
1 Distilled water 1000.0 cc. Preparation
2. Sodium ammonium phos- (1) Wash by immersing it
15.0 g. of agar
phate 4.0 g. in distilled water for about a week.
3. KH2PO4 2.0 g. Change the water each day.
4. Lactose (2) Add sufficient distilled water to make
5. Agar 30.0 g. a 1.5% agar solution.
6. Fuchsin (1.0% alcoholic (3) Steam (2) for one hour.
basic soln.) 5.0 cc. (4) Dissolve 0.05% Mg(NH4)P04 and
7. NajSO, (5.0% aque. solu- 0.05% K2HPO4 in (3).
tion) 5.0 cc. (5) Tube in 4 to 8.0 cc. quantities.
Preparation (6) Melt the sterile agar and pour an
(1) Dissolve 2, 3 and 4 in 500.0 cc. of 1. 8.0 cc. tube into a petri dish.
(2) Dissolve agar in 500.0 cc. distilled (7) Place a sterile disc of filter paper on
water and filter. the agar following inoculation and
(3) Mix equal parts (1) and (2) and flask gently press it down. Then pour
in 100.0 cc. lots. 4.0 cc. of sterile melted agar over the
(4) Prepare 1.0% alcoholic solution of filter paper.
basic fuchsin. Sterilization : Sterilize (5) intermittently in
(5) Prepare 5.0% H2O solution of so- steam. Sterilize, in the autoclave, pieces
dium sulfite. of filter paper cut to fit a petri dish and
(6) To every 100.0 cc. of sterile (3) while test tubes.
hottest, at time of plating add 0.5 cc. Use: Decomposition of cellulose by soil
of (4) and 0.5 cc. of (5) freshly pre- forms.
pared. Reference: Cunningham (1924 p. 140).
(7) Mix thoroughly.
1478. Bengis' Lactate Ammonium
not given. Phosphate Agar
Use: Enumeration of colon-aerogenes Constituents
group. Author reported the members of 1. Distilled water 100.0 cc.
the colon-aerogenes produced medium 2. (NH4)2HP04 0.2 g.
sized red colonies with a ring around the 3. Calcium lactate 1.0 g.
colony. Other colonies were pink. Puri- 4. Agar (powdered) 3.0 g.
fied litmus or brom cresol purple were not Preparation
as well suited as indicators as was the (1) Dissolve 4 in 1.
fuchsin sulphite mixture. (2) Add and dissolve 2 and 3 in (1).
Variants: Harvey prepared a similar me- (3) Filter into incubating flasks.
dium as follows: Sterilization: Sterilize in a Bramhall-Dean
(1) Dissolve 2.0 g. sodium ammonium autoclave.
phosphate, 1.0 g. KH2PO4, 5.0 g. lac- Use: To cultivate large quantities of
tose, 15.0 g. agar in 1000.0 cc. distilled B. coli. Author reported slight growth.
water. Reference: Bengis (1916 p. 392).
Tausz and Peter's Napthenate genes grew luxuriantly, forming large

1479.
Ammonium Phosphate Agar greenish blue colonies, producing a blue
color in the medium.
Constituents Variants: The dye used in the medium
Water 1000.0 cc.
1 above was a product of National Anilin
MgNH4P04 10 g.
2. (0.1%) and Chemical Company. A 0.4% aque-
0.1 g.
3. CaS04 (0.01%) ous solution of brom-thymol blue may
K2HPO4 (0.08%) 0.8 g.
4. be used in the proportions of 20.0 cc. per
FeCla trace
5. liter of agar. The aqueous solution is
ti-a'ê
6. KI prepared by grinding 0.1 g. of dry powder
Agar 15-0 g-
7. in an agate mortar with 3.2 cc. of N/20
Sodium salt of napthenic
8.
lO.Og.
NaOH and when solution is complete
acid The brom-
dilute to 25.0 cc. with water.
Preparation thymol blue distributed by the Lamotte
(1) Cut agar into small
pieces, mix with
Chemical Products Company gave satis-
water and allow to stand (time not factory results when used in these pro-
specified). portions.
(2) Wash with flowing water on a filter
Reference: Simmons (1926 p. 209).
for several days.
Dissolve 2, 3, 4, 5, 6 and (2) in 1.
Ammonium Phosphate
(3) 1481. Dolt's Lactose
(4) Add 1.0 g. of the sodium salt of
Agar
napthenic acid to each 100.0 cc. of (3).
Not specified. Constituents
Sterilization:
Use Cultivation of paraflBn bacteria, Bac-
:
1.
Agar (purified) 30.0 g.

terium aliphaticum, Bacterium alipha- 2.
3. Glycerol ^.0 g.
ticum liquefaciens
Reference: Tausz and Peters (1919 p. 49). 4. Ammonium phosphate 1.0 g.
5. Lactose 10-0 g.
1480. Simmons' Citrate Agar 6. Azolitmin 10 g.
Preparation
Constituents
1000.0 cc. (1) Purify agar
by cutting it in small
1. Distilled water
20.0 g. pieces and soaking it in distilled water
2. Agar
5.0 g. 24 hours.
3. NaCl
Dissolve (1), 2, 3 and 4 in 1.
4. MgS04 0.2 g. (2)
5. (NHOHsPOi 10 g- (3) Add NaOH until neutral to phenol-
10 phthalein.
6. K2HPO4 g-
Dissolve 5 in (3).
7. Sodium citrate (2.77 g. of (4)
Dissolve l.C g. of Kahlbaum's C. P.
sodium citrate, 5^ H2O) .
. . . 2.0 g. (5)
azolitmin in 100.0 cc. distilled water
8. Brom-thymol blue (1.5%
alcoholic) 10-0 cc. and boil, 15 minutes. This gives a
blue solution.
Preparation
and 7 in 1.
6 (6) Add 10.0 cc. of (5) to (4).
(1) Dissolve 3, 4, 5,
Method not given.
(2) Add 20.0 g. clean washed agar and Sterilization:
autoclave at 15 pounds pressure for Use Detection of acid production in water

:
analysis. Author reported that B.

coli
15 minutes.
Adjust to pH = 6.8. produced a red colony on this medium.
(3)
Bengis cultivated B. coli on a similar
(4) Add 8.
Tube and slant. medium.

(5)
Variants: Bengis prepared a medium as
Sterilization: See step (2) above.
follows: He employed the medium to
Use: Differentiation of typhoid-colon aero-
cultivate large numbers of B. coli:
genes group. The author reported that
(1) Dissolve 30.0 g.
of agar in distilled
Escherichia coli was markedly or com-
water.
pletely inhibited and does not change the
Aerobacter aero- Add 10.0 g. glycerol and 2.0 g.
color of the medium. (2)
(NH4)2HP04 to (1) and heat for phology of B. coli in the medium

10 minutes in a water bath. using only 15.0 g. agar per liter.
(3) Dissolve 10.0 g. of lactose in 2. References: Bengis (1916 p. 393), Scales
(4) Filter. (1921 p. 595).
(5) Sterilize in the autoclave.
References: Dolt (1908 p. 625), Bengis 1484. Cohn's Ammonium Tartrate Agar
(1916 p. 392). (Klimmer)
1482, Harrison and VanderLeck's Aesculin Constituents:

Ammonium Phosphate Agar 1. Water 1000.0 cc.
2. K2HPO4 5.0 g.
Constituents 3. MgS04 (Crystalline). 5.0 g.
1. Distilled water 1000.0 cc. Calcium phosphate.
4. 0.5 g.
2. Agar 15.0 g.
5. Ammonium tartrate.. 10.0 g.
3. Glycerol 5.0 g. Agar
6. 15.0 to 30.0 g.
4. Ammonium phosphate 1.0 g.
Preparation
5. Iron citrate 2.5 g.
6. Aesculin 1 .0 g. by dissolving
(2) Solidify 15.0 to 30.0 g.
Preparation of agar in (1).
(1) Purify agar by cutting it in small Sterilization: Not specified.
pieces and soaking it in distilled
water for 24 hours. Reference: Klimmer (1923 p. 172).
(2) Dis-solve (1), 3, 4 and 5 in 1.
(3) Adjust reaction to +0.4. 1485. Nelson's Ammonium Succinate Agar
Use: Detection of B. coli and B. typhosus. Constituents:
Authors reported that B. coli colonies 1. Distilled water 1000.0 cc.
were black, while typhoid colonies 2. K2HPO4 2.0 g.
were not. 3. Ammonium succinate 2.0 g.
Reference: Harrison and VanderLeck (1909 4. NaCl 2.0 g.
p. 622).
5. Glucose 2.0 g.
6. Agar 20.0 g.
1483. Bengis' Ammonium Lactate Agar Preparation
Constituents: (1) Dissolve 2, 3, 4, 5 and 6 in 1.
1. Distilled water 1000.0 cc. (2) Reaction of pH = 7.0 to 7.2 (un-

2. Agar 25.0 g. adjusted).
3. NajHFOi 2.0 g. Sterilization: Sterilize at 10 pounds pres-
4. CaCO, 10.0 g. sure for 20 minutes.
5. Ammonium lactate 10.0 g. Use: Cultivation of colon-typhoid group
Preparation: in mass culture.
(1) Dissolve 2 and 3 in 1 in the autoclave.

Reference: Nelson (1926 p. 373).
(2) Filter solution thru glass wool.
1486. Dolt's Lactose Lactate Agar
(3) Adjust reaction to +0.3.
(4) Distribute into liter Erlenmeyer Constituents:
flasks in 500.0 cc. lots. The flasks 1. Distilled water 1000.0 cc.
containing 5.0 g. ofCaCOs each. 2. Agar (purified) 30.0 g.
(5) Add 5.0 g. of ammonium lactate to 3. Ammonium lactate 5.0 g.
each flask. 4. Na2HP04 1.0 g.
(6) Shake thoroly. 5. Lactose 10.0 g.
Sterilization: Sterilize in autoclave at 6. Azolitmin (1.0% solution).. 10.0 cc.
12 pounds pressure for 15 minutes. Preparation
Use: To cultivate large amounts of B. coli. (1) Purify agar by cutting it into small
Variants pieces and soaking in distilled water
(a) The author omitted the CaCO 3. 24 hours.
(b) Scales studied the variation in mor- (2) Dissolve (1), 2, 3 and 4 in 1.
(3) Make neutral to phenolphthalein with 9. MgS04 (Cr.) (0.03%) 0.3 g.
NaOH. 10. NaCl (0.01%) 0.1 g.
11. FeoCle (0.001%) 0.01 g.

Prepare 6 by dissolving 1.0 g. Kahl- Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8,
(5)
baum's C. P. azolitmin in 100.0 cc. 9, and 11 in 1. (7, 8, 9, 10 and
10 11 con-
stitute Meyers mineral solution in the

distilled water. Boil 15 minutes.
The solution is blue. percentages given.)
Add to (4). Sterilization: Method not given.
(6) (5)
Sterilization: Method not given. Use: Bacterial count in soils.
Use: Detection of acid production in Reference: Fischer (1910 p. 457).
water analysis. Bengis used a similar Glucose Nitrate Agar
1489. Sackett's
medium for the cultivation of B. coli in
large quantities. Constituents
Water 1000 cc.
Variants : (a) Bengis omitted the azolitmin 1.
Na2HP04 instead of 2. Glucose 20.0 g.

solution, used 2.0 g.
1.0 g., and used 10.0 g. ammonium lactate
3. Agar 20.0 g.
instead of 5.0 g. He reported that the 4. NaXOs

medium gave excellent results without Preparation:
lactose. (1) Dissolve 2 and 3 in 1.
(2) Prepare a 10.0% NaNOs
Bengis (1916 solution in
References: Dolt (1908 p. 626),
distilled water.
p. 392).
(3) Add increasing amounts of (2) to (1)
1487. Harrison and VanderLeck's Aesculin so that the NaNOj concentration will
Lactate Agar be 0.0, 0.01, 0.03, 0.05, 0.1, 0.3 and
Constituents 0.5%.
1. Distilled water. 1000.0 cc. (4) Distribute in test tubes.
Agar 15.0 g. Sterilization: Sterilize in the autoclave for

2.
3. Ammonium lactate 5.0 g. 5 minutes at 120C.

Use: To study pigment production of
4. Na2HP04 10 g-
2.5 g. Azotobacter chroococcum. Author re-
5. Iron citrate
6. Aesculin 1-0 g. ported that NaNOa favored the produc-
tion of a brown pigment, NH4CI,
Preparation:
(1) Purify agar by cutting it
in small (NH 4)280 4, asparagin and peptone did not
pieces and soaking them in water for increase pigment production.
24 hours. Reference: Sackett (1913 p. 109).
(2) Dissolve (1), 3, 4, 5 and 6 in 1.
Conn and Breed's Carbohydrate
1490.
(3) Adjust reaction to 0.4.
Not specified.
Nitrate Agar
Sterilization:
Use: Detection of B. coli and B. typhosus. Constituents:
Water 1000.0 cc.
Author reported that B. coli produced 1.
Agar 15.0 g.
black colonies, typhoid organisms did not. 2.
Glucose or sucrose 10.0 g.
Reference Harrison and VanderLeck (1909
:
3.
p. 622).
4. KNO3 10 g-
5. CaCl.2 0.5 g.
1488. Fischer's Nitrate Tartrate Agar 6. MgS04 5.0 g.
Constituents 7. K2HPO4 5.0 g.

1000.0 cc. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
1. Water
2. Agar (1.25%) 12.5 g in 1.
3. Dextrose (0.1%) 1-0 g Sterilization: Not specified.
Ammonium tartrate (0.1%). 1.0 Use: Study of reduction of nitrates.

4. g
5. KNO3 (0.05%) 0.5 g Variants
6. Soda (crystalline) (0.15%). 1.5 g (a) The authors omitted the MgS04 and
7. K2HPO4 (0.1%) 1.0 g used only 0.5 g. K2HPO4 instead of
8. CaCl2 (0.01%) 0.1 g 5.0 g.

435
(b) Hucker and Wall omitted the MgSOi

and used 0.5 g. K2HPO4 instead of
5.0 g. They used the medium to
determine ammonia production from
inorganic sources. Add 1.0 cc. of a
10.0% phenol and 1.0 cc. of a 1.0%
(available chlorine) sodium hypo-
chlorite solution to the surface of an
agar slant. A
blue color forming
within 30 minutes denotes ammonia
production. Also 2.0 cc. of neutral
formaldehyde containing a few drops
of phenolphthalein may be added to
the slant. Presence of acid indicates
ammonia formation.
References: Conn and Breed (1919 p. 278),
Hucker and Wall (1922 p. 517), (1922
p. 485), Committee S. A. B. (1922 p. 525),
(1923 p. 27).
1491. Groenewege's Sucrose Nitrate Agar

Constituents:
1. Water agar 1000.0 cc.
2. Sucrose 20 g
3. KNO3 lOg"
4. K2HPO, 0.5 g.
Preparation: (1) Dissolve 2, 3 and 4 in
1000.0 cc. of tap water agar. (Composi-
tion not given.)
Use: Cultivation of Phytobacter lycoper-
sicum n. sp. (Causing tomato rot.)
Reference: Groenewege (1913 p. 30).
1492. Czapek's Sucrose Nitrate Agar (Conn)

Constituents:
1- Water lOOO.O cc.
2- Agar 15 ff.
3. MgS04 0.5
4. KCl
5. K2HPO4
6. FeS04
7. NaNOa
8. Sucrose
Preparation: (1) Dissolve 2,
7 and 8 in 1.

Use: Cultivation of actinomyces.
Reference: Conn (1921 p. 21).
1493. Pinoy's Dextrin Nitrate Agar

Constituents:
1- Water 600.0 cc.
2- Agar 9_0 g.
10.0 g. Giltay's Citric Acid Asparagin Agar

3. Glucose.
(Lohnis)
1^17
Lactose. 5.0 g.
4.
El. More than one type of organic carbon
KNO3... 1.0 g.
5.
0.5 g. added.
6. CaCh... 1518
K2HPO4 0.5 g. Conn's Asparaginate Agar
7.
and 7 Smith's Levulose Asparaginate
Preparation: (1) Dissolve 2, 3, 4, 5, 6
Agar 1519
in 1.
Higgins' Basal Lactose Asparagm
1520
Agar.
Use: For reduction of nitrate.
Sullivan's Glycerol-Nitrate Aspara-
Reference: Conn and Breed (1919 p. 278).
1521
gin Agar
SUBGROUP II-C. SECTION 2 Maassen's Malic Acid Asparagin

1522
Agar (Klimmer)
Uschinsky's Glycerol Asparaginate
together with nitrogen and carbon of known 1523
Agar (Klimmer)
chemical composition.
addition of Cj. Other amino acids employed.
Ai. Basal media used with the 1524
Berthelot's Nitrate Tyrosein Agar..
other materials. 1525
Jones' Histidin-hydro-chloride Agar.
Miehe's Basal Asparagin Agar. ..... 1496 their
Bj. Not containing amino acids, or
Fred and Peterson's Basal Asparagin
1497 salts.
Agar. Agar
Sohngen's Malate Urea
Stuart's Basal Asparagin Agar 1498
1526
(Percival)
Long's Basal Glycerol Asparagin
1499 Perotti's Glucose Dicyandiamide
Agar (Uyei) 1527
Agar
Aj. Complete media. 1528
or their salts. Stapp's Uric Acid Agar
Bi. Containing amino acids 1529
or sodium asparaginate Stapp's Hippurate Agar
Ci. Asparagin
employed.
1496. Miehe's Basal Asparagin Agar
Di. additional organic carbon added.
No
Noyes' Asparaginate Agar 1500 but solidified by the
Same as medium 379,
Agar
Sullivan's Salt Asparagin 1501
addition of agar.
Davis' Asparagin Phosphate Agar.. 1502
Bengis' Phosphate Asparagin Agar. 1503 Fred and Peterson's Basal Asparagin
1497.
Conn's Glucose Asparaginate Agar. 1504 Agar
Kisch's Glucose Asparagin Agar.. . .
1505
Constituents
Dolt's Lactose Asparagin Agar 1506
Water '
1000.0 cc.
D2. Additional organic carbon added.
1.
(NH4)2S04 3.0 g.
El. Only one type of organic
carbon added. 2.
3. Asparagin 1-5 g-
Fi. Carbohydrates added.
Groenewege's Starch Asparagin 4. K2HPO4 2.0 g.
CaCU 0-25 g.
Agar. 1507 5.
MgS04 0.25 g.
Noyes' Starch Asparaginate Agar 1508 . .
6.
Agar 150 g.
Stutzer & Hartleb's Glycerol As-
7.
1509 Preparation:
paragin Agar,
(1) Dissolve 2, 3, 4, 5, 6,
and 7 in 1.
Fj. Alcohols added.
(2) Dissolve one of
the sterile added
Sullivan's Glycerol Asparagin Agar. 1510
nutrients to sterile (1).
Peklo's Mannitol Asparagin Agar. 1511 .
(3) Reaction employed

varied from pH-
Thornton's Mannitol Asparagin
1512 6.5 to 7.2.
Agar
Conn's Glycerol Asparaginate Agar. 1513 sauer-
added. Use: To cultivate yeast found in
Fs. Organic acids or their salts
kraut.
Beijerinck's Malate Asparagin Agar. 1514
Added nutrients: Authors added 2.0%
Fraenkel's Lactate Asparagin Agar basic
1515 glucose, maltose or xylose to the
(Klimmer)
agar medium.
Voges' Lactate Asparaginate Agar.. 1516
Variants: Authors added 2.0 or 4.0% NaCl Preparation

to the basic agar solution. Dissolve
(1) 9 and 10
2, 3, 4, 5, 6, 7, 8, in 1.
Reference: Fred and Peterson (1922 p. (2) Add one of the added nutrients.
261). Sterilization: Not specified.
Use: Cultivation of tubercle bacilli. The
1498. Stuart's Basal Asparagin Agar
author reported that e.xtracts of tubercle
Constituents: show the presence of any
bacilli failed to
1. Agar solution 1000.0 cc. growth stimulating substances. Cod
2. Asparagin 10.0 g. liver oil concentrate e.xerted an indifferent
Preparation action, while concentrations of 1:1000
(1) Prepare an agar solution using Harris yeast vitamin, 1:100 tomato juice
washed agar prepared according to and orange juice, or 1:20 cabbage, exerted
medium 1398. an acceleration of growth.
(2) Dissolve one of the added nutrients in Added nutrients: The author added one of
(1). the following in various concentrations:
(3) Adjust the reaction between pH-7.0 (a) Cod liver oil concentrate (Oscodal of
and 7.2. Funk and Dubin).
Sterilization: Method not given. (b) Harris yeast vitamin.
Use: To study variation in Bacterium (c) Tomato juice.
typhosum. (d) Orange juice.
Added nutrients: The author added one (e) Cabbage.
of the following: (f) Tubercle bacilli extracts.
NaNOa 20g Variants: The author used the basic
KNO3 2.0 g."
medium without any additions.
NH4NO3 2.0 g. Reference: Uyei (1927 p. 427), (1927 p.
Bodium phosphate 2.0 g. 437).
potassium phosphate., 2.0 g.
Variants 1500. Noyes' Asparaginate Agar
(a) The author used asparagin and
4.0 g. Constituents:
added 2.0 g. sodium phosphate, 6.0 g. 1. Water 1000.0 cc.
ammonium lactate and 5.0 g. NaCl. 2. Agar (best) 15.0 g.
(b) The author used 3.4 g. asparagin in 3. Sodium asparaginate 1.0 g.
the basic agar solution and added Preparation
1.0 g. potassium phosphate, 10.0 g. (1) Dissolve 2 and 3 in 1.
ammonium lactate 5.0 g. NaCl, Sterilization: Method not given.
0.2 g. MgS04, 0.1 g. CaClj and 40.0 g. Use: General culture medium. Primarily
glycerol. used for study of soil organisms.
Reference: Stuart (1924 p. 586). Reference: Noyes (1916 pp. 93, 94).
1499. Long's Basal Glycerol Asparagin Agar 1501. Sullivan's Salt Asparagin Agar
(Uyei)
Constituents:
Constituents: 1- Water looO.O cc.
1- Water 1000.0 cc. 2. Asparagin IQ.O g.
2- Agar 25.0 g 3. NaCl 10.0 g.
3. Glycerol 5O.0 g 4. MgS04 3.0 g.
4. Ferrous ammonium ci- 5. Agar lo.O g.
trate 0.05 g Preparation
5. MgS04 10 g (1) Dissolve 2, 3, 4 and 5 in 1.
6. NaCl 2.0 cr
Sterilization Method not
: given.
7. Na.COa 3.0 Use: General synthetic culture medium.
8. KH2PO4... 3.0 Also used to study pigment production.
9. Ammonium citrate. 5.0 Author reported that most of the chromo-
10. Asparagin 5.0 genic bacteria did not produce color on
1504. Conn's Glucose Asparaginate Agar

thismedium. B. pyocijaneus produced
abundant pigment, however. Constituents
Reference: Sullivan (1905-C6 p. 117). 1. Distilled water 1000.0 cc.
2. Agar 12.0 g.
1502. Davis' Asparagin Phosphate Agar
3. Sodium asparaginate 1-0 g.
Constituents: 4. Glucose 1-0 g.
1. Water 1000.0 cc. 5. MgS04 0.2 g.
2. Agar 15.0 g. 6. NH4H2PO4 1-5 g.
3. Asparagin,. 20.0 g. 7. CaClj 0.1 g.
4. MgS04 10-0 g- 8. KCl 0.1 g.
5. K..>HP04 10-0 g- 9. FeCl3 trace..
(1) Dissolve 2, 3, 4 and 5 in 1. (1) Dissolve 2, 5, 6, 7, 8 and 9 in 1.
(2) Adjust to 0.8 or 1.0% normal
Sterilization: Method not given. acid to
Use To produce chlamydospores by Sporo-
:
phenolphthalein.
thrix schenckii. Author reported that (3) May be clarified with egg
or by
the growth of the organism was slightly simply heating for 30 minutes at 15
accelerated if pure or impure sugars be pounds pressure in such a way as not
added. to disturb the sediment and then
Reference: Davis (1914 p. 485), (1915 p. decanting thru cotton filter.
179). (4) Add 1.0 g. glucose and 1.0 g. sodium
asparaginate to (3).
1503. Bengis' Phosphate Asparagin Agar Sterilization: Method not given.
Use: Bacterial counts in soils. Author
Constituents:
1. Distilled water 1000.0 cc. reported that there may be other satis-
2. Asparagin 10.0 g. factory combinations of the salts than the
3. Agar (powdered) 30.0 g. one given. The amount of glucose and

asparaginate were increased without
4. Na2HP04 2.0 g.
Preparation varying the count.
References: Conn (1916 p. 722), Giltner
into incubating flasks. (1921 p. 372).

(2) Filter
Sterilization: Sterilize in Bramhall-Dean Agar
1505. Kisch's Glucose Asparagin
autoclave.
Use: To cultivate large quantities of B. Constituents
Water 1000.0 cc.
1.
coli.
2. K2HPO4 10 g.
Variants :
(a) The author used 2.0 g. (NH4)2HP04 3. MgS04 0.5 g.
NaCl 0.02 g.
instead of Na2HP04. 4.
reS04 trace
(b) Tanner described a similar medium 5.
Calcium phosphate trace
as Dolt's asparagin agar. One of 6.
Dextrose 10.0 g.
these media was composed of 100.0 7.
Asparagin 1-9 g-
cc. of distilled water, 350.0 cc. of a 8.
Agar (2.0%) 20.0 g.
3.0% purified agar solution in water, 9.
2.5 g. asparagin and 0.5 g. Na2HP04. Preparation

(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
The other medium contained 250.0
250.0 of a (2) Add NasCOa until the reaction is
cc. distilled water, cc.
purified agar solution, 6.25 g. slightly alkaline to litmus.

3.0%
asparagin and 1.25 g. Na2HP04. (3) Dissolve 9 in (2).
Tanner reported that these media (4) Dissolve 8 in (3).
would give a good growth of B. coli Sterilization: Not specified.
in 24 hours. Use: To study nitrogen requirements for
References: Bengis (1916 p. 392), Tanner colon typhoid group.
(1919 pp. 50, 65).

Reference: Kisch (1918-19 p. 32).
1506. Dolt's Lactose Asparagin Agar Use: General culture medium, primarily
Constituents: used to study organisms of the soil.
1. Distilled water Reference: Noyes (1916 pp. 93-94).
1000.0 ec.
2. Agar, purified 30.0 g. 1509. Stutzer and Hartleb's Glycerol
3. Asparagin 10.0 g. Asparagin Agar
4. NasHPO^ 2.0 g.
5. Lactose Constituents:
10.0 g.
6. Azolitmin (1.0% solution).. 10.0 cc.
1- Water 1000.0 cc.
Preparation 2. Agar 2O.O g.
(1) Purify agar by cutting it in small 3. Asparagin 10 g.
pieces and soaking it in distilled water 4. Glycerol 1.0 g.
for 24 hours. 5. Potassium phosphate 1-0 g.
Dissolve (1), 2, 3 and 4 in 1. Preparation

(2)
Make neutral to phenolphthalein (1) Dissolve 2, 3, 4, and 5 in 1.
(3)
with NaOH. Sterilization: Not specified.
Add 5.
Variants : Fraenkel used 15.0 g. agar instead
(4)
Prepare 6 by dissolving 1.0 g. of of 20.0 g.
(5)
Kahlbaum's C.P. azolitmin in 100.0 References: Stutzer and Hartleb (1897 p.
g. distilled water and boil for 15
403), Fraenkel (1898 p. 10).
minutes. It is a blue solution after

1510. Sullivan's Glycerol Asparagin Agar
boiling.
(6) Add to (4). Constituents
(5)
1. Water 1000.0 cc.
Use: Detection of acid forming bacteria in 2. Asparagin 10.0 g.
water analysis. Author reported that 3. Glycerol 35.0 g.
B. coli developed red colonies. 4. K2HPO4 1.0 g.
Reference: Dolt (1908 p. 621). 5. (NH4)3P04 10.0 g.

6. MgS04 2.0 g.
1507. Groenewege's Starch Asparagin Agar 7. Agar 10. g.
1. Water agar 1000.0 cc. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
2. Starch 10 g. (2) Medium is slightly acid due to evap-

3. Asparagin 1 -0 g oration of NH3 in sterilization.
4. K0HPO4 0.5 g^ Sterilization : Method not given.
Preparation Use: General culture medium. Author
(1) Dissolve 2, 3 and 4 in 1000.0 cc. tap reported that B. typhosus did not grow in
water agar. (Composition not this medium.
given.) Reference: Sullivan (1905-06 p. 119).
1511. Peklo's Mannitol Asparagin Agar
Use: Diastase production by Phytobacter
ly coper sicum n. sp. (causing tomato rot). Constituents
Author reported that when diastase was 1. Distilled water 500.0 cc.
produced, the starch disappeared. 2. KH2PO4 1.0 g-
Reference: Groenewege (1913 p. 28). 3. K2HPO4 1.0 g
4. MgS04 0.6 g
1508. Noyes' Starch Asparaginate Agar 5. NaCl 0.2 g
Constituents: 6. CaCl, 0.1 g
1- Water 1000.0 cc. 7. Asparagin 2.0 g
2. Agar (best) 15.0 g. 8. Mannitol 10.0 g,
3. Starch 2.0 g. Agar
9. 0.75%
4. Sodium asparaginate 1.0 g. 10. FeaCle trace
(1) Dissolve 2, 3 and 4 in 1. (1) Dissolve 2, 3, 4, 5, and 6 in 200.0 cc.
Sterilization: Method not specified. of distilled water.
(2) To 100.0 cc. of (1) add 400.0 cc. dis- 1514. Beijerinck's Malate Asparagin
tilled water and dissolve 2.0 g. as- Agar
paragin, 1.0 g. mannitol and a trace of
Constituents
Fe2Cl6 in the mixture.
1. Water.
(3) From sterile (2) prepare a 0.75% agar.
2. Agar.
(Method or final sterilization not
3. Sodium malate.
given.)
4. Asparagin.
Sterilization: times
Sterilize (2) several
5. Potassium phosphate.
(method not given). A turbidity forms
6. Mohr salt (NH4)2S04
but disappears when the medium cools.
Preparation
(1) Prepare a water solution of agar that
Author reported good growth.
has been washed free of soluble
material with distilled water.
(2) Pour distilled water over the solidified
1512. Thornton's Mannitol Asparagin Agar
mass and renew the water often.
Constituents Melt and boil.
(3)
1. Water 1000.0 cc.
(4) Add a small amount of nutrient solu-
2. K2HPO4 ,.
10 g.
tion consisting of sodium malate,
3. MgS04-7H20 0.2 g.
asparagin and potassium phosphate
4. CaCU 0.1 g.
(amounts not specified).
FeCls 0.002 g.
5.
(5) Boil again to remove all the air.
6. KNO3. 0.5 g.
(6) Add a drop of neutral solution of
Asparagin 0.5 g.
7. Mohr salt, (NH4)2S04, containing a
8. NaCl 0.1 g.
trace of Na^COs, there should be no
9. Agar., 15.0 g.
turbidity.
10. Mannitol 10 g.
Preparation Use: Cultivation of Spirillum desulfuri-
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9
in 1 by
cans, Spirillum tenue and other sulphate
steaming for 30 minutes. reducing organisms.
(2) Neutralize to turmeric paper. Reference: Beijerinck (1895 p. 108).
(3) Filter thru cotton-wool.
(4) Add 1.0 g. mannitol to (3). 1515. Fraenkel's Lactate Asparagin Agar
(5) Tube in 8.0 cc. quantities. (Klimmer)
22.5 pounds pressure. Constituents
Use: Cultivation of soil forms. 1. Water 1000.0 cc.
References: Thornton (1922 p. 241) taken 2. K2HPO4 2.0 g.
from (1923 p. 277), Cunningham (1924 3. NaCl 5.0 g.
p. 136). 4. Ammonium lactate... 6.0 g.
5. Asparagin 4.0 g.
1513. Conn's Glycerol Asparaginate Agar 6. Agar 15.0 to 30.0 g.
Preparation
Constituents and 6 in 1.
(1) Dissolve 2, 3, 4, 5,
1. Water 1000.0 cc. (indicator
(2) Make distinctly alkaline
2. Agar 15.0 g.
not specified).
3. Glvcerol 10.0 cc.
4. K2HPO4 0.5 g.
Use: Synthetic culture medium.
5. Sodium asparaginate 1-0 g.
Reference: Klimmer (1923 p. 172).
Preparation
(1) Dissolve 2, 3, 4, and 5 in 1. 1516. Voges' Lactate Asparaginate Agar
Same as medium 4416, but containing 10.0
Use : Cultivation of actinomycetes.
g. of agar per liter of medium.
Reference: Conn (1921 p. 7).
1517. Giltay's Citric Acid Asparagin Agar 3. Glycerol lo.o g.

(Lohnis) 4. Asparagin 1.0 g.
Constituents 5. Tannin (preferably from
1. Distilled water 1000.0 cc. Sumack) 1.0 g.
2. KNO3 2.0 g.
6. Potassium citrate 1.0 g.
3. Asparagin 10 7. Agar 2O.O g.
g.
Preparation
4. Citric acid 50g
5. KH2PO4 2^0 g
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
6. MgS04 2.0 g.
7. CaClj 0.2 g.
Use: Cultivation of B. Acaciae. Author
8- FeaCle trace reported that slime was produced.
9. Agar (1.5%) I5.0 g. Reference: Smith (1905 p. 383).
Preparation
1520. Higgins' Basic Lactose Asparagin
(1) Dissolve 2 and 3 in 250.0 cc. of 1.
(2) Dissolve 4, 5, 6, 7 and 8 in 500.0 Agar
cc.
of 1.
Solidify Basal Medium 386 by the addition
(3) Neutralize (2) by the addition of of agar.
KOH.
(4) Mix (1) and (3) and make up to a 1521. Sullivan's Glycerol Nitrate Asparagin
liter. Agar
(5) Dissolve 1.5% agar in (4).
Tube. Constituents:
(6)
Sterilization: Method not 1. Water 1000.0 cc.
given.
Use: To study denitrification. 2. Asparagin 10.0 g.
Reference: Lohnis (1913 p. 98). 3. MgS04 0.2 g.
4. K2HPO4 1.0 g.
1518. Conn's Asparaginate Agar 5. Ammonium lactate 0.5 g.

7. KNO3 0.2 g.
2. Agar 8. Glycerol 10.0 g.
12.0 g.
3. Glucose 9. Agar 20.0 g.
1.0 g
4. Sodium asparaginate Preparation
1.0 g
5. Glycerol (1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1.
10.0 g.
6. NH4H2PO4 Sterilization: Not specified.
1.5 g
7. CaCl2 Use: General Synthetic Medium.
0.1 g
8. MgS04 Reference: Sullivan (1905-06 p. 120).
0.2 g
9. KCl g 0.1
1522. Maassen's Malic Acid Asparagin Agar
10. FeCls trace
(Klimmer)
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7, 8, 9 and 10 in 1. 1. Water 1000.0 cc.
(2) Reaction adjusted to 1.0% acid to N 2. MgS04 (crystalline) 0.4 g.
phenolphthalein. 3. Asparagin 10.1 g.
Sterilization: Method not given. 4. CaCl2 0.01 g.
Use: Cultivation of soil microorganisms. 5. Na2HP04 2.0 g.
Variants: Giltner used 15.0 g. agar and 6. NasCOg (crystal-
specified the use of fused CaCU and line) 2.5 g.
MgS04-7H20. 7. Glucose 5.0 to 10.0 g.
References: Conn (1917 p. 42), Giltner 8. Malic acid 7.0 g.
(1921 p. 373), Harvey (1921-22 p. 102). 9. Agar 15.0 to 30.0 g.
Preparation
1519. Smith's Levulose Asparaginate Agar
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1.
1. Water 1000.0 cc. Use: Synthetic culture medium.
2. Levulose 20.0 g. Reference: Klimmer (1923 p. 172).
Use: Isolation of B. aminophilu. Author

1523. Uschinsky's Glycerol Asparaginate
Agar (Klimmer) reported that B. aminophilus developed a
colony 2.3 mm. in diameter, after 24 hours
Constituents
at 37C., having the appearance of a
1. Water 1000.0 cc.
clear colorless drop of water.
2. K2HPO4 2.0 to 2.5 g.
Reference: Jones (1918 p. 127).
3. MgS04 (Crystalline) . 0.2 to 0.4 g.
4. NaCl 5.0 to 7.0 g. 1526. Sohngen's Malate Urea Agar
5. Ammonium lactate... 6.0 to 7.0 g. (Percival)
6. CaCh 0.1 g.
Constituents:
7. Sodium asparaginate. 3.5 g.
1000.0 cc.
1. Water (tap)
8. Glycerol 30.0 to 40.0 g.
2. Calcium malate 5.0 g.
9. Agar 15.0 to 30.0 g.
3. K2HPO4 ,
0.5 g.
Preparation
4. Urea 10.0 g.
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1.
5. Agar 15.0 g.
Preparation
Use: Synthetic culture medium. 4 and 5 in
(1) Dissolve 2, 3, 1.
1524. Berthelot's Nitrate Tyrosine Agar Use: Cultivation of urea organisms from
by the soil and urine. Author reported that
Same as medium 402, but solidified
urea splitting organisms were surrounded
addition of agar.
with small white crystals of calcium
1525. Jones' Histidin-hydro-chloride Agar carbonate.
Constituents:
Dicyandiamide Agar
1.
1527. Perotti's Glucose
2. Histidin-hydro-chloride . 1.0 g
Same as medium 483 but solidified by the
3. KNO3 0-25 g
CaCl2 002g addition of 1.5% agar.
4.
K2SO4 0.2 g Agar

5. 1528. Stapp's Uric Acid
6. MgS04 0-2 g
5.0 Constituents:
7. K-2HP04 g
1. Water lOCO.O cc.
8. Agar 8.0 g
2. Uric acid 0.5 g.
NajSO (10.0% solution). 12.0 cc.
9. 3
3. Na2HP04 3.0 g.
10. Basic fuchsin solution
4. KH2PO4 10 g.
Preparation
5. CaCl2 0.1 g.
6. MgS04-7H20 0.3 g.
distilled water.
7. NaCl 0.1 g.
Dissolve 6 in 100.0 cc. distilled
(2)
8. Fe2Cl6 0.01 g.
water. Add to (1).
9. Agar (2.0%) 20.0 g.
(3) Dissolve 7 in distilled water.
and and make up to Preparation
(4) Mix (2) (3)
(1) Dissolve 4, 5, 6, 7 and 8
in 100.0 cc. of
1000.0 cc.
agar and add to (4). water. (Meyers solution).
(5) Wash 8.0 g.
450.0 cc. of water.
pounds pressure for (2) Dissolve 2 and 3 in
(6) Sterilize at 15
(3) Mix 50.0 cc. of (1) and (2) and solidify
15 minutes.
a 10.0% NajSOs by the addition of 2.0% agar.
(7) Add 12.0 cc. of
solution.
Erlenmeyer flasks.
(8) Titrate to neutral to litmus using
N/10 HCl.
of saturated alcoholic Use: Isolation of uric acid splitting bac-
(9) Add 30 drops cobayae,
teria from feces and soil, Bac.
solution of basic fuchsin.
Bac. capri, Bac. guano, Bac. musculi,
(10) Mix thoroly and pour
into plates.
step Bac. hollandicus.
Sterilization: Sterilization given in
preparation.
Reference: Stapp (1920 p. 3).
(6) of
1529. Stapp's Hippurate Agar Levine's Boric Acid Peptone Agar.. 1547
I3. Containing salts of divalent cations.
addition of 2.0% agar.
Bacto Lead Acetate Agar (Dehy-
drated) 1548
Lipman and Brown's Glucose Pep-
SUBGROUP II-C. SECTION 3
tone Agar 1549
Basal or complete media containing Waterman's Glucose Peptone Agar. 1550
agar and peptone (or other commercial Pfeiler and Lentz' Ringer Solution
digest); additional constituents, if any, of Agar 1551
known chemical composition. Harden's Glucose Peptone Agar 1552
Ai. Not containing additional materials. G2.* Disaccharides only added.
Jacobi's Peptone Agar 1530 Hi. Sucrose employed.
Molisch's Manganese Peptone Agar.. 1531 Greig-Smith's Sucrose Peptone
Roux and Rochaix' Peptone Agar 1532 . .
Agar 1553
Hesse and Niedner's Nahrstoff Hey- Owen's RawSugar Peptone Agar. . . 1554
den Agar 1533 Vierling's Sucrose Peptone Agar. . . . 1555
Lichtenstein's Yeast Extract Agar.. 1534 H2. Lactose employed.
A2. Containing additional materials. Levine's Eosine Methylene Blue
Bi. All additional materials inorganic. Agar (Dehydrated) 1556
Banning's Basal Salt Peptone Agar. 1535 Bacto Endo Agar (Dehydrated)
Matzuschita's Basal Sodium Chlo- Formula of Levine I557
ride Peptone Agar 1536 Levine's Eosine Methylene Blue
Glaessner's Nahrstoflf Heyden Agar. 1537 Agar 1558
Crendiropoulo and Panayotatou's Levine's Endo Agar 1559
Alkaline Peptone Agar 1538 Robin's Lactose Peptone Agar 1560
Stutzer and Hartleb's Phosphate Harvey's Lactose Peptone Agar 1561
Peptone Agar 1539 H3. Maltose employed.
Molisch's Salt Peptone Agar 1540 Sabouraud's Maltose Peptone Agar
B2. One or more of the added materials (Wurtz) 1562
organic. G3. Polysaccharides, only, added.
Ci.* Not containing nitrogen in addition to Molisch's Dextrin Peptone Agar. 1563 . . .
the peptone. Noyes' Starch Peptone Agar.. .

1564
.
Di.* Only one type of additional organic Vierling's Starch Peptone Agar 1565
carbon supplied. Gibson's Starch Peptone Agar
El. Carbohydrates only added. (Harvey) 1566
Fi. One carbohydrate used. F2. Two or more carbohydrates used.
Gi. Monosaccharides only added. Bacto Eosine Methylene Blue Agar
Hi. Inorganic salts not added. (Dehydrated) 1567
Bacto Sabouraud's De.xtrose Agar Levine's Rosolic Acid China Blue
(Dehydrated) , 1541 Peptone Agar 1.568
Sabouraud's Glucose Peptone Agar E2.* Alcohols only added.
(Anderson) 1542 Sabouraud's Basal Glycerol Peptone
Hj. Inorganic salts added. Agar (Park, Williams & Krum-
Ii. Containing salts of monovalent cations wiede) 1569
only. Omelianski's Alcohol Peptone Agar. 1570
Matzuschita's Glucose Peptone Robinson and Rettger's Glycerol
Agar 1543 Opsine Agar 1571
Glaessner's Glucose Peptone Agar.. 1544 Hesse's Glycerol NahrstofT Heyden
Hucker and Wall's Glucose Peptone Agar.... 1572
Agar 1545 Molisch's Glycerol Peptone Agar. 1573
. .
Hesse and Niedner's Glucose Pep- Spengler's Glycerol Somatose Agar. 1574
tone Agar 1546 E3. Organic acids or their salts added.
Ij. Containing salts of trivalent cations. Omelianski's Formate Peptone Agar. 1575
* See page 444 for C2 and D2. * See G3 and E3.

Boekhout and de Vries Tartrate Preparation

Peptone Agar 1576 (1) Dissolve 2, 3 and 4 in 1 by heating
Dawson's Butter Soap Peptone over a free flame.
Agar 1577 (2) Make up to the original volume by
Harvey's Phenol Peptone Agar 1578 the addition of water.
Da. More than one type of additional (3) Add Na2C03 or sodium phosphate
organic carbon supplied. until the reaction is slightly alkaline.
El. Carbohydrates and alcohols added. (4) Pour into a flask and steam until the
Bacto Malt Extract Agar (Synthe- albuminous material to be
still
tic) (Dehydrated) 1579 removed iscompletely separated.

Harvey's Glucose Glycerol Agar 1580 (Usually 2 hours if sodium phosphate
Ej. Carbohydrates and organic acids added. is used and longer if NajCOs.)
Beijerinck's Glucose Peptone Agar. 1581 (5) Filter thru cotton, using compressed
Sarbouraud's Glucose Peptone Agar air to effect a fast filtration.
(Serena) 1582 (6) Distribute into smaller flasks.
Chantemesse's Phenol Peptone Agar (7) Distribute sterile (6) into sterile test
(Bezan5on) 1583 tubes that have been autoclaved in
Boekhout and de Vries' Maltose the autoclave for 2J hours. (The
Peptone Agar 1584 tubes are contained in an enamel
Cheyney's Maltose Peptone Agar... 1585 container in the autoclave. The
Sabouraud's Glycerol Peptone Agar 1586 inner temperature reaching about
Ea. Other combinations of organic carbon 150C.)
added. Sterilization: Heat (6) in streaming vapor
Robinson and Rettger's Glycerol for two hours (if the glassware used has
Opsine Citrate Agar 1587 been sterilized in the autoclave).
Botelho's Lacto-phenol Peptone Use: General culture medium.
Agar 1588
Reference: Jacobi (1888 p. 538).
Cj. Containing nitrogen in addition to
peptone.
1531. Molisch's Manganese Peptone Agar
Di. Inorganic nitrogen added.
Sullivan's Ammonium Lactate Pep- Constituents:
tone Agar 1589 1. Water (Moldau River) 1000.0 cc.
Lipman and Brown's Nitrate Pep- 2. Manganese peptone 0.5 g.
tone Agar 1590 3. Agar 10.0 g.
Beijerinck's Ferric Ammonium Ci- Preparation
Agar (Janke)
trate Peptone 1591
Vierling's Nitrate Peptone Agar 1592
(2) Pour in plates.
Heinemann's Asparagin Peptone Sterilization: Method not given.
Agar. 1593
Use: Cultivation of iron bacteria, Lepto-
Dj. Inorganic nitrogen not added; addi-
thrix ochracea.
tional organic nitrogen supplied.
MacConkey's Basal Bile Salt Pep-
tone Agar 1594
Harrison and van der Leek's Aescul- 1532. Roux and Rochaix' Peptone Agar
in Bile Salt Agar 1595
Constituents:
Harvey's Brilliant Green Bile Salt
1596 1. Distilled water 1000.0 cc.
Agar
2. Agar 25.0 g.
1530. Jacobi's Peptone Agar 3. Peptone 10.0 to 15.0 g.
1. Water 1500.0 cc. (1) Soak 25.0 g. of chopped agar for 24
2. Meat peptone (Kem- hours in 500.0 cc. of water acidulated
merich's) 7.5 g. by the addition of 6.0% HCl. Stir
3. Peptone (siccum) 15.0 g. occasionally.
4. Agar-agar 15.0 to 22.5 g. (2) Wash thoroly with water.
(3) Soak the agar for 24 hours in 500.0 Sterilization: Not specified.
cc. of a 5.0% ammonia solution. Use: The authors used the medium pri-
(4) Wash thoroly. marily for water analysis, but various
(5) Place in a liter of distilled water and investigators have used the same or
heat until the agar is dissolved. similar media for a large variety of
(6) Add 50.0 cc. of water containing 10.0 purposes.
to 15.0 g. peptone. Variants
(7) Neutralize by the addition of a (a) Wimmer used the medium with only
saturated solution of NaHCOa. 980.0 cc. water instead of 1000.0 cc.
(8) Pass thru flannel and then filter using to study nitrification.
a hot water funnel. (b) Klimmer used 8.0 g. Nahrstoff-
(9) Distribute into flasks or tubes. Heyden and 13.0 g. agar.
Sterilization: Sterilize at 115 to 120 for References: Hesse and Niedner (1898 pp.
30 minutes. 454-462), Wimmer (1904 p. 139), Smith
Use: General culture medium. (1905 p. 196), Tanner (1919 p. 50), Percival
Variants (1920 p. 121), Klimmer (1923 p. 194).
(a) Noyes dissolved 15.0 g. of the best
1534. Lichtenstein's Yeast Extract Agar
agar and 0.05 g. of Witte's peptone in
1000.0 cc. of water. Same as medium 518, but solidified by the
(b) Harvey dissolved 18.0 g. of agar and addition of agar.
peptone in 1000.0 cc. of water.
30.0 g.
1535. Banning's Basal Salt Peptone Agar
This medium was used to cultivate
hyphomycetes. Constituents
(c) Mortensen solidified medium 560 by 1. Water 1000.0 cc.
the addition of 2.0 or 2.5% agar. 2. KH.PO4 3.0 g.
(d) Moll used a 2.0% Witte peptone 3. MgS04 2.0 g.
solution solidified with 2.0% agar as 4. Peptone 10.0 g.
a basal medium and added a variety 5. Agar 10.0 g.
of inorganic salts to study the effect Preparation
of salts on the growth of molds. He (1) Dissolve 2, 3, 4 and 5 in 1.
reported that the toxicity of salts is (2) Do not adjust the reaction.
an additive characteristic of the (3) Dissolve one of the added nutrients
ion. The toxicity depends on the in (1).
solubility and the ionization of the Sterilization: Heat to 75C. to sterilize
salt. (method not given).
References: Roux and Rochaix (1911 p. Use: To study o.xalic acid formation by
115), Noyes (1916 pp. 93-94), Harvey Bad. xylinum, Bad. aceti oxalici and
(1921-22 p. 102), Mortensen (1909 p. 523), Bad. diabeticum. Author reported that
Moll (1920 p. 258). when nitrogenous materials were added
oxalic acid was not produced.
1533. Hesse and Niedner's Nahrstoff- Added nutrients: The author added one
Heyden Agar of the following materials:
Constituents: methyl alcohol, 10.0 g.
"
1. Distilled water 1000.0 cc. ethyl alcohol 30.0 g.

2. Agar 12.5 g. propyl alcohol 20.0 g.
3. NahrstofT-Heyden 7.5 g. butyl alcohol 10.0 g.
Preparation amyl alcohol 5.0 g.
(1) Mix 3 with a little cold distilled water ethylene glycocol 10.0 g.
forming a paste. glycerol 10.0 g.
(2) Dissolve 2 and (1) in 1. erythritol 10.0 g.
(3) Filter thru cotton or paper in the mannitol 10.0 g.

steamer or hot water funnel. sodium salt of acetic acid 10.0 g.
(4) Adjustment of reaction not required. propionic acid 10.0 g.
(5) Pour in plates. butyric acid 5.0 g.

isobutyric acid 5.0 g. infusion from one of the following

valearic acid 5.0 g. materials:
glyoxalic acid 2.5 g. intestinal mucosa feces (human)
lactic acid 10.0 g. liver brain
malonic acid 2.5 g. pancreas rice
benzoic acid (potassium salt) 5.0 g. spleen pea
salicilic acid 5.0 g. Variants
pyrotaric acid 2.5 g. (a) The author used undiluted bile,
malic acid 10.0 g. urine or beer wort instead of infusion

tartaric acid. 10.0 g. listed above.
salicylic acid 5.0 g. (b) Matzuschita cultivated spore forming
glycocol (sodium salt) 10.0 g. bacilli, Clostridium butyricum,
leucine (sodium salt) 10.0 g. Bacillus oedematis maligni, Bacillus
hippuric acid 5.0 g. anthracis syniptomatici, Bacillus
sarcosine 2.5 g. sporogenes, Bacillus botulinus, on a
uric acid (potassium salt) 5.0 g. medium prepared as follows:
Urea 10.0 g. (1) Dissolve 10.0 g. Koch's meat pep-
creatin 2.5 g. tone, 20.0 g. agar, 5.0 g. NaCl,
creatinin 2.5 g. and 2.0% glucose (the glucose may
glucose 20.0 g. be omitted) in 1000.0 cc. water by
levulose 20.0 g. boiling in a steamer.
galactose 20.0 g. (2) Neutralize.
maltose 20.0 g. (3) Filter.
sucrose 20.0 g. (4) Sterilize in the steamer on from 2 to
lactose. 20.0 g. 5 successive days for 15 to 30
raffinose^. 20.0 g. minutes.
rhamnose 10.0 g. (5) Incubate for 2 days at 37C. to test
arabinose 20.0 g. sterility.
starch (wheat) 10.0 g. (c) Sullivan used the basal solution with
inulin 10.0 g. 20.0 g. peptone instead of 10.0 g.,
glycogen 10.0 g. as a general culture medium for

dextrin 10.0 g. Microspira comma, B. anthracis, B.
gum arable 10.0 g. pneumoniae, photogenic and chromo-
Reference: Banning (1902 p. 395-427). genic bacteria. He reported slow
growth and slight pigment pro-
1536. Matzuschita's Basal Sodium Chloride duction.
Peptone Agar Harvey used the basic solution with
(d)
Constituents: 10.0 to 20.0 g. peptone added. He
1. Water 1000.0 cc. merely stated that the solution be
2. Agar (2.0%) 20.0 g. solidified by the addition of agar, not
3. Peptone (1.0%) 10.0 g. giving the amount employed.
4. NaCl (0.5%) 5.0 g. (e) Teague and Deibert reported that a
Preparation: medium containing peptone, NaCI
(1) Prepare a watery infusion of one of and agar would not support the
the materials given under added growth of Unna-Ducrey bacillus.
nutrients. References: Matzuschita (1901-02 p. 214),
(2) Add 2.0% agar, 1.0% peptone and (1902 p. 288), Sullivan (1905-06 p. 114),
0.5%o NaCl to (1). Harvey (1921-22 p. 101), Teague and
(3) Neutralize (indicator not given). Deibert (1922 p. 70).
1537. Glaessner's Nahrstoff-Heyden Agar
Use: Cultivation of intestinal bacteria.
Similar media were used for the cultiva- Constituents:
tion of various other organisms. 1. Water 1000.0 cc.
Added nutrients: The author prepared 2. Agar (pulverized) 15.0 g.
CULTURE MEDIA FOR CULTIVATION" OF MICROORGANISMS 447
3. Nahrstoff-Heyden 10.0 g. Sterilization: Method not given.

4. XaCl,.., 5.0 g. Use: Used by Stutzer and Hartleb in an
Preparation attempt to cultivate the foot and mouth
(1) Dissolve 2, 3 and 4 in 1. disease bacterium. Temple and others
Sterilization: Method not given. used similar media to determine bacterial
Use: Cultivation of diphtheria bacilli. counts in soil.
Author reported better growth on Variants: Temple used 0.1 g. K2HPO4, 1.0
Loeffler's blood serum. g. peptone and 15.0 g. of agar per liter.
Reference: Glaessner (1900 p. 729). References: Stutzer and Hartleb (1897 p.
404), Temple (1912 p. 206), Brown (1913
1538. Crendiropoulo and Panayotatou's
p. 499).
Alkaline Peptone Agar
1540. Molisch's Salt Peptone Agar
Constituents
2. Peptone 5.0 g. 1. Distilled water 1000.0 cc.
3. NaOH (10.0%) 10.0 cc. 2. MgS04 0.5 g.
[agar 3.0 g. 3. K2HPO4 0.5 g.
4. Agar j peptone 1-0 g. 4. FeS04 trace
medium ]
NaCl 0.5 g. 5. Peptone 10.0 g.
[HjO 100.0 cc. 6. Agar 18.0 g.
(1) Dissolve 5.0 g. of peptone (Witte or (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Chepoteau) in 190.0 cc. of water. Sterilization: Not specified.
(2) Add 10.0 cc. of 10.0% solution of Use: Cultivation of purple bacteria.
NaOH (8.0 cc. if using Witte's pep- Reference: Molisch (1907 p. 11).
tone) and heat 3 to 5 minutes.
1541. Bacto Sabouraud's Dextrose Agar
(3) Cool and filter thru paper.
(Dehydrated)
(4) Prepare neutral peptone agar by
dissolving 3.0 g. agar, 1.0 g. peptone Constituents:
and 0.5 g. NaCl in 100.0 cc. water. 1. Water
(5) Mi.x 4 parts sterile (4) with 6 parts 2. Peptone (Bacto) 10.0 g.
sterile (5) and pour in sterile plates. 3. Glucose (Bacto) 40.0 g.
Sterilization: Sterilize (4) at 100C. for 30 4. Agar (Bacto) 15.0 g.
minutes. Method of sterilization of (5) Preparation
not given. (1) Dissolve 65.0 g. of Bacto Sabouraud's
Use: Diagnosis of cholera. Author re- Dextrose Agar (dehydrated) in 1000.0
ported that vibrio colonies are round, cc. of distilled water by boiling.
semi-transparent, bluish at first and later (2) Distribute as desired.
whitish. (3) If sterilized for 20 minutes at 15
Reference: Crendiropoula and Panayota- pounds pressure pH = 5.6.
tou(1910p. 249). Sterilization: Sterilize as desired. Avoid
e.xcess heat on account of high acidity of
1539. Stutzer and Hartleb's Phosphate
medium.
Peptone Agar
Constituents Reference: Digestive Ferments Co. (1925
1. Water 1000.0 cc. p. 14).
2. Agar 20.0 g.
1542. Sabouraud's Glucose Peptone Agar
3. Peptone 20.0 g.
(Anderson)
(1) Dissolve 2, 3 and 4 in 1. 1. Water 1000.0 cc.
(2) Neutralize by the addition of soda 2. Agar 20.0 g.
(indicator not specified). 3. Peptone 10.0 g.
(3) Add0.5g. NasCOsto (2). 4. Glucose 40.0 g.
Preparation 1543. Matzuschita's Glucose Peptone .4.gar

Constituents
(2) Adjust to +2 acid with N/1 HCl.
Add 4.
1. Water lOOO.O cc.
(3)
2. Peptone. 10.0 g.
(4) Tube in sterile tubes.
3. NaCL......" 5.0 g.
Sterilization: Sterilize for 10 minutes at 5
pounds pressure.
4. Agar 12.0 g.
5. Glucose (2.0%) 20.0 g.
Use: To isolate yeast found in human .
,
Preparation
intestinal tract. Different investigators
(1) Prepare ordinary nutrient agar using
used similar media to cultivate yeast,
water instead of bouillon. (Peptone
molds and other organisms.
and NaCl in amounts indicated were
Variants
assumed to be the constituents of
(a) BezanQon used agar instead of
18.0 g.
nutrient agar.)
20.0 g. The medium was used to
cultivate hyphomycetes, Sporotri- (2)Add 2.0% glucose to (1).
chum beurmanni Not specified.
Sterilization:
Use: Cultivation of mammalian and
(b) Harvey cultivated tineae, molds and
chicken tubercle bacilli. Author re-
eporothrix on a medium prepared as
ported that the mammalian types grew
follows:
poorly. Chicken types gave colonies of
(1) Prepare: Granulated peptone 10;
commercial glucose 40; agar 18;
1.5 mm. after six days.
Variants
water 1000.
(a) Truche and Cotoni solidified variant
(2) Raise slowly in the autoclave to a
(a) medium 596 by the addition of
temperature of 120C. and then
agar.
allow to fall to 100C.
(b) Harvey solidified medium 595 with
(3) Shake, to mix, at a temperature of
agar.
100 C.
References: Matzuschita (1899 p. 128),
(4) Have in readiness 2 one-liter flasks
Truche and Cotoni (1911 p. 480), Harvey
with funnels and thick, filter
(1921-22 p. 109).
paper.
(5) Place these flasks in the hot
1544. Glaessner's Glucose Peptone Agar
autoclave.
(6) Filter.
Same as medium 1537 but
contains 1.0%
(7) Keep the unfiltered medium hot. glucose. The author also described a
(8) Replace the funnels and filter medium the same as 1537, containing in
paper by new funnels with filter addition 10.0 g. peptone and 10.0 g. glucose.
paper as soon as filtration becomes
1545. Hucker and Wall's Glucose Peptone
slow.
Agar
Note: The filtration of this
medium is particularly difficult Constituents
and slow. 1. Water 1000.0 cc.
(9) Distribute the filtrate into test 2. Peptone 40.0 g.
tubes. 3. Glucose 2.0 g.
Note: The tubes should be 4. K2HPO4 5.0 g.
capped during incubation (which 5. Agar 15.0 g.
may be long) but may be placed Preparation
in a covered receptacle with its (1) Dissolve 2, 3, 4 and 5 in 1.
cover only just open. (2) Tube.

(10) No adjustment of reaction of this Method not given.
Sterilization:
medium required. Use To determine ammonia production by
:
References : Anderson (1917 p. 343), Tanner organisms. Add 1.0 cc. of a 10.0%
(1919 p. 51), Bezangon (1920 p. 646), phenol and 1.0 cc. of a 1.0% (available
Harvey (1921-22 pp. 101, 110). chlorine) sodium hypochlorite solution

A blue
to the surface of the agar slant. Sterilization: Sterilize in the usual manner.
color developing within 30 minutes Use: Primarily used for differentiating
denotes ammonia production. Sal. paratyphi from Sal. schotmuelleri
Reference: Hucker and Wall (1922 p. 516), Used also in the study of other organisms
(1922 p. 485). of this group.
1546. Hesse and Niedner's Glucose Peptone
p. 12).
Agar
Constituents
1549. Lipman and Brown's Glucose Peptone
Distilled water Agar
1. 1000.0 cc.
2. Agar 12.5 g. Constituents
3. Peptone (Gehe & Co.) 10.0 g. 1. Water (tap) 1000.0 cc.
4. De.xtrose 10 g. 2. Dextrose 10.0 g.
5. Sodium phosphate (ampho- 3. K2HPO4 0.5, 1.0 or 1.5 g.
teric) 0.5 g. 4. MgS04 0.2 g.
Preparation 5. Peptone 0.05 g.
(1) Dissolve 2, 3, 4 and 0.5 g. amphoteric 6. Agar 20.0 g.
reacting sodium phosphate in 1 by Preparation
boiling. (1) Dissolve 2, 4, 5 and 6 in 1.
Sterilization: Not specified. (2) To 1000.0 cc. of (1) add 0.5 g. of

Use: Used in water analysis. K2HPO4. This is medium A.
Reference: Hesse and Niedner (1898 pp. (3) To lOOO.O cc. of (1) add 1.0 g. K2HPO4.
454-462). This is medium B.
(4) To 1000.0 cc. of (1) add 1.5 g. K2HPO4.
1547. Levine's Boric Acid Peptone Agar
This is medium C.
Constituents: (5) To 1000.0 cc. of (1) add 0.5 g. K2HPO4,
1. Water 1000.0 g. and 0.5 cc. of N/1 HCl. This is
2. Peptone (1.0%) 10.0 g. medium D.
3. Agar (1.5%) 15.0 g. (6) To 1000.0 cc. of (1) add 0.5 g. K2HPO4
4. K2HPO4(0.3%) 3.0 g. and 1.0 cc. N/1 HCl. This is me-
5. Glucose (0.05%) 0.5 g. dium E.
6. Boric acid (0.63%) 6.3 g. Sterilization: Not specified.
Preparation: Use: Bacterial count of soil. Author re-
(1) Dissolve 2, 3, 4, 5 and 6 in 1. ported that Media A, B, D and E or
Sterilization: Not specified. combinations (2), (3), (5) and (6) gave
Use: Differentiation of Bad. coli and Bad. practically the same results. The re-
aerogenes. Author reported that Bad. action with 1.5 g. K2HPO4 seemed to
aerogenes did not show growth. Bad. inhibit development.
coli grew luxuriantly. Variants
Reference: Levine (1911 p. 22). (a) Authors used 0.5 g. K2HPO4 and 0.1
to 0.5 g. peptone, instead of amounts
1548. Bacto Lead Acetate Agar
used above.
(Dehydrated)
(b) Waksman used 1.0 g. KH2PO4, 5.0 g.
Constituents peptone and 25.0 g. agar instead of
1. Distilled water amounts used by Lipman and Brown.
2. Peptone (Bacto) 10.0 g References: Lipman and Brown (1910 pp.
3. Agar (Bacto) 12.0 g 447, 451, 592), Tanner (1919 pp. 49, 66),
4. Glucose (Bacto) 1.0 g Heinemann (1922 pp. 38, 40), Waksman
5. NaCl...., 5.0 g (1922 p. 340).
6. Lead acetate 0.1 g
1550. Waterman's Glucose Peptone Agar
Preparation
(1) Dissolve 20.0 g. of Bacto Lead Acetate Constituents
Agar (dehydrated) in 1000.0 cc. of 1. Agar solution 1000.0 cc.
distilled water. 2. Glucose (2.0%) 20.0 g.
3. Peptone (0.2%) 2.0 g. Variants: Park, Williams and Krumwiede

4. KH0PO4 (0.1%) 1.0 g. used 1.0 g. NaHCOa with 1.0 or 2.0%
5. CaCOa (0.2%) 2.0 g. peptone and 1.5 or 2.0% agar instead of
Preparation amounts indicated above.
(1) Dissolve 2, 3, 4 and 5 in 1000.0 cc.of References: Pfeiler and Lentz (1913 p. 123),
agar solution. Park, Williams and Krumwiede (1924 p.
Sterilization: Method not given. 122).
Use: To study acid production by acetic
1552. Harden's Glucose Peptone Agar
acid bacteria, Acetobacter melanogenum,
B. Pasteurianum, B. rancens, B. xylinum Constituents
and acetic acid bacteria from beer. B. 1. Water 500.0 cc.
xylinum and Acetobacter melanogedum 2. Glucose 10.0 g.
produced acid. The remaining bacteria 3. Peptone (Witte) 5.0 g.
listed produced no or very little acid. 4. Chalk 5.0 g.
Variants Janke gives this medium specify-
: Preparation
ing that the agar be a 2.0% agar solu- (1) Dissolve 2 and 3 in 1.
tion in water. (2) Add sterile 4 to sterile (1).

References: Waterman (1913 p. 453), Janke Sterilization: Sterilize (1); method not
(1916 p. 6). given.
Use: To study fermentation of glucose.
1551. Pfeiler and Lentz's Ringer Solution Reference: Harden (1905 p. 488).
Agar
1553. Greig-Smith's Sucrose Peptone Agar
Constituents:
1. Water 1000.0 cc. Same as medium 613 but solidified by the
2. NaCl 10.0 g. addition of 2.0% agar.
3. KCl 0.2 g.
1554. Owen's Raw Sugar Peptone Agar
4. CaCl2 0.2 g.
5. Sodium bicarbonate 0.1 g. Constituents
6. Glucose 1.0 g. 1. Water 1000.0 cc.
7. Agar 15.0 g. 2. Agar (2.0%) 20.0 g.
8. Peptone 10.0 g. 3. Sucrose (second 80)
Preparation (10.0%) 100.0 g.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. 4. Peptone (1.0%) 10.0 g.
(2) Add agar and peptone to (1) and boil Preparation:

three hours. (1) Dissolve 2, 3 and 4 in 1.
(3) Cool to 50C. and clarify with egg Sterilization: Not specified.
albumin powder. Add albumin, boil Use: Bacterial count of cane sugar
and filter. products.
Sterilization: Method not given. Reference: Owen (1914 p. 338).
Use: General inexpensive culture medium.
1555. Vierling's Sucrose Peptone Aâr
Author reported that organisms retained
their characteristic growth, their patho- Same as 622 but solidified by the addition
genicity, their ability to produce pigment, of 2.0% agar.
their agglutinative ability and their
1556. Levine's Eosine Methylene Blue Agar
gram stain on this medium. (Messer-
(Dehydrated)
schmidt in Centr. f. Bakt., 68: 107-111
1913, compares this medium with meat Constituents:
extract agar and finds meat extract 1. Distilled water
medium far superior. This medium does 2. Peptone, Bacto 10.0 g
not give characteristic reaction when 3. Agar, Bacto 15.0 g
used as a base for Endo Fuchsin agar, 4. K2HPO4 2.0 g

Conradi-Drigalski agar or Loeffler's Mala- 5. Lactose, Bacto 10.0 g
chite green agar according to Messer- 6. Eosine 0.4 g

schmidt.) 7. Methylene Blue 0.1 g
Preparation (4) Just prior to use add to each 100.0

(1) Dissolve 37.5 g. of Levine's Eosine cc. of sterile (3) 1.0 g. of sterile lactose
Methylene Blue Agar (Dehydrated) or 5.0 cc. of 20.0% sterile lactose
in 1000.0 cc. of water by boiling, or solution. Also to each 100.0 cc. add
better autoclaving. 2.0 cc. of a 2.0% aqueous solution of
(2) If sterilized at 15 pounds for 20 eosin (yellowish) and 2.0 cc. of a
0.5%
minutes pH = 7.1. aqueous methylene blue solution.
Sterilization : Sterilize in the usual manner. (5) Mix thoroly and pour into sterile
Use: Differentiation of the coli-aerogenes petri dishes. Allow to harden in the
group. incubator.
Reference: Digestive Ferments Co. (1925 Sterilization: Sterilize (3) at 15 pounds
p. 10). pressure for 15 minutes.
Use: To differentiate between B. coli and
1557. Bacto Endo Agar (Dehydrated)
B. aerogenes. Author reported that
Formula of Levine
Bad. coll colonies well isolated 3-4 mm.
Constituents: in diameter. Colonies flat, sometimes
1. Distilled water concave, dark and sometimes with a
2. K2HPO4 3.5 g. green metallic sheen. Bad. aerogenes
3. Peptone, Bacto 10.0 g. isolated colonies 4r-6 mm. in diameter;
4. Agar, Bacto 15.0 g. neighboring colonies run together
5. Lactose, Bacto 10.0 g. quickly, colonies raised and convex;
6. NasSOa 2.5 g. centers deep brown. By reflected light
7. Fuchsin, Basic, (10.0% ale. colonies are much lighter than Bad. coli.
soln.) 5.0 cc. Variants: Skinner and Murray added
Preparation: 1 : 100,000 crystal violet to the medium to
(1) Dissolve 41.5 g. of Bacto Endo Agar inhibit the development of spreaders.
(Dehydrated) in 1000.0 cc. of distilled References: Levine (1918 p. 43), Tanner
water by using as little heat as (1919 p. 54), Levine (1921 p. 117), Harvey
possible, autoclaving is recom- (1921-22 p. 92), A. P. H. A. (1923 p. 96),
mended. (1925 p. 99), Skinner and Murray (1924 p.
(2) If sterilized at 15 pounds for 20 590).
minutes pH = 7.5 .
1559. Levine's Endo Agar
Use: Differentiation of colon-typhoid Constituents
group. 1. Distilled water 1000.0 cc.
Reference: Digestive Ferments Co. (1925 2. Peptone (Difco)....,. 10.0 g.
p. 11). 3. Dipotassium phos-
phate (K2HPO4) 2.0 to 5.0 g
1558. Levine's Eosine Methylene Blue Agar
4. Agar 15.0 to 30.0 g.
Constituents: 5. Lactose 10.0 g.
1. Distilled water.., 1000.0 cc. 6. Fuchsin (10.0% soln.)
2. Peptone (Difco) 10.0 g. 7. Na.SOa (10.0% soln.)
4. Agar 15.0 g. (1) Boil 2, 3 and 4 in 1 until solution is
5. Lactose 10.0 g. or sterile complete.
20.0% solution) 50.0 cc. (2) Make up any loss due to evaporation.
6. Eosine (yellowish 2.0% aq. (3) Not necessary to filter or adjust the
solution) 20.0 cc. reaction.
7. Methylene blue (5% aq. (4) Distribute in measured quantities
solution) 20.0 cc. and sterilize.
Preparation (5) When ready for use add 1.0 g. of
(1) Dissolve 2, 3 and 4 in
1 by boiling. sterile lactose, or 5.0 cc. of a 20.0%
(2) Make up due to evaporation.
loss sterile lactose solution, 0.5 cc. of a
(3) Place measured quantities in flasks. 10.0% (saturated) alcoholic solution of
basic fuchsin, and 2.5 cc. of a freshly Preparation

prepared 10.0% Na2S03 solution to (1) Dissolve 2, 3 and 4 in 1 as in the
each 100.0 cc. of sterile (4). preparation of ordinary nutrient
(6) Pour into plates and allow to harden agar.
in the incubator. Sterilization: Method not given.
Use: Direct isolation or confirmation of Use: Cultivation of molds.
presumptive test for lactose fermenters. Variants
Variants : (a) Harvey used 18.0 g. agar, 40.0 g. of
(a) Harvey specified the use of 3.5 g. commercial French maltose and 10.0
K0HPO4 and 20.0 g. agar. g. of Chassaing peptone.
(b) Harris specified the use of 3.5 g. References: Wurtz (1897 p. 47), Harvey
K2HP04and 15.0 g. agar. (1921-22 p. 110), Stitt (1923 p. 208).
References: Levine (1921 p. 115), Harvey
(1921-22 p. 93), Harris (1925 p. 280). 1563. Molisch's Dextrin Peptone Agar
Constituents
1560. Robin's Lactose Peptone Agar Water (Moldau)
1. 1000.0 cc.
Constituents : 2. Agar... 18.0 g.
Water 3. Peptone 5.0 g.

1. 1000.0 cc.
4. De.xtrin. 5.0 g.
2. Agar 32.0 g. .,
Preparation
3. Peptone (Collas) 20.0 g.
5. Blue, soluble
6. Lactose
Use: Cultivation of purple bacteria.
40.0 g.
Preparation Reference: Molisch (1907 p. 11).
(1) Dissolve 2, 3 and 4 in 1 by heating at

1564. Noyes' Starch Peptone Agar
115 in the autoclave for 5 to 10
minutes. Constituents:
Add 1.0 cc. of a 1.0% aqueous solution 1. Water.,, 1000.0 cc.
(2)
of soluble blue to boiling (1). 2. Agar (best) 15.0 g.
Starch
(3) Add sufficient tenth normal KOH 3. 2.0 g.
solution to decolorize 4. Peptone...., 0.05 g.

(2).
Add 40.0 Preparation:
(4) g. lactose to (3).
Filter. (1) Dissolve 2, 3 and 4 in 1.
(5)
(6) Distribute in tubes. Sterilization: Not specified.

Use: General culture medium. Used pri-
minutes. marily to study soil organisms.
Use: Differentiation of coli and typhoid Reference Noyes (1916 pp. 93, 94).
:
bacilli. Author reported that coli colo- 1565. Vierling's Starch Peptone Agar
nies colored the medium blue while ty-
phoid colonies were colorless. Constituents:
Reference: Robin (1897 p. 50). 1. Water 1000.0 cc.
2. K2HPO4 1.0 g.
1561. Harvey's Lactose Peptone Agar 3. CaClo 0.1 g.
4. MgS04 0.1 g.
Same as medium 607 but solidified by the 5. FeCls,. trace
addition of agar. 6. NaCl..,.., trace
7. Starch 5.0 g.
1562. Sabouraud's Maltose Peptone Agar
8. Peptone 10.0 g.
(Wurtz)
9. Agar
1. Water..,., 1000.0 cc. (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
2. Maltose ^, 38.0 g. (2) Solidify with agar (amount not
3. Peptone 5.0 to 8.0 g. specified).
4. Agar 14.0 g. Sterilization: Not given.
Use: To study amylase production by Reference: Digestive Ferments Co. (1925

mycobacteria. Author reported that the p. 11).
agar was slightly turbid. The medium
1568. Levine's Rosolic Acid China Blue
was slightly cleared around mycobacteria
Peptone Agar
colonies. The was more notice-
clearing
able if the starch content was increased Constituents
1.5 or 2.0%. The starch was dissolved. 1. Distilled water 1000.0 cc.
No color was produced near the colonies 2. Agar 15.0 g.
when Lugol's solution was added. 3. Peptone 10.0 g.
Reference: Vierling (1920 p. 204). 4. K2HPO4 4.0 g.
5. Lactose, 20% soln 50.0 cc.
1566. Gibson's Starch Peptone Agar Glucose, 5% soln
6. 10.0 cc.
(Harvey) Rosolic acid (1.0% in 90.0%
7.
Constituents alcohol) 10.0 cc.

1. Water. 1000.0 cc. 8. China-blue (0.5% in water).. 10.0 cc.
2. Peptone 20.0 g. Preparation
3. NaHCOs 1.5 g. (1) Dis.solve 2, 3 and 4 in 1.
4. Agar 30.0 g. (2) pH = 7.4 to 7.5, requires no adjust-

5. Potato starch 10.0 g. ment.
6. Litmus (3) Need not be filtered if used on plates.
Preparation (4) Distribute in 100.0 cc. lots.
(1) Di.s.solve 2, 3 and 4 in 1. (5) To melted sterile (4) add 5.0 cc.
(2) Filter, while hot, thru well-wetted sterile 5, 1.0 cc. of sterile 6, 1.0 cc. of
thick filter paper. 7 and 1.0 cc. of 8 per 100.0 cc.
(3) Make a suspension of 10.0 g. potato (6) Mix thoroly and pour in sterile
starch with a little of the hot filtrate plates.
and add to the bulk of the filtrate. Sterilization: Sterilize (4), method not
(4) Mix. given.
(5) Add sterile litmus solution to sterile Use: To isolate dysentery bacilli. The
(4) to give the desired color. author reported that the dyes such as
Sterilization: Sterilize (4) at 100C. on eosin, methylene blue, the fuchsin-
each of three successive days. sulphite indicator, and an excess of
Use: Detection of V. cholerae. Rosolic acid and china blue inhibited
Reference: Harvey (1921-22 p. 112). many dysentery types.
Variants: Harvey used 5.0 g. peptone
1567. Bacto Eosine Methylene Blue Agar
instead of 10.0 g.
(Dehydrated)
Reference: Levine (1920 p. 39), Harvey
Constituents: (1921-22 p. 88).
1. Distilled water
1569. Sabouraud's Basal Glycerol Peptone
2. Peptone, Bacto 10.0 g
3. Agar, Bacto 15.0
Aâr (Park, Williams & Krumwiede)
g
4. Lactose, Bacto 5.0 g Constituents
5. Sucrose, Bacto 5.0 g 1. Water 1000.0 cc.
6. K2HPO4 2.0 g 2. Peptone (1.0 to
7. Eosine 0.2 g 2.0%) 10.0 to 20.0 g.
8. Methylene Blue 0.05 g 3. Glycerol (0.5%) 5.0 g.
Preparation 4. Agar to solidify
(1) Dissolve 37.0 g. of Bacto Eosine Preparation
Methylene Blue Agar (dehydrated) (1) Dissolve 2, 3 and one of the added
in 1000.0 cc. water by boiling or nutrients in 1.
autoclaving. (2) Solidify by the addition of agar.
Sterilization: Sterilize in the usual manner. (3) Do not adjust the reaction.
Use: Isolation of Esch. coli, Esch. Ebert, Sterilization: Not specified.
Esch. typhi, etc. Use : Cultivation of molds.
Added nutrients: The author added 2.0% Preparation

glucose or 2.0% maltose. (1) Mix the Nahrstoff Heyden with a
Reference: Park, Williams and Krumwiede little water in a beaker, forming a
(1924 p. 134). paste. Stir until all the Nahrstoff-
Heyden has dissolved.
1570. Omelianski's Alcohol Peptone Agar (2) Dissolve 3, 4, 5 and 5.0 cc. of a normal
crystal soda solution in 1 by boiling
Constituents
2 hours.
(3) Mix (1) and (2).
2. Peptone 5.0 to 10.0 g.
(4) Boil for 15 minutes, stirring con-
stantly.
4. Agar-agar 20.0 g.
(5) Filter thru folded moistened filter
5. Ethyl alcohol few drops
paper in a steamer. Five funnels
Preparation
may be used to hasten the process.
(1) Dissolve 2, 3, 4 and 5 in 1. (Details
of preparation not given).
References: Hess (1899 p. 505), Kolle and
Use: Study aroma producing microorgan-
Wasserman (1912 p. 412), Abel (1912 p.
isms, Bad. ester oar omaticum. Author
94), Klimmer (1923 p. 224).
reported that more aroma was produced
if little alcohol be added.
1573. Molisch's Glycerol Peptone Agar
Reference: Omelianski (1923 p. 408).
Constituents
1571. Robinson and Rettger's Glycerol 1. Water, Moldau 1000.0 cc.
Opsine Agar 2. Agar 18.0 g.
Constituents: 3. Peptone 5.0 g.
1. Water 1000.0 cc. 4. Gh'cerol 5.0 g.
2. Opsine 10.0 g. Preparation

3. NaCl 5.0 g. (1) Dissolve 2, 3 and 4 in 1.
4. Glycerol 50.0 g. Sterilization: Not specified.
Preparation Use : Cultivation of purple bacteria.

(1) Dissolve 2, 3, and 4 in 1 by heating. Variants: Harvey prepared Sabouraud's
Adjust to faintly acid or basic to Glycerol Agar by dissolving 20.0 g.
(2)
litmus. Chassaing's peptone, 40.0 g. glycerol and
Boil over flame a few minutes. 20.0 g. agar in 1000 cc. water.
(3)
(4) Filter and tube. Reference: Molisch (1907 p. 11), Harvey
Sterilization: Autoclave at 12 pounds pres- (1921-22 p. 101).

1574. Spengler's Glycerol Somatose Agar
ported that the medium would support Constituents
the growth of some of the pathogenic 1. Distilled water 1000.0 cc
forms. 2. Nahrstoff Heyden. 5.0 g
Reference: Robinson and Rettger (1918 3. Somatose 5.0 g
p. 212). 4. NaCl 5.0 g
5. Glycerol., 30.0 g
1572. Hesse's Glycerol Kahrstoff Heyden Agar 15.0 g
6.
Agar 7. Soda 2.0 to 4.0 g
1. Distilled water 1000.0 cc. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
2. Nahrstoff -Heyden 5.0 g. Sterilization: Not specified.

3. Glycerol 30.0 g. Use: Cultivation of tubercle bacilli.
4. Agar 10.0 g. References: Spengler (1903 p. 92), Kolle
5. NaCl 5.0 g. and Wasserman (1912 p. 413).
1575. Omelianski's Formate Peptone Agar Preparation:

Constituents: (1) Dissolve 2 and 3 in 1.
1. Water 1000.0 cc.

(2) Add 0.05 cc. of a 5.0% phenol solution
to sterile melted (1) just before use.
2. Calcium formate 20.0 g.
3. Peptone 5.0 g.
4. Agar 15.0 g.
Preparation Reference: Harvey (1921-22 p. 90).

1579. Bacto Malt Extract Agar (Synthetic)
(Dehydrated)
Use: To study decomposition of formic
acid by Bacterium formicicum.
Constituents:
Variants: Author used potassium foymate 1. Distilled water
instead of calcium formate. 2. Maltose, technical, Difco .... 12.75 g.
Reference: Omelianski (1903-04 3. Dextrin, Difco 2.75 g.

pp. 185,
186).
4. Glycerol, C. P 2.35 g.
1576. Boekhout and de Vries Tartrate 6. Agar, Bacto 15.0 g.
Peptone Agar Preparation:
(1) Dissolve 33.63 g. of Bacto Malt Ex-
Constituents
tract Agar (Synthetic), (Dehydrated)
1. Water 1000.0 cc.
in 1000.0 cc. water by boiling or better
2. Peptone 10.0 g.
autoclaving.
3. Sodium tartrate 5.0 g.
manner.
4. Agar 15.0 g.
Use: Cultivation of yeasts and molds.
Preparation
p. 17).
Use: Cultivation of Bacillus fuchsinus. 1580. Harvey's Glucose Glycerol Agar
Author reported that after 18 hours
Constituents
growth was white; after 27 hours red; and
1. Water 1000.0 cc.
a metallic sheen developed after two days.
2. Peptone 15.0 g.
Reference: Boekhout and de Vries (1898 p.
3. Glucose 10.0 g.
498).
4. Glycerol 5.9 g.
1577. Dawson's Butter Soap Peptone Agar 5. NaCl 5.0 g.
6. Agar 20.0 g.
1. Water 1000.0 cc.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
2. Peptone 2.5 g.
(2) Do not adjust the reaction.
3. Butter soap 10.0 g. Sterilization: Not specified.
4. Agar 20.0 g. Use: Cultivation of hyphomycetes. Klim-
Preparation: mer cultivated favus and trichophyton
on a similar medium.
Not specified.
Sterilization: Variants: Klimmer used 10.0 to 20.0 g.
Use: Culture medium, to study variation peptone and 20.0 g. of NaCl. He cred-
of B. coli. ited Plaust with having described this
Reference: Dawson (1919 p. 142). medium previously.
References: Harvey (1921-22 p. 101), Klim-
1578. Harvey's Phenol Peptone Agar
mer (1923 p. 228).
Constituents
1581. Beijerinck's Glucose Peptone Agar
1. Water 1000.0 cc.
3. Agar 20.0 g. 1. Water 1000.0 cc.
4. Carbolic acid 5.0% 0.05 g. 2. Agar 20.0 g.
3. Glucose 50.0 g. (2) Add 5 volumes 90.0% alcohol.

4. Peptone 20.0 g. (3) Boil on a water bath.
5. KH2PO4 0.5 g. (4) Decant the supernatant fluid.
6. Ferric ammonium citrate... 0.1 g. (5) Add 6 parts distilled water to the
7. Chalk 20.0 g. residue.
Preparation (6) Boil.
(1) Dissolve 2, 3, 4, 5, 6, and 7 in 1. (7) Allow to cool.
(2) Pour into plates. (8) Divide into two portions.
Sterilization: Not specified. (9) Render one portion slightly red with
Use: To pigment production by
study dilute sulphuric acid.
vinegar bacteria. Author reported that (10) Add to this reddened portion the
the medium was blackened. other untreated portion little by
Reference: Beijerinck (1911 p. 172). little until the mixture becomes blue
again.
1582. Sarbouraud's Glucose Peptone Agar
(11) Filter thru paper when cool.
(Serena)
Constituents (13) Sterilize at 110C.
1. Water 1000.0 cc. (14) Keep for use.
2. Glucose 40.0 g. References: Bezangon (1920 p. 341), Harvey
3. Agar 18 g. (1921-22 p. 90).
4. Peptone 10.0 g.
1584. Boekhout and de Vries' Maltose
5. Tartaric acid 3.0 to 5.0 g.
Peptone Agar
Preparation
(1) Dissolve 2, 3, 4 and 5 in 1. Constituents:
Use: Cultivation of Oidium suis. 2. Peptone 5.0 g.
Reference: Serena (1913 p. 280). 3. Sodium tartrate 5.0 g.
4. Maltose 5.0 g.
1583. Chantemesse's Phenol Peptone Agar 5. Agar 15.0 g.
(Bezanfon) Preparation
Constituents (1) Dissolve 2, 3, 4 and 5 in 1.
1. Distilled water 1000.0 cc. (2) Add 0.0, 2.0 or 5.0 g. of lactic acid or
2. Peptone 30.0 g. 0.0, 1.0, 2.0 or 5.0 g. NaOH to obtain a
3. Lactose 20.0 g. neutral acid or alkaline reaction.
4. Agar 20.0 g. Sterilization: Not specified.
5. Phenol Use: Cultivation of Bacillus fuchsinus.
6. Litmus Author reported that the colonies were
Preparation first red and then a metallic sheen de-
(1) Dissolve 2, 3 and 4 in 1. veloped. Organism grew only on a

(2) When ready for use add 4 drops of a neutral or slightly alkaline medium.
3.0% aqueous phenol solution and 1.0 Reference: Boekhout and de Vries (1898
cc. of litmus solution to each 10.0 cc. p. 500).
sterile (1).
1585. Cheyney's Maltose Peptone Agar
Use: Differentiation of colon-typhoid
group. Bezanfon reported that the colon Constituents
colonies were red, typhoid colonies color- 1. Distilled water 1000.0 cc.
less. 2. Agar (2.0%) 20.0 g.
Variants: Harvey prepared the medium as 3. Maltose (1.0%) 10.0 g.
did Bezangon but added just before use, 4. Peptone (1.0%) 10.0 g.
0.05 cc. of a 5.0% phenol solution and 5. Lactic acid

100.0 cc. of litmus solution to the entire Preparation:
lot of sterile medium. The litmus (1) Dissolve 2.0% agar in distilled water.
solution was prepared as follows: (2) Filter thru a thin layer of cotton.
(1) Grind up litmus in a mortar. (3) Add 1.0% maltose and 1.0% peptone.
(4) Make up to exactly 2.0% acidity by 5. Cotton blue ("Poirier C^B")

the addition of lactic acid. 6. Sugar (white crystallized)
(5) Tube. 7. NaCl 5.0 g.
Sterilization: Autoclave for 10 minutes at 8. Peptone (Chapoteaut) 10.0 g.
15 pounds pressure. 9. Agar 15.0 g.
Use: Cultivation of organisms found in Preparation
canned food. (1) Prepare a lactophenol solution by
Reference: Cheyney (1919 p. 183). mixing the following materials:

Distilled water 1 part, glycerol 2
1586. Sabouraud's Glycerol Peptone Agar parts, pure lactic acid 1 part, snowy
phenol 1 part.
Constituents
(2) Add 0.5 centigrams of cotton blue
1.Water 1000.0 cc.
"Poirier C^B" to 30.0 g. of (1).
2 Peptone 20.0 g.
(3) Mix (2) and dilute to i with distilled
3. Glycerol 20.0 g.
water.
4. Acetic acid (glacial) 5 drops
(4) Mix 200.0 g. of crystalline white
5. Agar 13.0 g.
sugar in 100.0 cc. of cold water.
Preparation:
(5) Boil (4) and stir until solution is
complete.
(6) Filter thru flannel.
Use: Cultivation of Seborrheaie's bacilli
(7) Dissolve 15.0 g. agar, 10.0 g. Chapo-
(Bacillus acne).
teaut's peptone, and 5.0 g. of NaCI
Reference: Sabouraud (1897 p. 144).
in 1000.0 cc. ofwater by boiling.
1587. Robinson and Rettger's Glycerol (8) Filter, but do not neutralize.
Opsine Citrate Agar (9) Measure 100.0 cc. of melted sterile

(8) into a balloon flask.
Constituents: (10) Add 2.0 cc. of (3) and 10.0 cc. of (9)
1. Water 1000.0 cc. and mix well.
2. Opsine 20.0 g. (11) Boil.
3. NaCl 5 0g. (12) Add 7.0 cc. of a 2.0% NaOH solution
4. KH2PO4 5 0g. in distilled water. This completely
5. Sodium citrate 2.0 g. decolorizes the agar.
6. MgS04 2 g. (13) Distribute in 3.0 cc. lots into small
7. Glucose 5.0 g. sterile Wassermann tubes.
8. Glycerol 60.0 g. Sterilization: Sterilize (8) in the autoclave.
9. Agar 15 g. Use: Isolation of members of colon typhoid
Preparation group. Author reported that members
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1. of the colon-typhoid group color the
(2) Medium adjusted to very faintly medium blue.
alkaline to litmus. Reference: Botelho (1917 p. 436).
minutes. 1589. Sullivan's Ammonium Lactate
Use: General culture media. Author re- Peptone Agar

ported that the medium supported the Constituents
growth of some pathogenic organisms. 1. Water 1000.0 cc.
Reference: Robinson and Rettger (1918 pp. 2. Peptone, Witte's 15.0 g.
214, 215). 3. NaCl 3.0 g.
4. K2HPO4 1.5 g.
1588. Botelho's Lacto-phenol Peptone Agar MgS04 0.5 g.
5.
Constituents: 6. Fe2(S04)3 trace

1. Distilled water 7. CaCls trace
2. Lactic acid 8. Ammonium lactate 0.5 g.
3. Phenol (snowy) 9. Glycerol 20.0 g.
4. Glycerol 10. Agar 10.0 g.
Preparation 1592. Vierling's Nitrate Peptone Agar

(1) Dissolve 2, 3, 4, 5, 6, 7, 8. 9 and 10 in 1.
Constituents
1. Water 1000.0 cc.
2. K2HPO4 1.0 g.
ported that putrefactive, phosphorescent,
3. CaCl2 1.0 g.
chromogenic and pathogenic varieties
4. MgS04 0.1 g.
developed. If the salts CaCh, Fe2(S04)3
5. FeCU trace
and NaCl be omitted number of organisms 6. NaCl trace
grown was not markedly decreased. 7. Agar (2.0%) 20.0 g.
Reference: Sullivan (1905-06 p. 113).
Peptone (1.0%) 10.0 g.
8.
9. Dextrose (1.0%) 10.0 g.

1590. Lipman and Brown's Nitrate Peptone
Agar
10. KNO3 (0.75%) 7.5 g.
Preparation
Constituents: (1) Dissolve 2, 3, 4, 5 and 6 in 1000.0 cc.
1. Water 1000.0 cc. water.
2. Dextrose 10.0 g. (2) Dissolve 2.0% agar, 1.0%, peptone,
3. K2HPO4 0.5 g. 1.0% de.xtrose and 1.0% KNO3 in
4. MgS04 0.2 g. 100.0 cc. of (1).
5. Agar 20.0 g. (3) Pour in petri dishes.
6. Peptone 0.1 to 0.5 g. Sterilization: Method not given.
7. KNO3 0.05 to 0.5 g. Use: To study nitrate reduction by myco-
Preparation: bacteria. To test nitrate production,
(1) Dissolve 2, 3, 4, 5 and 6 in 1. pour 2.0% KI solution over the plates
(2) To (1) containing 0.5 g. peptone, add which have been acidified with dilute
0.05 to 0.1, 0.2, or 0.5 g. KNO3. acetic acid, li KNO2 is formed, the
Sterilization: Method not specified. iodine will be freed, coloring the starch
Use: Determination of bacterial count of blue. The addition of starch to the
soil. Author reported that different soils medium is not specified however.
showed different results with the same References: Vierling (1920 p. 201).
peptone and nitrate concentration.
Variants 1593. Heinemann's Asparagin Peptone Agar
(a) See medium 1549.
Constituents:
(b) Harvey used 0.06 g. KNO3.
1. Water 1000.0 cc.
References: Lipman and Brown (1910 p.
2. Agar 15 g.
451), Harvey (1921-22 p. 105).
3. Asparagin 5.0 g.
Ammonium 4. K2HPO4 2.0 g.

1591. Beijerinck's Ferric
5. Na2HP04 2.0 g.
Citrate Peptone Agar (Janke)
6. MgS04 2 0g.
Constituents 7. CaCl2 2.0 g.
1. Water 1000.0 cc 8. Ammonium lactate 2.0 g.
2. Agar 2.0 g 9. Peptone 10.0 g.
3. Glucose 5.0 g, 10. Starch (washed) 30.0 g.
4. Peptone 2.0 g Preparation
5. KH2PO4 0.05 g (1) Dissolve agar in 600.0 cc. of water by
6. Ferric ammonium citrate . . 0.01 g heating.
7. Chalk 2.0 g. (2) Dissolve 3, 4, 5, 6, 7 and 8 in 200.0 cc.
Preparation: of water. (A fine precipitate is
(1) Dissolve 2, 3, 4, 5, 6, and 7 in 1. formed)

Sterilization: Method not given. (3) Add (2) to hot (1).
Use: Cultivation of acetic acid bacteria in (4) Add 9.
beers. (5) Adjust to neutral with phenol-

Reference: Janke (1916 p. 6). phthalein.
(6) Filter. (6) When the solution is complete,

(7) To hot gradually add a suspension
(6) filter thru cotton.
of 30.0 g. washed starch made per- (7) Tube.
fectly homogeneous in a mortar. (8) Sterilize in the steam sterilizer
Stir constantly. once for 25 or 30 minutes.
(8) Bring near the boiling point and (b) MacConkey 2.0%
specified the use of
weigh. Total weight should be Witte's 0.5% commercial
peptone,
1000.0 g. sodium taurocholate, added 0.5% of a
(9) Tube. 1.0% solution of neutral red, 1.0%
Sterilization: Sterilize in autoclave at 120 glucose or any other carbohydrate
for 5 minutes and 0.034% CaCl., 0.5% KNO3 or
Use: Substitute for potato as a culture 0.5% KI might be added. The
medium. Author reported that the me- medium was clarified with white of
dium was superior to potato for the com- egg before the addition of neutral red
position is always the same, reaction can and carbohydate, and sterilized in the
be adjusted and pigment formation can steamer for 10 minutes on each of two
be better studied. successive days. The salts, CaCU,
References: Heinemann (1907 p. 283), (1922 KXO3 and KI, in the concentrations
p. 29), Besson (1920 p. 57). given, stimulated the growth of
lactose fermenters.
1594. MacConkey's Basal Bile Salt Peptone
Agar
(c) MacConkey used litmus in the above
variant instead of neutral red.
Constituents: (d) MacConkey used 2.0% agar in
1.5 to
1. Water 1000.0 cc. the preparation of variant (b) and
2. Sodium glycocholate added 1.0% serum or 1.0% alkali-
(0.5%) 5.0 g. haematin solution, (CaCl2, KNO3 or
3. Peptone (1.5%) 15.0 g. KI were not used in this medium).
. Agar (1.5%) 15.0 g.
(e) Abel used 0.4% of a 1.0% neutral red
Preparation: solution in MacConkey's medium,
(1) Dissolve 2, 3, and 4 in 1.
variant (b) and specified the use of
(2) Filter. lactose as a carbohydrate.
(3) Dissolve one of the added nutrients
(f) Ball prepared a medium containing
in (2).
5.0 g. ofsodium taurocholate, 15.0 g.
peptone, 35.0 g. lactose and 15.0 g. of
Use: Differentiation Bacillus coli commune agar per liter.
and Bacillus typhi abdominalis. medium
(g) Fercival prepared a as
Added nutrients: The author added 0.3 to
follows:
0.5% lactose or glucose.
(1) Dissolve 5.0 g. sodium tauro-
Variants
cholate, 20.0 g. peptone, and 15.0
(a) Frost prepared a similar medium as
g. of agar in 1000.0 cc. of water by
follows:
heating in a water bath for one
(1) Dissolve 20.0 g. of agar in a liter of
hour.
boiling water.
(2) Cool to 50C.
(2) Dissolve 20.0 g. of peptone in a
liter of boiling water. (3) Add the white of an egg and heat in
the water bath for 90 minutes.
(3) Mix (1) and (2).
Make alkaline by the addition of (4) Filter.
(4)
4.0 cc. of a normal sodium hydrate (5) Add 1.0 g. lactose, 1.0 cc. of a
solution, after neutralizing the 0.5% solution of neutral red and
litmus. 1.0 cc. of a 0.1% solution of crys-
While hot add 5.0 g. of sodium tal violet, per 100.0 cc. of the
(5)
taurocholate, 10.0 g. of lactose and filtrate.
10.0 cc. of a 5.0% watery solution (6) Tube in sterile tubes in 10.0 cc.
of neutral red. quantities.
(7) Sterilize by heating 20 minutes on (4) Filter while hot thru cotton wool.
3 successive days. (5) Add 1.0% lactose dissolved in a
(h) Levine gave the following medium as little water and 1.0% Andrades
Bile Salt (Rebipel) Agar (After indicator.
Savage): Sodium taurocholate 5 (6) Sterilize intermittently.
grams, Witte's peptone 20 grams and References: MacConkey (1900 p. 20), (1901
distilled water 1 liter, are boiled up p. 740), Frost (1903 p. 342), MacConkey
together, 20 grams of agar are added (1905 pp. 334, 336), (1908 p. 325), Abel
and dissolved in the solution in the (1912 p. 227), Ball (1919 p. 81), Tanner
autoclave in the ordinary way. The (1919 p. 49), Percival (1920 p. 307),
medium is cleared with white of egg Harvey (1921-22 p. 89), Klimmer (1923
and filtered. After filtration, 10 p. 214), Levine (1921 p. 116), Cunningham
grams of lactose and 5 cc. of recently (1924 p. 98).
prepared per cent neutral red solu-
1
1595. Harrison and VanderLeck's Aesculin
tion are added. The medium is then
Agar
Bile Salt
tubed and sterilized for 15 minutes on
three successive days. Constituents:
(i) Harvey gave the following method 1. Water 1000.0 cc.
of preparation 2. Peptone (Witte (1.0 or
(1) Dissolve 20.0 g. peptone, 5.0 g. 2.0%) 10.0 or 20.0 g.
sodium taurocholate in 1000.0 cc. 3. Sodium taurocholate
tap water and make faintly alkaline (Commercial) (0.5%) 5.0 g.
to litmus. 4. Aesculin (0.1%) 1.0 g.
(2) Steam 45 minutes. 5. Iron citrate (0.05%) 0.5 g.
(3) Add 15.0 g. powdered agar, making 6. Agar 15.0 g.
it into a paste or suspension, Preparation
before addition, with alittle of the (1) Dissolve the agar in part of 1.
taurocholate peptone solution. (2) Add 2, 3 and 4 to the rest of 1.
(4) Steam gently 2^ hours to bring the (3) Mix (1)and (2).
agar thoroughly into solution. (4) Boil and filter (may be clarified with
(5) Bring the volume up to 1000.0 cc. egg white).
by the addition of water. (5) Tube.
(6) Cool to 60C., clarify by the addi- Sterilization: Sterilize by steaming on 3
tion of egg and filter. successive days or autoclaving at 15
(7) Dissolve 10.0 g. lactose (or other pounds for 15 minutes.
sugar if desired) in 15.0 cc. sterile Use: Presumptive test for B. coli in water
water and steam 15 minutes. analysis. Author reported that B. coli
(8) Add the lactose solution to the colonies were black, typhoid colonies
hot, clear, nutrient agar. produced no blackening.
(9) Add 5.0 cc. freshly prepared sterile Variants
1 per cent neutral red by means (a) The authors used 0.25% commercial
of a sterile pipette. bile salt, 1.0% Witte's peptone and
(10) Distribute the resulting agar, 0.1% iron citrate instead of amounts
which is deep red, into flasks or used above.
test tubes. (b) Levine gave Eyre's method of prep-
(11) Steam 25 minutes. aration as follows:
(j) Cunningham prepared the medium as (1) Measure out 400.0 cc. distilled water
follows: into a tared 2 liter flask.
(1) Steam 5.0 g. sodium taurocholate (2) Weigh out 15 grams agar, 10 grams
and 20.0 g. peptone in 1000.0 cc. peptone, 5 grams sodium taurocholate
water for one hour. and make into a thick paste with 150.0
(2) Filter. cc. distilled water.
(3) Add 1.5% agar and steam for one (3) Add this paste to the distilled water
hour. in the flask.
(4) Dissolve the ingredients by bubbling Ickert's Yeast Infusion Peptone

live steam thru the mixture. Agar 1599
(5) Weigh out 1.0 g. aesculin and 0.5 g. Sherman's Yeast Extract Peptone
ferric citrate and dissolve in a second Agar 1600
flask containing 100.0 cc. distilled Bj. Containing additional constituents of
water. unknown chemical composition.
(6) Mix the contents of the two flasks Rivers and Kohn's Basal Yeast Ex-
adjust the weight to the calculated tract Peptone Agar 1601
medium figure (in this case 1031. 5g.) Sturtevant's Egg Yolk Yeast Agar . 1602
by the addition of distilled water at A2. Containing bacterial or fungus de-
100C. rivatives.
(7) Clarify with egg and filter. Savini and Savini-Castano's Hemo-
(8) Tube and sterilize as for nutrient globin Bacterial Extract Agar. 1603 . . .
agar. Mankowski's Fungus Infusion Pep-

References: and
Harrison VanderLeck tone Agar 1604
(1909 p. 549, 607), Committee Am. Public A3. Containing derivatives of plants other
Health Asso. (1909 p. 286), Harvey (1921- than Ai and A2 above.
22 p. 89), Levine (1921 p. 117),Heinemann Bi. Plant juices or tissues (not infusions)
(1922 p. 33). employed.
Owens' Cane Juice Peptone Agar. . . 1605
1596. Harvey's Brilliant Green Bile Salt Plaisance and Hammer's Corn Juice
Agar
Peptone Agar 1606
Constituents: Peglion's Grape Must Peptone
1. Water 1000.0 cc. Agar 1607
2. Sodium taurocholate 5.0 g. Harvey's Banana Agar 1608
3. Peptone 30.0 g. B2. Plant infusions, extracts, etc., em-
4. NaCl 5.0 g. ployed.
5. Lactose 10.0 g. Ci. Grain derivatives used.
6. Powdered agar 30.0 g. Meacham et al. Malt Extract Agar. 1609 .
7. 1-1000 Brilliant-green 20.0 cc. Marpmann's Flour Casein Agar .... 1610
8. Picric acid (1.0%) 20.0 cc. Williams and Povitzky's Flour Pep-
Preparation tone Agar 1611
<1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1000.0 Otabe's Wheat Peptone Agar 1612
cc. of water. Dawson's Flour Peptone Agar 1613
(2) Reaction to be 1.5% acid to phenol- Plaisance and Hammer's Stover
phthalein. Infusion Peptone Agar 1614
Sterilization: Method not given. C2. Potato derivatives used.
Use: Enrichment medium for colon- De Gaetano's Potato Extract Agar . . 1615
typhoid group. Jochmann's Potato Bouillon Agar.. 1616
Reference: Harvey (1921-22 p. 91). Nicholle and Alilaire's Potato In-
fusion Agar 1617
Shiga et al. Potato Blood Agar 1618
Basal or complete media containing agar Gaehtgen's Potato Peptone Agar. . . 1619
and peptone (or other commercial digest) C3. Derivatives of legumes used.
together with additional constituents of Matzuschita's Pea Bile Agar 1620
unknown chemical composition of plant or Tanner's Pea Extract Tryp. Agar... 1621
soil origin. Behrens' Pea Blood Agar 1622
Ai. Containing yeast or yeast derivatives. De Rossi's V. Faba Infusion Pep-
Bi. All additional constituents of known tone Agar 1623
chemical composition. C4. Miscellaneous plant derivatives used.
Gassner's Yeast E.xtract Peptone Owens' Molasses Peptone Agar 1624
Agar 1597 Seiffert and Bamberger's Chloro-
Cohen and Clark's Yeast E.xtract phyll Bouillon Agar 1625
Peptone Agar 1598 Dawson's Edestin Peptone Agar. 1626 . . .
A4. Containing soil or its derivatives. 1600. Sherman's Yeast Extract Peptone
Temple's Soil Infusion Peptone Agar
Agar 1627
Constituents:
1. Water 1000.0 cc.
1597. Gassner's Yeast Extract Peptone
2. Peptone 20.0 g.
Agar Yeast
3. 10.0 g.
Constituents 4. Lactic acid (as sodimn

1. Water 900.0 cc. lactate) 10.0 g.
2 Yeast extract water 900.0 cc. 5. Agar 1.5 g.
3. Agar 30.0 g. Preparation:

4. NaCl 5.0 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
5. Peptone 20.0 g. (2) Details of method of preparation not

Preparation: given.
(1) Exact method of yeast extract water Sterilization: Method not given.
not given. Use: Isolation of organisms causing flavors
Mix 1 and in cheese.
(2) (1).
(3) Dissolve 3 and 4. Reference: Sherman (1921 p. 388).
(4) Adjust to slightly alkaline to litmus.

1601. Rivers and Kohn's Basal Yeast
Extract Peptone Agar
(6) To each 80.0 cc. of (5) add 20.0 cc.of
a 0.5% solution of water blue and 0.5,
Constituents:
0.1 or 1.0% dextrose. 1. Distilled water 1000.0 cc.
Sterilization: Not specified. 2. Peptone 20 g.
Use: To study nitrogen requirements of 3. Agar agar 15.0 g.
colon-typhoid and dysentery group. 4. NaCl 5 0g.

Variants: The author omitted the glucose. 5. Blood clot extract
Reference: Gassner (1917-18 p. 260).
6. Yeast extract
7. Brom Cresol Purple (25 0%
1598. Cohen and Clark's Yeast Extract alcoholic soln.)
Peptone Agar Preparation

(1) Dissolve 2, 3 and 4 in 1 by boiling
Constituents (2) Adjust the reaction to pH = 7.4.
1. Water 1000.0 cc. (3) Filter.
2. Yeast, Brewer's 10.0 g. (4) To 15.0 cc. of sterile (3) add 20.0 cc. of
3. Peptone, Difco 10 g. sterile blood clot extract (method
4. K2HPO4 5.0 g. of preparation not given) and 15 to
5. Agar (amount not given) 20.0 cc. of yeast extract (preparation
6. Glucose 10.0 g. not given).
Preparation: (5) Add 10.0 cc. of a 10.0% solution
(1) Dissolve 5 in an extract of 10.0 g. of of any one of the added nutrients, and
Brewer's yeast in 1000.0 cc. water 25.0% alcoholic solution of brom
containing 3, 4 and 6. cresol purple to give a good color.
(2) Filter. (6) Incubate to test sterility.
(3) Adjust with NaOH and HCl to desired Sterilization: Sterilize (3) and the blood
pH. extract in autoclave. Sterilization of
Sterilization: Method not given. other materials not given.
Use: To study growth of B. vulgaricvs and Use: To study fermentation ability of
B. coll at different H-ion concentrations. influenza bacillus producing meningitis.
Reference: Cohen and Clark (1919 p. 421). Added The authors added 10.0
nutrients:
cc. 10.0% solution of any desired
of a
1599. Ickert's Yeast Infusion Peptone Agar
carbohydrate, alcohol, etc., to each 100.0
Same as medium 695 but solidified by the cc. of medium.
addition of agar. Reference: Rivers and Kohn (1921 p. 381).
1602. Sturtevant's Egg Yolk Yeast Agar (16) Adjust the reaction of (13) so that
upon the addition of 1.0 cc. of (15)
Constituents:
to 10.0 cc. of the agar, the final pH
1. Water 1000.0 cc.
value will be about 6.8.
2. Yeast (dry) 10.0 g.
(17) Tube the agar (without the addition
3. Peptone 10.0 g.
of egg yolk suspension) in 10.0 cc.
4. Sodium glycero-phosphate
lots.
(buffer)....' 5.0 g.
(18) When desired for use add 1.0 to 2.0
5. Agar 15.0 g.
cc. (15) to each tube of melted
of
6. Egg yolk
agar under aseptic conditions, mix
Preparation
M'ell and slant. The agar should be
(1) Heat 10.0 g. dry yeast, 10.0 g. pep-
cooled to about 55C.
tone and 5.0 g. buffer (sodium glyc-
ero-phosphate) in 500.0 cc. of water
given. Sterilize (17) by autoclaving at
with flowing steam for 30 minutes.
Use: Isolation of organism causing
(3) Boil for one minute over a free
American foul brood, Bacillus larvae. To
flame.
isolate the organism, drop some infected
(4) Filter thru paper on a perforated
material into the water of condensation
porcelain funnel, using siliceous
and smear over the surface of the agar.
earth to clarify.
Author reported that best growth was
(5) Place 15.0 shred agar in 1000.0
g. of
obtained when 0.5% glucose was added.
water and allow to stand
cc. distilled
To make counts, add only 10 or 15 drops
for 24 hours at room temperature.
of the egg yolk suspension, or 1.0 cc. of
(6) Pour off as much of the water as
the supernatant fluid of the suspension
possible by placing a piece of cheese
to 10.0 cc. yeast extract agar containing
cloth over the top of the flask.
varying amounts of glucose. Mix thoroly
(7) Add as much distilled water as was
and pour in plates. Add a dilute solution
poured off and soak for another 24
of fuchsin to the plates when counting.
hours.
This colors the colonies.
(8) Filter thru a cotton flannel cloth,
Variants: Author added from 0.5 to 10.0%
and wash the agar with 500.0 cc. of
glucose.
distilled water.
Reference: Sturtevant (1924 p. 136).
(9) Allow to drain, and squeeze the agar
as free from water as possible. and Savini-Gastano's
1603. Savini
(10) Add enough water to (9) so that the Hemoglobin Bacterial Extract
total weight will be 515.0 g.
Agar
(11) Dissolve the agar in the water by
heating. Same as medium 977 but agar employed
instead of bouillon.
(12) Filter.
(13) Mix equal parts of (4) and (12)
1604. Mankowski's Fungus Infusion
(double strength agar).
Peptone Agar
(14) Prepare a series of flasks containing
200.0 cc. of water. 0.5 to 1.0% Constituents:
neutral buffer salt may be added to 1. Fungus infusion 1000.0 cc.
keep the acidity down which 2. Agar 15 g.
develops on long standing. A small 3. Peptone 10.0 g.

amount of normal NaOH 2 to 3.0 cc. 4. NaCl 5.0 g.
added. This tends to keep the
is Preparation
egg yolk suspension at the desired (1) Method of preparation of Fungus
pH. Plug the flasks with cotton and infusion not given.
protect the plug with a paper cap. (2) Dissolve 2, 3 and 4 in (1).
(15) Add under aseptic conditions the (3) Clarify with egg white.
yolk of one egg to each sterile flask (4) The reaction is neutral.
of (14). Mix well. Sterilization: Not specified.
Use: Differentiation of typhoid bacilli and 1606. Plaisance and Hammer's Corn Juice
Bacterium coli communis. Author re- Peptone Agar
ported that Bacterium coli grew in a
Constituents
silver white, solid and dry film. Typhoid
1. Corn juice 1000.0 cc.
bacilli grew slower, appeared as trans-
2. Agar 15.0 g.
parent, shining, damp strips. When 3. Peptone 10.0 g.
fuchsin indigo carmine be added typhoid
Preparation
bacilli colored the blue medium red,
(1) Obtain corn juice by pressing green
Bacterium coli communis gave a bluish
corn.
green color and finally completely de-
colorized the medium.
Variants
(a) The author prepared a medium con-
Use: Study mannitol producing organisms
taining fuchsin and indigo carmine
from silage.
by continuing from step (4) above as
Reference: Plaisance and Hammer (1921 p.
follows:
432).
(5) Prepare a saturated solution of
acid fuchsin in a 1.0% solu- KOH 1607. Peglion's Grape Must Peptone Agar
tion (acid fuchsin may be added Constituents
to a 1.0% KOH solution until a 1. Grape must 500.0 cc.
dark black brown color is reached). 2. Peptone 10.0 g.
(6) Prepare a watery saturated solu- 3. Ammonium phosphate 2.0 g.
tion of indigo carmine. 4. Glycerol 10.0 cc.
(7) Add 2.0 cc. of (2) and 1.0 cc. of 5. Agar 10.0 g.
(3) to 22.0 cc. of distilled water. Preparation
This solution is dark blue and Dissolve 4 and 5 in 1.
(1) 2, 3,
reaction slightly alkaline.
(2) Pour into petri dishes or plates.
(8) Add (7) drop by drop to (4) until Sterilization: Not specified.
the agar is colored blue and then Use: Cultivation of Saccharomyces ellip-
violet blue.
soideus.
(9) Distribute into test tubes. Reference: Peglion (1898 p. 477).
(10) Add to each test tube a drop of
watery
saturated solution of
1608. Harvey's Banana Agar
indigo carmine. Constituents:
(b) Wiegert solidified medium 701 by the 1. Agar (nutrient) 1000.0 cc.
addition of agar. 2. Banana (10.0%) 100.0 g.
References: Mankowski (1900 p. 23), Preparation
Wiegert (1921-22 p. 110). (1) Use banana in the form of cut cylinder
or as 10.0% pulp incorporated with
1605. Owens' Cane Juice Peptone Agar
nutrient agar. (Medium 779 variant
Constituents: (bb) solidified by the addition of
1. Cane juice 1000.0 cc. agar.)
2. Agar (2.0%) 20.0 g. Sterilization: Not specified.
3. Peptone (1.0%) 10.0 g. Use: General culture medium.
Preparation Reference: Harvey (1921-22 p. 119).
(1) Heat fresh raw cane juice.
1609. Meacham et al. Malt Extract Agar
(2) Filter thru cotton.
(3) Add 1.0% peptone (peptone may be Constituents
omitted). 1. Malt extract (2.5%) agar. . . . 1000.0 cc.
(4) Solidify by the addition of 2.0% agar. 2. K2HPO4 M/50
Sterilization: Not specified. 3. Acetic acid N/50
Use: Bacterial count of cane sugar Preparation

products. (1) Preparation of malt extract agar not
Reference: Owen (1914 p. 337). given.
(2) Dissolve 2 and 4 in (1). Add sodium

(7) chloride, 5 g. and pep-
(3) Reaction about pH = 5.7. tone 10.0 g.
Sterilization: Method not given. (8) Boil. Reaction adjusted to pH-7.4.
Use: To study phosphate-phthalate and (9) Add agar 1.5% and autoclave at 15
phosphate-acetate as buffers for cultiva- pounds pressure for half hour.
tion of E. parasitica (Murr). Author (10) On removal from autoclave add
reported that acid phthalate gave best gelatin 2.0%. Adjust reaction.
growth of E. parasitica. (11) Cool down to 45C. and clarify with
Variants Author used M/50 acid potassium
: white of egg.
phthalate instead of acetic acid. (12) Heat in the Arnold steamer 45
Reference: Meacham, Hopfield and Acree minutes. Correct reaction to pH-7.6.
(1920 p. 305). This will give desired end reaction
(pH-7.4), after autoclaving.
1610. Marpmann's Flour Casein Agar
(13) Filter thru thin cotton (3 times) and
Constituents: tube.
1. Bouillon 1000.0 cc Sterilization: Sterilize the tubes of agar for
2. Glycerol 30.0 g 30 minutes at 15 pounds pressure.
3. Casein, dissolved 50.0 g Use: Cultivation of B. influenzae. Author
4. Sodium phosphate 30 g, reported that growth of the influenza
5. Rye flour 50.0 g organism was obtained when mixed with
6. Gelatin 40.0 g other organisms.
7- Agar 3.0 g Reference: Williams and Povitzky (1920-
Preparation 21 p. 407).
(1) Method of preparation or exact
composition of bouillon not given. 1612. Otabe's Wheat Peptone Agar
(2) Dissolve 2, 3, 4, 5, 6 and 7 in (1). Solidify medium 707 by the addition of 1.5
(3) Adjustment of reaction not given. to 2.0% agar.
(4) Distribute into flat bottomed flasks.
Sterilization: Method not given. 1613. Dawson's Flour Peptone Agar
Use: Cultivation of tubercle bacilli. Constituents
Reference: Marpmann (1903 p. 636). 1. Water 1000.0 cc.
2. Peptone 2.5 g.
1611. Williams and Povitzky's Flour
3. Flour protein 10.0 g.
Peptone Agar
4. Agar 20.0 g.
1. Water (tap) 1300 cc. (1) Dissolve 2, 3 and 4 in 1.
2. Wheat 1.0 lb. (2) Reaction to be alkaline.
3. Diastase 0.5 g. Sterilization: Method not given.
4. NaCl 5.0 g. Use: Culture medium to study variation of
5. Peptone 10.0 g. B. coli.
6. Agar (1.5%) 15.0 g.'
Reference: Dawson (1919 p. 142).
7. Gelatin (2.0%) 20.0 g.
Preparation 1614. Plaisance and Hammer's Stover
(1) Roast whole wheat (with or without Infusion Peptone Agar
the husk) in pan until brown. Constituents
(2) To one pound roasted wheat add 1. Stover juice 1000.0 cc.
1300.0 cc. tap water. 2. Agar 15.0 g.
(3) Steam in Arnold for one-half hour. 3. Peptone 10.0 g.
(4) Strain thru fine wire strainer. 4. Sucrose 5.0 g.
(5) Make up to 1000.0 cc. with tap water. Preparation:
(6) Cool to 35C. and add Taka diastase (1) Prepare stover juice by soaking corn
or ordinary diastase, 0.5 gm. and stover in water for 12 hours and then
shake well. Keep flask at 30-40 C. pressing.
for half hour. (2) Dissolve 2, 3 and 4 in (1).
Use: Diagnosis of typhoid fever. Author

(3) May be cleared with egg.
Not specified. reported that typhoid colonies after 12
Sterilization :
hours were typical, small, silver gray,

Use: Study mannitol producing organisms
reported this glistening, fine fibered; coli colonies
from silage. Authors
larger, light yellow and mostly round.
all
medium to be inferior to 1606.
Hammer Medium was dark yellow or dark brown
Reference: Plaisance and (1921
in color.
p. 432).
Reference: Jochmann (1902 p. 461).
De Gaetano's Potato Extract Agar

1615. 1617. KicoUe and Alilaire's Potato Infusion
Agar
Constituents
1. Potato infusion 1000.0 cc. Constituents
2. Meat extract (Lie- 1. Water 2000.0 cc.
big's) 20.0 g. 2. Potato 500.0 g.
3. Peptone 10.0 g. 3. Beef 500.0 g.

4. Agar 20.0 to 30.0 g. 4. Peptone (Chapoteaut) 30.0 g.
5. Tartaric acid 5. NaCl 10.0 g.
6. Glucose 6. Glycerol 20.0 g.
Preparation 7. Agar 60.0 g.
(1) Preparation of
potato infusion not Preparation
given. (1) Extract 500.0 g. of
finely chopped beef
(2) Dissolve 2, 3, and 4 in (1) by boiling.

with one liter of water over night.
(3) Filter. (2) Extract 500.0 g. of
potatoes cut in
(4) Distribute in 6.0 cc. lots in tubes. large pieces in a second liter of water
(5) To each tube add 5.0 to 6.0 drops of over night.
10.0 to 15 0% tartaric acid solution
(3) Mix (1) and (2).
and 5.0 to 6.0 drops of a 50.0% glucose (4) Dissolve 4, 5, 6 and 7 in (3).
solution. (5) Proceed in the ordinary manner for

Sterilization: Method not given. the rest of the preparation for nu-
Use: Cultivation of Saccharomyces trient agar (Method not given)
septicus. Sterilization: Not specified.
References: De Gaetano (1897 ^200), Use : Mass cultivation of B. encapsulas and
Kamen (1899 p. 833). Cholera vibrio.
Variants: Lubinski solidified medium 873
1616. Jochmann's Potato Bouillon Agar by the addition of I'.O to 1.5% agar.
Constituents References: NicoUe and Alilaire (1909 p.
200 cc. 548), Lubinski (1895 p. 126).

1. Bouillon
Potato 600.0 g. Agar

2. 1618. Shiga et al Potato Blood
3. Agar 3.75 g.
Constituents:
Preparation
Glvcerol (4.0% soln) 250 cc.
(1) Grate 600.0 g. of potatoes and allow 1
Potato 125.0 g.
to stand in a glass flask at about
2.
3. Meat water 750.0 cc.

15C. for 12 hours.
Agar 20.0 g.
Press the juice thru a filter cloth using 4.
(2)
the juice. 5. Peptone 100 g.
the hands to expell all
NaCl 5.0 g.
(3) To 300.0 g. add 200.0 g. of
of (2) 6.
Blood (beef or horse) 4000.0 cc.
weakly alkaline bouillon (exact compo- 7.
sition or method of preparation not Preparation

(1) Peel 500.0 g. of
potatoes and cut into
given).
completely 3.75 finely small pieces.
(4) Dissolve g.
using the (2) Add (1) to 1000.0 cc. of 4.0% glycerol
divided agar in (3)
water.
autoclave.
slightly acid. (3) Boil for 30 minutes in the autoclave.
(5) The reaction is
Filter thru gauze.
Sterilization: Not specified. (4)
(5) Method of preparation of meat water 3. Pea flour 30.0 g.

not given. 4. Peptone (0.7%) 7.0 g.
(6) Mix 250.0 cc. of (4) and 750.0 cc. of (5). 5. NaCl (0.5%) 5.0 g.
(7) Add 20.0 g. of agar. 6. Bile, ox 600.0 g.
(8) Boil in the autoclave for one hour. 7. Agar (2.0%) 32.0 g.
(9) Make slightly alkaline. Preparation
(10) Filter. (1) Boil 500.0 g. of finely chopped liver
(11) Add 1.0% Witte's peptone and 0.5% with 30.0 g. of pea flour and one liter
NaCl. of water.
(12) Distribute into tubes. (2) Allow to cool and add 0.5% peptone,
(13) Melt and cool to 50C. when ready 0.5% NaCl and 0.02% HCl.
for use. (3) Mix thoroly and allow to stand for 3
(14) Add agar to beef or horse blood in hours at 37C.
the ration of 1:4. (4) Add 600.0 g. of ox bile and allow to
(15) Tube and slant or pour into petri stand another 3 hours at 37C.
dishes. (5) Proceed as in the preparation of
(16) After heat
solidification the agar regular nutrient agar by boiling,
at 56C. for 30 minutes. filtering, adding 2.0% agar and
Sterilization: Not specified. filtering again.
Use Cultivation of whooping cough bacilli
: (6) Reaction is slightly acid.
and influenza bacilli. Sterilization: Method not given.
Reference: Shiga, Imai and Eguchi (1913 Use: Cultivation of intestinal bacteria.
p. 104). Reference: Matzuschita (1901-02 p. 214).
1619. Gaehtgen's Potato Peptone Agar

1621. Tanner's Pea Extract Tryp. Agar
Constituents:
1. Water 1000.0 cc, Constituents
2. Potato 500.0 g. 1. Medium 1116 1000.0 cc.
3. Agar 2 Agar (2.0%) 20.0 g.
4. Peptone 3. CaClo 0.125 g.
5. NaCl 4. Pea extract (5.0%) 50.0 cc.
(1) Carefully wash and peel 500.0 g. (1) Add 2.0% agar fiber and 0.125 g. of
potatoes. CaCl2 to 1000.0 cc. of trypsinized
(2) Grind the potatoes by means of a
fine broth, medium 1116.
porcelain This is to be
mortar. (2) Autoclave at 118C. for 45 minutes to
carried out under the water as far as dissolve the agar.
possible. (3) Mix together in a sauce pan.
(3) Boil for one hour. (4) Titrate with N/10 KOH to give an
(4) Dissolve 3, 4 and 5 (amounts not absolutely neutral reaction.
given) in the paste. (5) Cool to 60C.
(5) Add soda to obtain the desired (6) Add the whites of two eggs beaten
reaction. up with the crushed shells.
Sterilization:Method not given. (7) Autoclave again at 118C. for 75
Use: Inexpensive culture Medium. Author minutes (or in the steamer for two
reported that the medium was not trans- hours).
parent. (8) Filter.
Reference: Gaehtgens (1916 p. 47). (9) Add 5.0% sterile pea extract (prepara-
tion not given).
1620. Matzuschita's Pea Bile Agar
Sterilization: Sterilize in the ordinary way
Constituents (method not given).
1. Water 1000.0 cc. Use: General culture medium.
2. Liver, ox 500.0 g. Reference: Tanner (1919 p. 49).
1622. Behrens' Pea Blood Agar (4) The reaction is slightly acid or
alkaline.
Constituents
Use: Cultivation of bacteria found in the
2. Peas 10.0 g.
nodules of leguminous plants.
3. Peptone 20.0 g.
Variants: The author used 2.0% glucose
4. NaCl 5.0 g.
instead of sucrose.
5. CaClj 0.1 g.
Reference: de'Rossi (1907 p. 301).
6. N/1 NajCO, soln 10.0 cc.
7. Agar 20.0 g.
1624. Owen's Molasses Peptone Agar
8. Blood (rabbit) 2000.0 cc.
Preparation: Constituents
(1) Boil 10.0g. peas in 1000.0 cc. distilled 1. Water 1000.0 cc.
water to obtain extract. Length of 2. Molasses (Final 75 Brix) . . 160.0 g.
boiling not specified. 3. Agar 20.0 g.
(2) Strain, boil the extract and filter. 4. Peptone 10.0 g.
(3) Dialyze the filtrate in a large collo- Preparation
dium sac against running distilled (1) Dissolve 160.0 of final molasses,
g.
water for 24 to 48 hours. 75 Brix, 20.0 agar and 10.0 g. pep-
g.
(4) Dilute (3) to 1000.0 cc. tone in 1000.0 cc. water.

(5) Dissolve 3, 4, 5, 6 and 7 in (4). Sterilization: Not specified.
(6) Distribute in test tubes in 1.0 cc. lots. Use : Bacterial counts of organisms found in
(7) Shortly before use the desired number corn juice and cane sugar products.
of sterile agar tubes are melted in Reference: Owen (1914 p. 338).
the water bath, cooled to 60C. and
1625. Seiffert and Bamberger's Chlorophyll
two volumes of defibrinated rabbit's
Bouillon Agar
blood are added.
(8) Mix well and solidify in slanting Constituents
position. 1. Neutral agar 1000.0 cc.
Sterilization: Sterilize by autoclaving at 2. Chlorophyll solution ("So-
105 to 108C. for 15 minutes. lutio spirituosa," Merck)... 25.0 cc.
Use: Special culture medium for Trypano- 3. Sucrose

soma Brucei. 4. Dextrin
Variants The author used beans instead
: of 5. Diamond fuchsin solution
peas. 6. NasSOs
Reference: Behrens (1914 p. 28). 7. Soda
Preparation
1623. De'Rossi's V.Faba Infusion Peptone
(1) Prepare 60.0 cc. of a 10.0% soda
Agar solution using water free soda.
Constituents (2) Add 25.0 cc. chlorophyll solution
1. Water 1000.0 cc. (Solutio spirituose Merck) to (1).
2. V. Faba leaves 100.0 g. (3) Heat for one hour in the steamer.
3. Agar (1.5%) 15.0 g. (4) Prepare a 20.0% sucrose solution.
4. NaCl 5.0 g. (5) Prepare a 20.0% dextrin solution.
5. Sucrose (2.0%) 20.0 g. (6) Add 50.0 cc. sterile (4) and 50.0 cc.
6. Peptone (1.0%) 10.0 g. sterile (5) to (3).

Preparation (7) Mix with one liter sterile neutral
(1) Boil 8 to 10 parts water with Vicia agar.
Faba leaves. (8) Prepare a diamond fuchsin add-
(2) Filter and add 0.5% NaCl, 2.0% ing an excess of diamond fuchsin
sucrose and 1.0% peptone. (NaCl, to absolute alcohol and incubating
sucrose and peptone may be omitted.) for 24 hours. Shake often.
(3) Continue in the same manner as in (9) Add 4.0 cc. of (8) to (7) when ready
the preparation of nutrient agar. for use.
(10) Add about 15.0 cc. of a 10.0% sterile Ai. Animal cells and tissues or their deriva-
Na2S0j solution to decolorize (9). tives added.
(11) Pour in sterile plates. Rivers and Kohn's Basal Blood Cell
(12) Dry the plates in the air. Peptone Agar 1628
Sterilization: Method of sterilization of Krumwiede, Pratt and Grund's Egg
(4), (5) or neutral agar not given. Peptone Agar 1629
Use: Selective medium for cholera. Au- Weiss' Nahrstoff Heyden Gelatin
thor reported that the cholera colonies Agar 1630
were red. Other organisms were inhibited. Deycke's Alkaline Albumin Gelatin
Reference: Seiffert and Bamberger (1916 p. Agar 1631
288). Deycke's Albuminate Glycerol
Agar :.... 1631a
1626. Dawson's Edestin Peptone Agar
Krumwiede and Pratt's Gelatin
Constituents: Serum Agar 1632
1. Water 1000.0 cc. Dunschmann's Bile Salt Peptone
2. Peptone 5.0 g. Agar (Bezanyon) 1633
3. Edestin 5.0 g. Capaldi's Peptone Gelatin Agar 1634
4. Agar 20.0 g. Smyth's Egg Trypsinized Peptone
Preparation: Agar 1635
(1) Dissolve 2, 3 and 4 in 1, Aj. Animal fluids added.
(2)Reaction to be alkaline. Bi. Blood employed.
Method not given.
Sterilization: Czaplewski's Blood Nahrstoff Hey-
Use: Culture medium to study variation of den Agar (Klimmer) 1636
B. coll. Norris et al. Blood Peptone Agar. 1637 . .
Reference: Dawson (1919 p. 142). Nicolle's Blood Peptone Agar

(Behrens) 1638
1627. Temple's Soil Infusion Peptone Agar
Kelser's Blood Peptone Agar 1639
Constituents Harvey's Blood Peptone Agar 1640
1. Water 1000.0 cc. Tildes' Blood Peptone Agar 1641
2. Soil 100.0 g. Golowkoff's Blood Peptone Agar
3. K2HPO4 1.0 g. (Uche) 1642
4. Peptone 10.0 g. Harvey's Blood Serum Peptone
5. Agar 15.0 g. Agar 1643
Preparation: Harvey's Litmus Blood Peptone
(1) Prepare a soil infusion by adding Agar 1644
1000.0 cc of water to 100.0 g. soil and B2. Serum or ascitic fluid employed.
bringing to a boil. Tochtermann's Serum Peptone
(2) Filter. Agar 1645
(3) Dissolve 3, 4 and 5 in (2). Browning's Telluric Acid Serum
(4) Adjust the reaction to +0.5. Agar (Wood) 1646
Sterilization: Not specified. Teague and Deibert's Serum Pep-
Use: Determination of bacterial counts tone Agar 1647
from soil. Emile-Weil's Pleuritic Serum Pep-
Variants: Brown suggested the use of 0.05 tone Agar 1648
g.peptone instead of 10.0 g. B3. Bile or its derivatives employed.
References: Temple (1912 p. 205), Brown Bacto MacConkey's Agar (De-
(1913 p. 499). hydrated) 1649
Jordan's Basal Peptone Bile Agar.. 1650
Padlewsky's Bile Peptone Agar
Basal or complete media containing agar (Bezanôn) 1651
and peptone (or other commercial digest) Northrup's Peptone Bile Agar 1652
and at least one other organic material of A3. Animal secretions, excretions or their
unknown composition. derivatives added.
Bacto Whey Agar (Dehydrated).. . 1653 3. NasCOa 3.6 to 4.05 g.

Sabouraud and Noire's Basal Whey 4. Peptone 7.0 g.
Peptone Agar (Weil & Noire) .... 1654 5. NaCl 3.5 g.
Tanner's Whey Peptone Agar 1655 6. Agar 21.0 g.
Committee A. P. H. A. Whey Pep- Preparation
tone Agar 1656 (1) Mix 150.0 cc. water and whole eggs in
Huss' Whey Peptone Agar 1657 equal parts, add 3 and shake thor-
Heller's Peptone Urine Agar 1658 oughly. (May be filtered thru thin
Loeffler's Malachite Green Nutrose layer of cotton to remove any thick
Peptone Agar 1659 part of egg.)
Raskin's Whey Peptone Agar 1660 (2) Steam (1) for 20 minutes.
(3) Dissolve 4, 5 and 6 in 700.0 cc. of
1628. Rivers and Kohn's Basic Blood Cell
water.
Peptone Agar
(4) Adjustment of reaction of (3) not
Constituents specified.
1. Distilled water 1000.0 cc. (5) Mix (2) and (3) while (3) is boiling
2. Peptone (Fairchild) 20.0 g. hot.
3. NaCl 5.0 g. (6) Pour medium thick plates and allow
4. Agar 15.0 g. to stand open for 20 to 30 minutes.
5. Red Blood Cells 10.0 cc. Sterilization: Method not given.
6. Brom cresol purple (25.0% Use: Isolation of cholera vibrio. Author
ale. soln.) reported that colonies by transmitted
Preparation : light appeared to be deep in agar, with
(1) Dissolve 2, 3 and 4 in 1 by boiling. hazy appearance.
(2) Titrate to pH = 7.4. References: Krumwiede, Pratt and Grund
(3) Filter and distribute in 100.0 cc. lots. (1912 p. 137), Stitt (1923 p. 50).
(4) At the time the agar is taken from the
1630. Weiss' Nahrstoff Heyden Gelatin Agar
autoclave while still at 95 C. add 1.0
cc. of washed red blood cells, 10.0 cc. Constituents
of a 10.0% solution of one of the added 1. Water 1000.0 cc.
nutrients and enough 25.0% alcoholic 2. Gelatin 75.0 g.
solution of brom cresol purple to each 3. Nahrstoff Heyden 4.0 g.
100.0 cc. to give a good color. 4. Agar 7.5 g.
(5) Tube while still warm. Preparation

(6) Incubate to test sterility. (1) Dissolve 15.0% (75.0 g.) gelatin and
Sterilization: Sterilize (3) in the autoclave. 0.8% (4.0 g.) Nahrstoff Heyden in
Use: To study fermentation. Authors re- 500.0 cc. water.
ported that the medium had a dirty look- (2) Dissolve 1.5% (7.5 g.) agar in 500.0
ing appearance but this did not interfere cc. water.
with the results. (3) Mix (1) and (2).
Added nutrients: The authors added 10.0 Sterilization: Not specified.

cc. of a 10.0% solution of one of the Use: Bacterial count of water.
following to each 100.0 cc. of agar: Reference: Weiss (1920 p. 25).
glucose fructose
1631, Deycke's Alkaline Albumin Gelatin
galactose maltose
Agar
xylose
Reference: Rivers & Kohn (1921 p. 481). Constituents:
1. Water 1000.0 cc.
1629. Krumwiede, Pratt and Grund's Egg 2. Gelatin 50.0 g.
Peptone Agar
3. Agar 20.0 g.
Constituents 4. .\lkaline albumin 25.0 g.
1. Water 850.0 cc. 5. NaCl 10.0 g.
2. Whole egg 150.0 cc. 6. Peptone 10.0 g.
Preparation by drop, using litmus paper as an

(1) Add2, 3, 4, 5and6to(l). indicator.
(2) Heat slowly until the gelatin is (3) Make alkaline by adding 1.0% of a
dissolved. Stir. soda solution which contains two
(3) Neutralize the alkaline mixture by parts water and one part soda.
the careful addition of HCl, drop by (4) Soak at room temperature for one
drop, using litmus as an indicator. or more hours.
(4) Add 2% of a soda solution containing (5) Boil in the steamer for I to one
two parts water to one part soda. hour.
(5) Boil two hours in the steamer. (6) Filter thru a layer of sterile cotton
(6) Filter thru a thin layer of sterile (may be filtered thru paper in a
cotton. Unna steam funnel).
(7) Distribute into sterile petri dishes (7) Distribute into sterile test tubes.
and solidify. (8) Sterilize once more in streaming
Sterilization: Sterilization given in the steam for one-half hour.
preparation. (9) Slant.
Use: Diagnosis of cholera. Author re- (b) Deycke and Voigtlander prepared a
ported that cholera colonies after 4 or 5 similar medium as follows:
hours appeared similar to colonies 15 to 20 (1) Pour 300.0 cc. Ba(OH)ii solution on
hours old in gelatin, having the same finely chopped horse heart (or other
characteristics as the cholera colonies fat free horse meat) and place in
described by Koch, shiny with irregular the incubator at 37C. for 48 hours.
edge. Liquefaction took place after 24 This solution is usually complete
to 30 hours at 20-22C. at the end of this time.
Reference: Deycke (1895 p. 244). (2) Dilute (1) with 600.0 cc. of water.
(3) Filter.
1631a. Deycke's Albuminate Glycerol Agar
(4) Heat to avoid the splitting of
Constituents: ammonia.
1. Distilled water 1000.0 cc. (5) Neutralize the filtrate with HCl
2. Alkali albuminate (from using litmus as an indicator.
veal) 10.0 g. (6) To each 100.0 cc. of (5) add 400.0
3. Peptone 10.0 g. cc. of water, NaCl, 5.0 g.
1.5 g.
4. NaCl 5.0 g. peptone, 25.0 g. glycerol and 10.0
5. Agar 20.0 g. g. agar. 1.5 to 2.0% glucose may be
6. Glycerol 10.0 g. added.
Preparation (7) Sterilization not specified.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. References: Deycke (1894 p. 528), (1896
(2) Make alkaline by adding 33.0% soda. p. 242), Deycke and Voigtlander (1901
(3) Filter. p. 621).
(4) Tube.
Sterilization: Method not specified. 1632. Krumwiede and Pratt's Gelatin
Use: Isolation of cholera vibrios. Author Serum Agar
reported that only cholera, anthrax,
diphtheria and tubercle bacilli grew. Constituents:
Variants : 1. Water 1000.0 cc.
(a) The author used the following me- 2. Peptone 20.0 g.
dium for the cultivation of diphtheria 3. Agar 10.0 g.
bacilli. 4. Gelatin SO.O g.
(1) Dissolve 10.0 g. alkaline albumi- 5. Serum, horse 500.0 cc.
nate, 10.0 g. peptone, 5.0 g. NaCl, Preparation
'^'"^
20.0 g. agar-agar and 5.0 g. glycerol (1) Dissolve 2, 3 and 4 in 1.
in 1000.0 cc. of distilled water. (2) Mix one part of 5 with two parts (1).
(2) Neutralize the alkaline mixture by (3) Adjust to slightly alkaline to litmus.
the careful addition of HCl, drop Sterilization: Method not specified.
Use: Cultivation and preservation of stock (c) Weiss made bacterial counts of water
cultures of fusiform bacilli. in a medium prepared as follows:
Variants: The authors substituted ascitic (1) Dissolve 15.0% gelatin and 1.0%
fluid for horse serum. peptone in 500.0 cc. of water.

Reference: Krumwiede and Pratt (1913 (2) Add 0.15% NasCOa to (1).
p. 200). (3) Dissolve 1.5% agar in 500.0 cc. of
distilled water.
1633. Dunschmann's Bile Salt Peptone Agar
(4) Mix 500.0 cc. (2) with 500.0 cc. (3).
(Bezanfon) Sterilization not specified.
(5)
Constituents References: Capaldi (1896 p. 475), Abt

1. Water 1000.0 cc. (1914 p. 151), Tanner (1919 p. 64), Weiss
2. Agar 30.0 to 40.0 g. (1920 p. 25), Heinemann (1922 p. 35).
3. Gelatin 5.0 g.
1635. Smyth's Egg Trypsinized Peptone
4. Sodium taurocholate. 15.0 to 25.0 g.
Agar
5. Lactose 40.0 g.
6. Peptone (vegetable) 10.0 g. Constituents:
Preparation: Dissolve 2, 3, 4, 5 and 6
(1) 1. Ringer's solution (see
in 1 (method not given). medium 180) 740.0 cc.
Sterilization: Method not given. 2. Egg albumin 250.0 cc.
Use: Enrichment of colon-typhoid group. 3 Peptone 10.0 g.
Reference: Bezangon (1920 p. 341). 4. Agar 7.5 g.

Preparation
1634. Capaldi's Peptone Gelatin Agar eggs in bichloride of
(1) Sterilize fresh
Constituents mercury solution followed by alco-
1. Distilled water 1000.0 cc. hol to remove the bichloride.
2. Peptone (Witte's) 20.0 g. (2) Cut a small window in the side of
3. Gelatin 10.0 g. an egg with a small sharp pointed
4. Glucose 10.0 g. scissors, in the same manner as one
5. NaCl 5.0 g. opens an egg to obtain the embryo.

6. KCl 5.0 g. (3) Remove the white with a sterile glass
7. Agar (2.0%) 20.0 g. pipette (3.0 to 5.0 cc.) to a sterile

Preparation Erlenmeyer flask, without breaking
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. the yolk.
(2) Make alkalineby the addition of (4) Dissolve 10.0 g. of Witte's peptone
10.0 cc. N/1 NaOH. in 200.0 cc. of Ringer's solution
(3) Filter. (See medium 180) at 80 to 90C.

Sterilization: Method not given. (5) Cool to 45C. and add 0.5 cc. of tryp-
Use: Differentiation between colon and sin powder suspended by shaking in
typhoid bacilli. The author reported 15.0 cc. of distilled water or Ringer's
that coli colonies were large milky colored solution and digest for 3 hours at
by reflected and brown by transmitted 40 to 45 C.
light. Typhoid colonies were small, (6) Dilute (5) to one liter with Ringer's
glistening and colorless. Abt used a solution (see medium 180) and boil
similar medium to produce anthrax for 20 minutes and replace loss with
spores. distilled water.
Variants (7) Filter and sterilize (The peptone

(a) Abt used 2.0 g. Chapoteaut's peptone, solution so prepared may be kept
2.0 g. glucose, 20.0 g. gelatin and for some time in a sterile flask).
15.0 g. agar instead of the amounts (8) To (3) add an equal quantity of (7).
specified by Capaldi and omitted the (9) Dissolve 15.0 g. agar in one liter
KCl. Ringer's solution in the Arnold
(b) Tanner used either 10.0 g. glucose or steam sterilizer or autoclave
mannitol. (10) Clarify with egg.
(11) Distribute in small flasks. Preparation

(12) When ready for use, melt sterile (11) (1) Dissolve 2, 3 and 4 in 1.
at 100C., cool to 45C. in a water (2) Distribute in test tubes in 1.0 cc. lots.
bath and then add an equal volume (3) Shortly before use the desired number
of (8) (albumen peptone solution). of sterile agar tubes are melted in the
(13) Keep at 45C. to prevent solidifica- water bath, cooled to 60C. and two
tion before use. volumes of defibrinated rabbit's blood
Sterilization: Method of sterilization not are added.
given. (4) Mi.x well and solidify in slanting
Use: To cultivate chick tissue in vitro. position.
Reference: Smyth (1914-15 p. 255). Sterilization: Sterilize by heating in the
autoclave at 105 to 108C. for 15 minutes.
1636. Czaplewski's Blood Nahrstoff Heyden
Agar (Klimmer) Use: Cultivation of Trypanosoma Brucei.
Variants: Harvey prepared a similar me-
Constituents:
dium by dissolving 0.5% peptone, 1.0%
1. Agar 1000.0 cc.
NaCl and 1.5% agar in water and mixing
2. Nahrstoff Heyden (1.0%) . . . 10.0 g.
one part of this sterile melted agar cooled
3. Blood, pigeon or dog.
to 45C. with one part defibrinated rab-
Preparation
bit's blood also at 45C.
(1) Add 1.0% Nahrstoff Heyden to
References: Behrens (1914 p. 28), Harvey
neutral agar.
(1921-22 p. 74).
(2) Add several drops of pigeon or dog
blood to (1). 1639. Kelser's Blood Peptone Agar
(3) Solidify in a thin layer.
Same as medium 724 but contains no glu-
cose and contains 1.5% agar.
Use: Cultivation of influenza bacilli.
Reference: Klimmer (1923 p. 227). 1640. Harvey's Blood Peptone Agar
1637. Norris et al. Blood Peptone Agar Same as variant (b) 724, but solidified by
Constituents: the addition of 3.0% agar.
1. Water 1000.0 cc.
1641. Fildes' Blood Peptone Agar
2. Peptone 20.0 g.
3. NaCl (5.0%) 50.0 g. Constituents:
4. Glucose 20.0 g. 1. Water.
5. Agar 20.0 g. 2. Agar.
6. Citrated blood (human). 100.0 cc.
. .
3. NaCl.
Preparation 4. Peptone (Morson's).
5. Laked blood.
(2) Adjust slightly alkaline to phenol-
6. Serum.
phthalein. Preparation
(3) Distribute in 20.0 cc. lots. (1) Collect ox or horse blood from a
(4) Add 2.0 cc. human citrated blood per

slaughter house.
20.0 cc. lot of medium. (2) Defibrinate the blood by stirring
Sterilization: Method not given. with a sterilized large wooden stick
Use: Cultivation of spirochetes. wrapped in gauze.
Reference: Norris, Pappenheimer and (3) Lake the blood by the addition of an
Flournoy (1906 p. 281). equal volume of distilled water.
(4) Distribute in 200.0 cc. lots into per-
1638. Nicolle's Blood Peptone Agar fectly fitting glass stoppered bottles.
(Behrens) (The author mentions that the me-
Constituents dium should contain no suspended
1. Distilled water 1000.0 cc. material, or it will not sterilize with
2. Agar 20.0 g. the method employed. However, he
3. Peptone 20.0 g. does not specify any sedimentation
4. NaCl 5.0 g. of the mixture after the addition of
5. Defibrinated blood, rabbit. . 2000.0 cc. the water.)
(5) Add chloroform to each bottle until and streptococci developed abnormally
0.5% chloroform has been added. large colonies on this medium.
(6) Add a drop of sterile oil to the stop- Reference: Fildes (1917 p. 492).
per and fasten a dust cover tightly Agar

1642. GolowkofE's Blood Peptone
over the stopper. (Uche)
(7) Place in the air incubator at 37C.
for 24 hours shaking constantly. Constituents:
Water 500.0 cc.
(8) After this time, remove a sample 1.
under aseptic conditions and test its 2. Agar (2.0%) 10.0 g.

Peptone 5.0 g.
sterility by mixing it with agar and 3.
incubating. (Time not specified.) 4. NaCl 2.5 g.

Blood 500.0 cc.
If sterile, do not heat longer for 5.
Sugar (0.5%) 2.5 g.

heating tends to darken the medium. 6.
Collect horse or ox blood in tall Preparation

(9)
(1) Prepare a 2.0% agar solution in
water.
sterile jars from the slaughter house.
(10) Allow to stand until all the cor- (2) Dissolve 3 and 4 in (1).
(3) Add an equal volume of
blood (with-
puscles have deposited. It may re-
quire 14 days for this to take place. out any preparation) heated to
After five days examine the jars and 80C., to (2).
pipette or siphon off the clear serum. (4) Boil for 15-20 minutes.
Do not disturb the sediment. (5) While hot, separate the coagulum by
the clear serum in pressing thru marly (a type of em-
(11) Distribute
200.0 cc. lots in perfectly fitting glass broidery cloth).
stoppered flasks. (6) Filter.
(12) Add chloroform to each

bottle until (7) Add 0.5% sugar.
0.5% chloroform has been added. Sterilization: Not specified.
(13) Add a drop of sterile oil to

the stop- Use: Diagnosis of diphtheria.
per and fasten a dust cover tightly Reference: Uche (1899 p. 393).
over the stopper. Serum Peptone Agar
1643. Harvey's Blood
(14) Place the bottles in a water
bath for
one hour at 45"C. Shake occa- Constituents:
sionally.
Peptone 10.0 g.
(15) When the bottles have cooled, re- 2.
move a sample under aseptic condi- 3. NaCl ,,..... 5.0 g.
Incubate Lactose 10.0 g.

tions and mix with agar. 4.
Neutral red (sat. soln.).... 10.0 cc.

the mixture at 37 for five days to 5.
6. Agar to solidify
test sterility.
Prepare a 2.5 to 3.0% agar solution in 7. Blood (defibrinated ox)
(16)
water containing salt and Morson's 8. Serum (ox)
peptone only (meat infusion or Preparation

derivatives not employed to decrease (1) Prepare a medium from 1, 2, 3 and 4
color).
(see medium 607) and solidify with
Add 1.0 cc. of (8) to 50.0 cc. of (15) agar.
(17)
under aseptic conditions, and add (2) Mix one part chloroform with 200.0
this mixture to 400.0 cc. of (16) and to 250.0 cc. of ox serum obtained
mix well. under as nearly aseptic conditions

Tube thru a "hooded pipette." as possible.
(18)
Sterilization: Method of sterilization of (3) Place in incubator 48 hours with
occasional shaking.
agar not specified.
Cultivation of meningococci and other (4) Test sterility.
Use :
Mix equal parts distilled water and

pathogenic forms, pneumococci and strep- (5)
defibrinated ox blood obtained under
tococci. The author reported that this
as nearly aseptic conditions as
medium was very good for routine work
with the meningococci. Pneumococci possible.
(6) Mix one part chloroform with 200.0 3. Peptone 10.0 g.

to 250 parts (5). 4. NaCl 5.0 g.
(7) Place (5) in incubator 48 hours with 5. Glucose 3.0 to 5.0 g.

occasional shaking. Preparation:
(8) Test sterility. (1) Dissolve 2, 3, 4 and 5 in 1.
(9) Add one part (8) to 20 parts (4) (2) Filter.
with sterile precautions, without (3) Mix (2) with equal parts sheep blood
shaking. serum or 3 parts serum to 2 parts agar,
(10) Mix with sterile precautions at 45 C. (4) Filter.
one part (9) with seven parts (5) Tube.
sterile (1). Sterilization: Sterilize in the usual manner
(11) Distribute with sterile precautions (method not given).
into test tubes. Use: Diagnosis of diphtheria. The author
(12) Slope. reported that diphtheria colonies after
(13) Stack the test tubes in the horizontal 24 hours were white.
position. Variants
(14) Test sterility bj' incubating 48 hours. (a) Dieudonne gave the following method
Sterilization: Method not given. of preparation:
Use: Cultivation of meningococci. The (1) Allow sheep blood to stand for
author reported that the medium should 24 hours.
be clear and free from all traces of red (2) Pour off the serum.
color. (3) Dissolve 10.0 g. peptone, 5.0 g.
Variants Levinthal (Harvey) used
: one NaCl and 20.0 g. agar in 1000.0 cc.
part (9) with 3 parts sterile agar. water.
Reference: Harvey (1921-22 p. 75). (4) Filter and add 3.0 to 5.0 g. glucose
to (3).
1644. Harvey's Litmus Blood Peptone Agar (5) Add an equal volume (or 3 parts
serum to 2 parts agar) of (2) and
Constituents
boil for 15 or 30 minutes.
1. Distilled water
(6) Filter.
2. Peptone 5.0 g.
(7) Distribute into test tubes and
3. NaCl 2.5 g.
streaming steam for 1 to
sterilize in
4. Glucose 5.0 g.
I5 hours on each of 3 successive
5. Litmus solution 20.0 cc.
days.
6. Defibrinated blood
(b) Kolle and Wasserman prepared the
Preparation:
medium as follows:
(1) Lake defibrinated blood with the
(1) Dissolve 20.0 g. agar, 10.0 g. pep-
smallest possible amount of sterile
tone, 5.0 g. NaCl, and 3.0 to 5.0 g.
distilled water.
glucose in 1000.0 cc. water.
(2) Prepare a medium from 1, 2, 3, 4 and 5
(2) Mix (1) with equal parts sheep
as indicated in medium 595, and
serum (or in the ratio of 2:3).
solidify with agar.
(3) Boil for 75 to 90 minutes.
(3) Add a sufficient amount of (1) to (2)
(4) Filter.
to give a deep color to the medium.
(5) Tube.
Sterilization: See medium 595 for steriliza-
(c) Besson specified the use of Chapo-
tion of the agar.
teaut's or Witte's peptone, heated in
Use: Cultivation of meningococci.
the autoclave at 115 to 120C. for
30 minutes and sterilized the medium
at 115C.
1645. Tochtermann's Serum Peptone Agar
(d) Harvey specified the use of calf serum,
Constituents boiled for 20 minutes and sterilized
1. Water 1000.0 cc. for 50 minutes at 100C. or 30 minutes
2. Agar 20.0 g. at 117C.
References: Tochtermann (1895 p. 965), blood cell extract was added. Otherwise
Kolle and Wassermann (1912 p. 406), Bes- no growth.
son (1920 p. 53), Harvey (1921-22 p. 79), Reference: Teague and Deibert (1922
Klimmer (1923 p. 221). p. 70).
1646. Browning's Telluric Acid Serum Agar 1648. Emlle-Weil's Pleuritic Serum
(Wood) Peptone Agar
1. Peptone water agar 1000.0 cc. 1. Water 1000.0 cc.
2. Serum, sheep 50.0 cc. 2. Peptone (Chapoteaut) 20.0 g.
3. Telluric acid (1.0%) 9.0 cc. 3. Glucose 8.0 g.
Preparation: (1) Add 50.0 cc. of sterile 4. Glycerol 20.0 g.
eheep serum, and 9.0 cc. of a 1.0% solution 5. Agar 24.0 g.
of telluric acid in distilled water to 6. Pleuritic serum (human) . . 250.0 cc.
1000.0 cc. of peptone water agar. Preparation
Sterilization : Sterilize the serum by heating (1) Dissolve 2, 3, 4 and 5 in 1.
at 57C. on several occasions. Method of (2) Tube.

sterilizing peptone water agar or telluric (3) To luke warm fluid (2) add one part
acid solution not given. human pleuritic serum to four parts
Use: Isolation of diphtheria bacilli. agar.
Reference: Wood (1921 p. 562). Sterilization: Not specified.
Use: Cultivation of Bacillus leprae.
1647. Teague and Deibert's Serum Peptone Variants: Emile- Weil gave the following
Agar variants:
Constituents (a) Used 40.0 g. glycerol, 4.0 g. glucose,
1. Distilled water. 20.0 peptone, 20.0 g. agar, and

g.
2. Peptone. mixed one part human pleuritic serum

3. Agar. with two parts agar.
4. NaCl. (b) Dissolved 10.0 g. NaCI, 8.0 g. glucose,
5. Serum. 16.0 g. peptone and 16.0 g. agar in
6. Red Blood Cells. 1000.0 cc. water and mixed 2 parts of

Preparation this solution with one part human
(1) Obtain sheep or rabbit serum from pleuritjc serum.
freshly drawn defibrinated blood. Reference: Emile-Weil (1905 p. 798).
(2) Heat (1) for 15 minutes at 55C.
1649. Bacto MacConkey's Agar
(3) Add 2.0 cc. of red blood cells (obtained
(Dehydrated)
by centrifuging defibrinated rabbit
blood and removing the serum) to Constituents:
10.0 cc. of physiological salt solution. 1. Water
(4) Keep the temperature of (3) at 100C. 2. Bile, Ox, Bacto 5.0 g.
for 3 minutes. 3. Lactose, Bacto 10.0 g.
(5) Shake and allow to cool.

(4) 4. Agar, Bacto 5.0 g.
(6) Centrifuge (5) and obtain the super- 5. Peptone, Bacto 20.0 g.
natant fluid. 6. Neutral red 0.05 g.
(7) Prepare a 5.0% peptone solution. Preparation

(8) Mix various amounts of (2), the super- (1) Dissolve 50.0 g. of Bacto MacConkey's
natant fluid from (6) and (7) and add Agar (Dehydrated) in 1000.0 cc. dis-
an agar solution containing 5.0 g. tilled water by boiling or autoclaving,
NaCl and 20.0 g. agar per 1000.0 cc. preferably the latter.
Sterilization: Not specified. (2) Restore the loss by the addition of
Use: To growth requirements of

study water if necessary.
Unna-Ducrey bacillus. Author reported (3) If sterilized at 15 pounds for 20 min-
that excellent growth was obtained if red utes pH = 7.0.
Sterilization: Sterilize in the autoclave at (2) Dissolve 10.0 g. lactose and 10.0 g.
15 pounds pressure for 20 minutes. peptone in 1000.0 cc. of (1).
Use: General culture medium, and in water (3) Dissolve 15.0 g. of agar in (2).
analysis. (4) Filter. Great care must be taken

Reference: Digestive Ferments Co. (1925 in filtering so that medium be free
p. 12). from precipitate, or colonies can-

not be counted.
1650. Jordan's Basal Peptone Bile Agar
Constituents References: Jordan (1912 p. 327), Ecker
1. Bile (ox) 1000.0 cc. (1918 p. 97), Obst (1919 p. 76).
2. Peptone (Witte) , 10.0 g.
3. Agar 15.0 g. 1651. Padlewsky's Bile Peptone Agar
Preparation: (Bezangon)
nutrients in 1000.0 cc. of undiluted Constituents:
fresh neutral ox bile. Avoid exces- 1. Water 1000.0 cc.
sive heating. 2. Peptone 20.0 g.
without adjusting the reaction. 3. Bile 35.0 cc.

(2) Filter
Tube. 4. Lactose 10.0 g.
(3)
Sterilization: Sterilize in the autoclave for 5. Malachite green
(1.0%) 5.0 cc.
Use: To show inhibitory action of bile on 6. Agar 30.0 g.
B. coli. Author reported that both 7. NasSOj (10.0%

soln.) 7.5 to 10.0 cc.
freshly isolated and strains of B. coli
long under cultivation were inhibited. Preparation:
(1) Dissolve 20.0 g. peptone, 30.0 g. agar
Added nutrients: Jordan added 10.0 g.
lactose.
and 30.0 cc. beef bile in a liter of
Variants
water (method not given).
(a) Ecker specified the use of Difco pep- (2) To 100.0 cc. of one add 0.5 cc. of a
tone, used 10.0 g. lactose as an added 1.0% aqueous solution of malachite
nutrient and used the medium to green and 0.5 cc. of bile.
study effect of bile on growth of (3) Add 0.75 cc. to 1.0 cc. of a 10.0%
He reported that some Na^SOa to each 100.0 cc. of medium.

B. typhosus.
strains were inhibited to a greater
degree than others. Approximately Use: Cultivation of colon-typhoid group.

the same results were obtained using Reference: Bezangon (1920 p. 344).
50.0% or 10.0% bile (diluted with dis-
1652. Northrup's Peptone Bile Agar
tilled water).
(b) Ecker used a medium containing Constituents
10.0 g. glycerol instead of lactose as 1. Water 1000.0 cc.
an added nutrient. The same re- 2. Agar 15.0 g.
sults were reported as under variant 3. Peptone 20.0 g.
(a) above. 4. NaCl. 5.0 g.
(c) Ecker used the basal medium with- 5. Lactose 20.0 g.
out any added nutrients. The same 6. Litmus (Standard solution

results were reported as under Merck) 10.0 cc.
variant (a) above. 7. Bile (ox or sheep) 60.0 cc.
(d) Obst used the following medium to Preparation

determine total count of B. coli in (1) Soak 15.0 g. of agar in 500.0 cc. of
water analysis. water over night.

(1) Sterilize freshly collected ox bile (2) Dissolve 20.0 g. of peptone and 5.0 g.
and store at 1C. until ready for NaCl in 100.0 cc. hot water.
use. Never use bile after one week (3) After the agar has been digested add
following collection. (2) to the sugar.
(4) Dissolve 20.0 g. lactose in 400.0 cc. Sterilization: Sterilize at 15 pounds pres-
hot water. sure for 20 minutes.
(5) Add (4) to (3). Use: Culture medium, particularly for bac-
Add the contents of two ox sheep bile teria in dairy products.
(6)
(about 60.0 cc). The bile having Reference: Digestive Ferments Co. (1925
been removed under aseptic condi- p. 14).
tions.
1654. Sabouraud and Noire's Whey Peptone
(7) Add 1.0% of a standard solution of
Agar (Weil and Noire)
Merck's litmus (Brown,
purified
C. W., Litmus media, 47th Ann. Rep., Constituents
Michigan Board of Agriculture, pp. 1. Water 500.0 cc.
127-129). 2. Milk., 1000.0 cc.
(8) Filter. 3. Peptone... 10.0 g.
Sterilization: Not specified. 4. Sucrose 5.0 g.
Use: Isolation of typhoid bacillus. 5. Urea 1.75 to 2.0 g.
Variants: (a) Rector reported that colon 6. Agar 24.0 g.
colonies on the following medium were Preparation

surrounded by a purplish red zone. (1) Precipitate the casein from one liter
(1) Add 100.0 g, of dried

ox bile and milk by the addition of 2.0 cc. of HCl.
10.0 g. Witte's peptone to 1000.0 cc. (2) Filter thru a wet cloth.
distilled water and boil. (3) Dissolve 10.0 g. peptone, 5.0 g.

sucrose, 1.75 to 2.0 g. urea in 500.0 cc.
(2) Filter.
(3) Add 1.5% shredded agar to (2) and water.
heat for an hour in the Arnold (4) Add one-half volume of (3) to one
sterilizer. volume of the filtrate from (2).
Dissolve 10.0 g. glucose in a small (5) Dissolve 1.6% agar in (4).

(4)
quantity of hot water. (6) Sterilize(method not given).
(5) Filter (3) thru absorbent cotton (7) Filter.
and cheese cloth. (8) Tube.

Prepare a 1.0% solution of neutral Sterilization: Sterilize again (method not
(6)
red. given).
(7) Add (4) and (6) to the filtrate of (5). Use: Cultivation of gonococcus.
(8) Mix well. Variants Bezangon prepared the medium
:
(9) Tube in 6 to 10.0 cc. quantities. as follows:
(10) Autoclave for 10 minutes at (1) Boil 1000.0 cc. of milk for 5 minutes.
pounds pressure.
12 (2) Add 2.0 cc. of HCl.
References: Northrup (1912 p. 420), Rector (3) Filter thru linen.
(1913p. 154),Stitt(1923p. 47). (4) Add one-half the volume of water to
the whey.
1653. Bacto Whey Agar (Dehydrated) (5) Neutralize by the addition of a
Constituents 10.0% soda solution.
1. Distilled water 1000,0 cc. (6) Autoclave (5) for 10 minutes at
2. Whey (dry) 13,0 g. 120C.
3. Peptone (Bacto) 10.0 g. (7) Filter.
4. NaCl 5.0 g. (8) Dissolve 1.0% peptone, 1.0% sucrose

5. Agar, Bacto 12.0 g. or glucose,0.3% urea and 1.6% agar
Preparation: in the filtrate by heating in the
(1) Dissolve 40.0 g. Bacto Whey Agar autoclave.

(Dehydrated) in 1000.0 cc. of dis- (9) Filter thru paper.
tilled water by boiling or preferably (10) Distribute in tubes.
autoclaving. (11) Sterilize for 10 minutes at 110C.
If sterilized at 15 pounds pressure References: Weil and Noire (1913 p. 1322),

(2)
for 20 minutes pH = 6.5. Bezangon (1920 p. 119).
1655. Tanner's Whey Peptone Agar Variants

(a) Fulmer and Grimes cultivated yeast
Constituents found in cream and butter on a me-
1. Whey. 1000.0 cc.
dium prepared as follows:
2. Agar (1.5%), 15.0 g.
(1) Dissolve 1.5%o agar and 0.5%, pep-
3. Peptone (Witte) (1.0%) .... 10.0 g.
tone (or omit peptone) in 1000.0 cc.
Glucose (2.0%) 20.0 g.
4.
of whey obtained from skim milk
Preparation by coagulation with rennet.
(1) Add a few drops of acetic
acid to boil-
(2) Filter thru absorbent cotton.
ing milk until the casein is pre- Tube in 10.0 cc. portions.
(3)
cipitated. for 20 minutes at
(4) Sterilize
(2) Filter. 15 pounds.
(3) Neutralize or bring to -t-1.0%. When used as a plate medium, add
(5)
(4) Dissolve 1.0% peptone, 2.0%,
glucose
1.0 cc. of 1.0% tartaric acid solu-
and 1.5%o agar in (2). tion to each petri dish, to keep
Clarify with whites of eggs or dry
(5) down bacterial growth. For use
albumin.
as solvents add 1.0 cc. of (4) to
(6) Tube. each tube after cooling to not
above 45C.
Use: Isolation of B. vulgaricus. (b) Cunningham gave the following
References: Heinemann and Hefferan (1909 method of preparation:
p. 310), Tanner (1919 p. 71), Stitt (1923
(1) Warm two liters of clean fresh
p. 38). skim milk pot at 37C.
in a large
1656. Committee A. P. H. A. Whey Peptone (2) Add sufficient rennet to curdle.

Agar (3) Allow to settle for 10 minutes.
(4) Break the curd into large pieces
Constituents:
by means of a stirring rod.
1. Whey 1000.0 cc.
Heat to SO'^C. to contract the clot
(5)
2. Peptone (1.0%) 10.0 g.
and to express the whey.
3. Agar (1.5%) 15.0 g.
(6) Strain thru a cheese cloth.
Preparation: NaCl.
(7) Add 1.0%o peptone and 0.5%o
(1) sufhcient rennet to a liter of
Add
(8) Steam for 30 minutes.
skimmed milk at 41C. to cause Neutralize to tumeric paper.
(9)
coagulation (1.0 cc. of liquid rennet
(10) Steam for one hour.
in 20.0 cc. distilled water).
(11) Dissolve 1.5% agar in (10).
(2) When the curd is firm cut in fine
(12) Filter thru paper until clear.
pieces and steam for 40 minutes.
(13) Add 1.0%, Andrades indicator.
Strain thru muslin.
(3) References: Committee A. P. H. A. (1909
(4) Adjust the reaction of the whey
Fulmer and Grimes (1923 p.
p. 287), 585),
+1.5 and add 1.0%, of dry peptone Klimmer (1923 p. 172), Cunningham (1924
and 1.5% shredded agar.
p. 102).
(5) Steam for one hour.
(6) Readjust to an acidity of 1.5%. 1657. Huss' Whey Peptone Agar
(7) Cool to 60C.
Clarify with egg. Constituents:
(8)
(9) Counterpose and boil over a free 1. Whey 1000.0 g
Agar 20.0 g
flame for 5 minutes. 2.
NaCl..... 5.0 g
(10) Filter thru cotton or a hot washed
3.
Peptone 1.0%, 10.0 g
plaited filter paper. 4.
Tube. Preparation: (1) Dissolve 2, 3 and 4 in 1

(11)
Sterilization: Sterilize 15 minutes for 3 (The peptone may be omitted.)
successive days in steam. Sterilization: Method not given.

culture medium for milk Use: Cultivation of aroma producing bac-
Use: General
analysis. teria. Bacillus esterificans, Maassen and
Pseudomonas trifolii. Other investiga- (3) Dissolve 10.0 to 12.0% gelatin or

tors cultivated bacteria found in milk in agar in (2).
similar media. (4) Filter, using a hot water funnel or
Variants in a steamer.
(a) Miiller used 1.5% agar instead of (5) Distribute into 10.0 cc. lots and
2.0%. sterilize for 10 to 15 minutes on
(b) Meier mi.xed equal parts (whey each of two days using the frac-
from goat milk (see medium 2277)) tional method.
and water and dissolved in the mix- (c) Piorkowski also gave the following
ture 1.5% agar, 1.0% Witte's pep- medium for the differentiation of
tone (the peptone may be omitted) colon and typhoid organisms. He
and 0.5% NaCl. The reaction was reported that the medium was blue.
adjusted by the addition of KOH Bad. coli communi appeared as
was turned a
until turmeric paper bluish-grey opaque damp colonies.
weak brownish red. Typhoid bacillus colonies delicate,
References: Huss (1907 p. 58), Miiller transparent and appeared blue.
(1917 p. 390), Meier (1918 p. 435). (1) Add 100.0 cc. of urine and 0.5 g.
peptone to a flask and plug with
1658. Heller's Peptone Urine Agar
cotton. A urine with a specific
Constituents gravity of 1012 gave best results.
1. Urine 1000.0 cc. (2) Steam for 15 minutes in a steamer.
2. Peptone 10.0 g. (3) Dissolve 2.0 g. of agar in (2).
3. NaCl 5.0 g. (4) Filter, using a hot water funnel or
4. Agar (1.5%) 15.0 g. filter in a steamer.
(1) Secure human urine as near average (6) To each tube add 8 drops of
specified gravity as possible. Bohmer's Hamato.xylyn solution
(2) Make weakly alkaline with soda. (strength not given).
End point indicated by precipitation minutes on each of
(7) Sterilize for 15
of salts. two successive days.
(3) Filter. (d) Matzuschita dissolved 2.0% agar,
(4) Add peptone, NaCI and 1.5% agar. 1.0% peptone and 0.5% NaCl in urine.
(5) Boil. References: Heller (1890 p. 893), Pior-
(6) Filter. kowski (1896 pp. 687, 694), Matzuschita
(7) Tube. (1901-2 p. 214).
Sterilization: Sterilize once in streaming
1659. LoefHer's Malachite Green Nutrose
steam.
Use: General culture medium. Peptone Agar (Harvey)
Variants Constituents:
(a) The author any carbo-
specified that 1. Distilled water 1000.0 cc.
hydrate, alcohol, etc., might be 2. Peptone 20.0 g.
added. The color and some in- 3. Lactose 50. g,
hibitory substances may be removed 4. Glucose 10.0 g.
by animal charcoal. 5. Nutrose 10.0 g.
(b) Piorkowski used the following me- 6. Malachite green (2.0%
dium to differentiate between Bac- chem. pure) lO.O cc.
terium coli and Bacillus typhi ab- 7. NaOH (N/1) 15.0 cc.
domin. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
(1) Add 100.0 cc. urine and 0.5 g. pep- in 1.
tone to a flask and plug with cot- Sterilization: Not specified.
ton. Urine should be fresh, clear, Use: Cultivation of colon-typhoid group.
bright yellow, and of acid reaction. Variants: Klimmer gave the following
A urine of specific gravity of 1.012 method of preparation:
gives the best medium. (1) Prepare a 0.5% peptone solution.
(2) Steam for 15 minutes in a steamer. (2) Add 9.5 cc. of HCl to (1).
(3) Soak 3 in (2). (9) Add soda to neutralize the reaction

(4) Boil for 30 minutes. (10) Filter until clear thru a paper in a
(5) Add 7.5 cc. of normal KOH. hot water funnel.
(6) Neutralize to litmus by the addi- Sterilization: Method not given.
tion of soda solution. Use Cultivation of pathogenic organisms.
:
(7) Add 5.0 cc. of a normal soda solution Variants

(143.0 g. crystalline soda per liter). (a) Raskin added 0.5% XaCl.
(8) Prepare a 10.0% nutrose solution. References: Raskin (1887 p. 358).

(9) Add 100.0 cc. of (6) to (5).
(10) Flask. SUBGROUP II-C. SECTION 6
(11) Sterilize on each of two successive
days. Basal or complete media containing agar
(12) Decant the clear agar from the and peptone (or other commercial digests)
sediment. and extracts or infusions of animal origin.
(13) Prepare a 2.0% solution of malachite Ai.* Not containing additional organic ma-
green 120 (Hochst) in sterile distilled terials (exclusive of indicators).
water (do not boil). Bi.* Infusions specified.
(15) Pour into Petri dishes. Ci. Indicators or dyes not added.
(16) Leave the plates open until the agar Di. Meat infusion used.
has solidified. Jacobi's Meat Infusion Salt Agar.. 1661
References: Harvey (1921-22 p. 91), Klim- Zettnow's Meat Infusion Agar 1662
mer(1923p.212). Besson's Phosphate Infusion Agar. . 1663
Smith's Infusion Agar 1664
1660. Raskin's Whey Peptone A.gar
Francis' Basal Infusion Agar 1665
Constituents: Deycke's Alkali Albuminate Agar 1666. . .
1. Whey 1000.0 cc. Harvey's Basal Infusion Agar 1667

2. Glycerol 50.0 cc. Krasnow's et al. Meat Infusion Agar 1668
3. Agar 5.0 to 7.0 g. Dj. Infusions of other tissues or special
4. Peptone 10.0 g. organs, etc. used.
Preparation Dopter's Liver Infusion Agar
(1) Add 50.0 cc. of glycerol and 5.0 to (White) 1669
7.0 g. finely dried agar to 1000.0 cc. Richardson's Mucose Infusion Agar. 1670
fresh milk. Lichtenstein's Blood Clot Infusion
(2) Allow to stand for 12 to 14 hours (at Agar 1671
room temperature in the winter) and Stuart's Stomach-Liver Infusion
then boil for 75 to 90 minutes over a Agar 1672
free flame. In order to prevent loss Bailey's Hormone Agar 1673
of water during the boiling place a Fasiani & Zironi's Veal Autolysate
lid over the container or steam for Peptone Agar 1674
3 to 3j hours in a steamer. The Cj. Indicators or dyes added.
coagulation of the casein takes place Harvey's Basal Indicator Infusion
slowly. Agar 1675
(3) Press thru four folds of linen cloth Jordan and Victorson's Lead Ace-
separating the casein and liquid. tate Infusion Agar 1676
(4) The reaction of the fluid is slightly Elser and Huntoon's Basal Litmus
acid. Infusion Agar 1677
(5) Pour into a tall glass cylinder and Emile-Weil's Neutral Red Infusion
place in a warm thermostat. Agar 1678
(6) After about 20 or 30 minutes two Lentz and Tietz's Malachite Green
layers are formed, the lower layer Infusion Agar 1679
being transparent and fat free, the Zielleczky's Phenolphthalein Infu-
upper layer yellowish white, con- sion Agar 1680
taining the fat. Worbitzki's China Green Infusion
(7) Allow to cool, and remove the fat Agar..,. 1681
by means of a spoon.
(8) Heat the fat free portion to boiling
and add 1.0% peptone. See page 482 for A2 and B2.
Teague's Victoria Blue Infusion Warden's Salt Agar 1711

Agar 1682 Stroszner's Regenerated Agar 1712
Drennan and Teague's Crystal C2. Indicators or dyes added.
Violet Infusion Agar 1683 Besson's Basal Litmus Agar 1713
Meyer & Batchelder's Sulphite Gen- Mandelbaum's Basal Rosolic Acid
tian Violet Infusion Agar 1684 Agar 1714
MuUer's Indicator Infusion Agar. . . 1685 Zipfel'sRegeneratedDrigalski'sAgar 1715
Friedberger & Joachimoglu's Tellu- Rothberger's Indicator Agar 1716
rite Placenta Infusion Agar 1687 Omelianski's Indicator Agar 1717
Ragit Agar 1688 Burnet and Weissenbach's Lead Ace-
Bj.* Extracts specified. tate Agar 1718
Ci. Indicators or dyes not added. Noeggerath's Indicator Agar (Bes-
Di. Complete dehydrated media commer- son) 1719
cially prepared. Gasser's Fuchsin Agar 1720
Bacto Nutrient Agar (Dehydrated) 1689 . Krumwiede and Pratt's Dahlia Agar 1721
Bacto Nutrient Phosphate Agar A2. Containing additional organic ma-
(Dehydrated) 1690 terials.
Bacto Nutrient Agar 1.5% (Dehy- Bi.* All additional material of known chem-
drated) 1691 icalcomposition.
Bacto Nitrate Agar (Dehydrated) 1692 . . Ci.t Infusions specified.
D2. Media prepared from its constituents. Di.f Only one additional source of carbon
Bannings' Basal Meat Extract Agar. 1693 added.
Heim's Meat Extract Salt Agar 1694 El. Carbohydrates employed.
Heinemann's Meat Extract Agar. . . 1695 Fi. Monosaccharides, only, used.
Glaessner's Nahrstoff Heyden Ex- Gi. Indicators or dyes not added.
tract Agar 1696 Meier's Glucose Infusion Agar 1722
Guth's Selenicate Extract Agar 1697 Kitchens' Glucose Infusion Agar 1723
Conn and Breed's Nitrate Extract Jackson and Muer's Liver Infusion
Agar 1698 Agar 1724
Kotlar's Pancreas Peptone Agar 1699 Hall's Testicular Infusion Agar 1725
C2. Indicators or dyes added. G2. Indicators or dyes added.
Bacto Andrade Maltose Agar (De- Kligler's Lead Acetate Infusion
hydrated) 1700 Agar 1726
Bacto Andrade Mannite Agar (De- Gage and Phelps' Neutral Red Infu-
hydrated) 1701 sion Agar 1727
Bacto Andrade Dextrose Agar (De- Harvey's Malachite Green Infusion
hydrated) 1702 Agar 1728
Bacto Andrade Lactose Agar (De- F2. Disaccharides, only, used.
hydrated) 1703 Endo's Fuchsin Sulphite Infusion
Bacto Andrade Saccharose Agar Agar 1729
(Dehydrated) 1704 Wurtz' Litmus Lactose Agar 1730
Percival's Basal Litmus Extract Bitter's China Blue Malachite Green
Agar 1705 Agar 1731
Committee S. A. B. Lead Acetate F3. Polysaccharides used.
Extract Agar 1706 Guth's Alizarine Lactose Agar
Conradi's Brilliant Green Picric (Klimmer) 1732
Acid Extract Agar (Bezan9on) 1707
. . . Harvey's Starch Agar 1733
Klinger's Malachite Green Extract Gassner's Metachrone Yellow Water
Agar 1708 Blue Infusion Agar 1734
B3. Whether infusions or extracts employed Torrey's Brom Cresol Purple Lac-
not definitely specified. tose Agar 1735
Ci. Indicators or dyes not added. E2.t Alcohols employed.

Kohler's Basal Agar 1709 Abbott's Glycerol Infusion Agar 1736
Wurtz's Nutrient Agar 1710
* See page 484 for B2.
t See page 483 for C2 and Dj.
See B3. t See E3, page 483.
Kowalski's Glycerol Lung Infusion Salomonsen's Sucrose Extract Agar

Agar (Dittrich) 1737 (Besson) 1762
Henssen's Glycerol Kidney Agar. .. 1738 Fj. Polysaccharides, only, added.
Harvey's Mannitol Infusion Agar. 1739
. .
Hesse's Starch Extract Agar (Stokes
E3. Organic acids or their salts employed. and Hachtel) 1763
Omelianski's Formate Agar 1740 Committee S. A. B. Starch Extract
Harvey's Salicylate Infusion Agar. 1741 .
Agar 1764
D2. More than one additional source of E2. Alcohols, acids or their salts employed.
carbon added. Coplin and Bevan's Glycerol Ex-
El. Two or more carbohydrates employed, tract Agar 1765
de Gasperi and Savini's Glucose
E3. Other organic materials employed.
Lactose Agar 1742
_.
Piettre and de Souza's Citric Acid

Teague and Clurman's Eosin Bril-
Extract Agar 1766
Green Agar
liant 1743
Fawcus' Dye Bile Salt Agar (Be-
Kan-Ichiro, Morishima's Lead Ace-
zangon) 1767
tate China Blue Agar 1744
Percival's Urea Extract Agar 1768
Aronson's Fuchsin Sulphite Agar
D2. More than one additional source of
(Harvey) 1745
carbon added.
E2. Carbohydrates and alcohols employed.
V. Szaboky's Glycerol Lung Agar. 1746
. .
El. Organic nitrogen added.
Hulton-Frankel's Inositol Infusion Gaehtgen's Caffeine Endo Agar. 1769 . . .
Agar 1747
Wilson and Darling's Brilliant
Robinson and Rettger's Opsine In- Green Bile Salt Agar 1770
fusion Agar 1748

Olszewski and Kohler's Endo Bile
E3. Carbohydrates with an organic nitrog-
Salt Agar 1771
enous compound of known chemical com- E2. Organic nitrogen not added.
position. Fi. Containing carbohydrates and alcohols.
Lubenau's Lactose Caffeine Agar.. 1749 Bacto Saccharose-Mannitol Agar
Harvey's Caffeine Endo Agar 1750 (Dehydrated) 1772
Gathgen's Caffeine Fuchsin Sulphite Hesse's Lactose Glycerol Agar
Agar (Bezangon) 1751 (Stokes and Hachtel) 1773
C2.* Extracts specified. Schniirer's Saponin Glycerol Agar. 1774 .
Di. Only one additional source of carbon F2. Containing carbohydrates, without
added. alcohols.
El. Carbohydrates employed. Gi. Containing two carbohydrates.
Fi. Monosaccharides, only, added. Bacto Russell Double Sugar Agar
Viehoever's Basal Glucose Extract (Dehydrated)., 1775
Agar 1752 Nichols and Woods' Russell Double
Bacto Dextrose Agar (Dehydrated) . 1753 Sugar Agar 1776
Henneberg's Glucose Extract Agar. . 1754 Bailey and Lacey's Phenol Red Lead
Oldekop's Neutral Red Glucose Ex- Acetate Agar ^. 1777
tract Agar 1755 Holt-Harris and Teague's Eosine
F2. Disaccharides, only, added. Methylene Blue Agar 1778
Bacto Purple Lactose Agar (Dehy- Krumwiede, Pratt and McWilliams'
drated) 1756 Brilliant Green Agar 1778a
Bacto Litmus Lactose Agar (Dehy- Aronson's Fuchsin Sulphite Agar. 1779 . .
drated) 1757 Hesse's Malachite Green Agar

A. P. H. A. Litmus Lactose Extract (Klimmer) 1780
Agar (1917) 1758
G2. Containing more than two carbo-
Endo's Fuchsin Sulphite Agar hydrates.
(Heinemann) 1759
Bacto Krumwiede Triple Sugar
Hirschbruch & Schwer's Crystal
Agar (Dehydrated) 1781
Violet Litmus Lactose Agar 1760
Krumwiede and Kohn's Triple Sugar
Chesney's Indicator Lactose Agar. 1761 .
Agar 1782
* See C3, page 484. Amoss' Four Sugar Agar 1783
Amoss' Sucrose Salicin Agar (Top- Penf old's Monochlorhydrin Agar. 1814 . .
ley and Ayrton , 1784 Jacobson's Ethylcinnamic Ether

C3. Whether infusions or extracts employed Agar 1815
not definitely specified. Hurler's Caffeine Agar 1816
Di. Only one additional source of carbon Finger, Ghon and Schlagenhaufer's
added. Urea Agar 1817
El. Carbohydrates employed. D2. More than one additional source of
Monosaccharides only added. carbon added.
Fi.
Frost's Glucose Agar 1785 El. Containing two or more carbohy-
Mankowski's Indigo Carmine Glu- drates.
cose Agar 1786

Russell's Double Sugar Agar 1818
Rivas' Glucose Agar 1787 E2. Containing carbohydrates and alcohols.
Hall and EUiefson's Gentian Violet Thoinot and Masselin's Glucose
Glucose Agar 1788 Glycerol Agar 1819
Wilson and Blair's Sulphite Glucose Thoinot and Masselin's Sucrose
Agar 1789 Glycerol Agar 1820
Scheffler's Neutral Red Glucose Kligler and Defandorfer's Double
Agar 1790 Sugar Agar 1821
Martin and Loiseau's Glucose Lit- Kendall and Ryan's Sucrose Man-
mus Agar 1791 nitol Agar 1822
F2. Dissaccharides, only, added.
Hulton-Frankel and MacDonald's
Inositol Dextrin Agar 1823
Frost's Lactose Agar 1792
E3. Containing carbohydrates and organic
Wurtz's Litmus Lactose Agar 1793
acids or their salts.
Gassner's Metachrome Yellow
Water Blue Lactose Agar (Klim- Kitasato's Glucose Formate Agar
(Tanner).. 1824
mer) 1794
Hirschbruch and Schwer's Azolit- Sohngen and Fol's Glucose Butyrate
min Crystal Violet Lactose Agar. Agar 1825
. 1795
Ramond's Rubine Acid Lactose Agar MacDonald's Glucose Lactic Acid
1796
Delta's Fuchsin Lactose Agar Agar 1826
1797
Kindberg's E4. Containing carbohydrates and organic
Fuchsin Malachite
Green Agar nitrogenous compounds.
1798
Miiller's Lactose Tartrate Agar 1827
China Blue Malachite-green
Bitters'
Kligler's Lead Acetate Glucose Agar. 1828
Agar (Klimmer) , 1799
Liebermann and Acels' Congo Red MacConkey's Lactose Bile Salt
Agar (Klimmer) 1800
Agar (Heinemann) 1829
Massini's Triple Dye Lactose Agar. Es. Containing alcohols and organic acids
1801
or their salts.
F3. Polysaccharides, only, added.
Fleming's Oleic Acid Glycerol Agar. 1830
Lange's Starch Agar 1802
B2. Containing material of unknown chem-
Hoffman's Nitrate Starch Agar 1803
ical composition.
Hoffman's Nitrite Starch Agar 1804
Ci.* Extracts specified.
Khouvine's Cellulose Agar 1805
Di. Containing animal tissue or cells, or
Scales' Salt Cellulose Agar 1806
their derivatives.
E2. Alcohols employed.
Rosenow and Towne's Ascitic Fluid
Cantani's Basal Glycerol Agar 1807
Kidney Agar 1831
Wurtz's Glycerol Agar 1808
Rosenow's Glucose Brain Agar
Scheffler's Indicator Glycerol Agar. 1809
(Haden) 1832
Mandelbaum's Rosolic Acid Glyc-
Goldberger's Glucose Alkaline Egg
Agar
erol 1810
Agar (Abbott) 1833
Heinemann's Litmus Mannitol Agar. 1811
Goldberg's Meat Infusion Extract
E3. Organic materials other than carbo-
Agar (Stitt) 1834
hydrates or alcohols employed. Dimitroff's Egg Agar 1835
Penf old's Phenylacetate Agar 1812
Wurtz's Phenol Agar (Copeland). . . 1813 * See page 485 for C2.
Dunschmann's Bile Salt Gelatin F3. Egg or other materials employed.

Agar 1836 Sacquepee and Delater's Egg Albu-
D2. Containing animal fluids. min Infusion Agar 1861
El. Ascitic fluid specified. Emile-Weil Egg Yolk Infusion Agar . 1862
Paneth's Glucose Ascitic Fluid Agar. 1837 Huntoon's Hormone Agar 1863
E2. Blood specified. Harvey's Alkaline Egg Agar 1864
Baehr and Plotz's Blood Ascitic Meyers' Basal MoUusk Infusion
Fluid Agar 1838 Agar 1865
Reed and Orr's Blood Agar 1839 Faroy and Chavaillon's Egg Albu-
Becker's Defibrinated Blood Agar. 1840 min Serum Agar 1866
Esch's Alkaline Hemoglobin Ragit Huntoon's Hormone Blood Agar. . . 1867
Agar 1841 Torrey and Buckell's Ascitic Fluid
E3. Bile specified. Egg Agar 186S
Padlewsky's Malachite Green Bile Williams-Burdick's Modified Pet-
Agar 1842 roff's Egg Infusion Agar (Roddy) . 1869
E4. Variety of body fluids specified. Chapin's Egg Yolk Urine Agar 1870
Fildes'Body Fluid Agar 1843 Harvey's Sucrose Egg Agar 1871
Haner and Frost's Milk Body Fluid Leboeuf's Egg White Liver Infusion
Agar 1844 Agar 1872
Bacto Conradi-Drigalski Agar (De- F4. Tissue derivatives employed.
hydrated) 1845 Besson's Gelatin Infusion Agar 1873
D3. Containing animal secretions or excre- Frothingham's Gelatin Infusion
tions or their derivatives. Agar 1874
Haner and Frost's :\Iilk Agar 1846 Krause's Gelatin Urea Agar 1875
Eldredge and Roger's Whey Agar. 1847 . .
MacXeal and Kerr's Gelatin Agar. 1876 .
Ayers, Courtland and Mudge's Milk Supplee's Nutrose Gelatin Agar

Powder Agar 1848 (Ayers and Johnson) 1877
Conradi-Drigalski's Crystal Violet Kinsella, Brown and Garcia's Nu-
Litmus Agar (Park, Williams and trose Gelatin Agar 1878
Krumwiede) 1849 Ayers and Johnson's Casein Gelatin
Tausz and Peter's Ragit Nutrose Agar 1879
Agar 1850 Frazier's Gelatin Agar 1880
Hunter's Trypsinized Casein Extract E2. Containing animal fluids.
Agar 1851
Fi.* Blood or its derivatives employed.
Zoller's Citrate Milk Agar 1852
Gi.* Defibrinated blood specified.
C2.* Infusions specified.
Hi. Additional organic constituents (if any)
Di.f Containing unknown constituents of
of known chemical composition.
animal origin.
Duval and Lewis' Glucose Blood
El. Containing animal tissues or cells or
Agar 1881
their derivatives.
Torrey's Glucose Blood Agar 1882
Fi. Tissue employed.
Agar Erickson and Albert's Testicular
Smillie's Tissue Infusion 1853
Blood Agar 1883
vanRiemsdijk's Liver Infusion Agar. 1854
Harvey's Lactose Blood Agar 1884
Williams' Tissue Infusion Agar 1855
Harvey's Saponin Blood Agar 1885
Olitsky and Gates' Ascitic Fluid
Warden's Blood Veal Agar 1886
Kidney Agar 1856
Pelouze & Viteri's Brain Veal Infu-
Wherry and Ervin's Glycerol Blood
Agar 1887
Agar
sion .
, 1857
Elser and Huntoon's Basal Blood
F2.I: Blood Cells employed.
Agar Infusion Agar 1888
Avery's Oleate Blood Cell
(Harvey) 1858 North's Gelatin Blood Agar
Brown and Orcutt's Blood Cell Agar . 1859 (Kligler) 1889
Behren's Blood Cell Agar 1860 Dieudonne's Alkaline Blood Agar
(Harvey) 1890
* See C3, page 487.
t See page 486 for D2.
X See F3 and F4. See page 485 for F2 and Gj.
Fildes' Blood Digest Agar (Kristen- Harvey's Telluric Acid Serum Agar. 1923
sen) 1891 Francis' Cystine Serum Agar (Stitt) 1924 .
MacNeal's Blood Infusion Agar 1892 employed.

F3. Ascitic fluid
Smedley's Blood Infusion Agar... 1893 Elser and Huntoon's Basal Ascitic
Harvey's Peptic Blood Digest Agar. 1894 Fluid Agar 1925
Bailey's Hormone Blood Agar 1895 Gilbert and Humphrey's Tellurite
H2. One or more of additional organic con- Serum Agar 1926
stituents of unknown chemical com- Scholtz's Ascitic Fluid Agar 1927
position. Kiefer's Ascitic Fluid Agar (Abel) . . 1928
Harvey's Ascitic Fluid Blood Agar. . 1896 Tx)rrey and Buckell's Urine Ascitic
G2. Whole blood specified. Fluid Agar 1929
Hi. Blood citrated or oxalated. Watabiki's Whey Ascitic Fluid Agar. 1930
Harvey's Oxalated Blood Agar 1897 Esch's Maltose Ascitic Fluid Agar. 1931 .
Wolbach, Chapman and Stevens' Ci- F4. Animal than blood, serum,
fluids other
trated Blood Agar 1898 or ascitic fluid employed.
Noguchi's Serum Plasma Agar 1899 Steinschneider's Hydrocele Fluid
Soparkar's Citrated Blood Agar Agar 1932
(Liston) 1900 Heiman's Pleuritic Serum Agar 1933
Hirsch and McKinney's Chocolate Lentz and Tietz's Malachite Green
Agar 1901 Bile Agar (Klimmer) 1934
Harvey's Glucose Blood Agar 1902 E3. Containing animal secretions, excre-
H2. Blood neither citrated nor oxalated. tions or their derivatives.
Grassberger's Blood Agar 1903
Fi. Milk or its derivatives employed.
Ruediger's Blood Agar 1904
Gi. Nutrose used.
Dieudonne's Alkaline Blood Agar
Lentz and Tietz's Malachite Green
(Tanner) 1905
Nutrose Bile Agar (Klimmer) . . . 1935
Sherwood and Downs' Glucose
V. Drigalski and Conradi's Crystal
Blood Agar 1906
Violet Nutrose Agar 1936
Hall's Testicular Infusion Blood
Galli-Vallerio's Neutral Red Nu-
Agar (Stitt) 1907
trose Agar 1937
Cutler's Blood Clot Infusion Agar
Teague and Travis' Eosin Bismark
(Klimmer) 1908
Brown Nutrose Agar 1938
Harvey's Trypsinized Blood Agar. 1909 .
Gassner's Nutrose Agar 1939

Harvey's Hydrolyzed Blood Agar.. 1910
LoefHer's Malachite Green Nutrose
G3. Hemoglobin specified.
Agar (Roddy) 1940
Kristensen's Hemoglobin Infusion
Schmitz's Nutrose Blood Clot Infu-
Agar 1911
sion Agar 1941
Brown and Orcutt's Hemoglobin In-
fusion Agar 1912 G2. Nutrose not used.
Esch's Alkaline Hemoglobin Infu- Harvey's Milk Agar 1942
sion Agar (Tanner) 1913

Lubenau's Caffeine Whey Agar 1943
Harvey's Whey Infusion Agar 1944
r2. Serum employed.
Sherwood and Downs' Basal Serum F2. Milk or its derivatives not employed.
Agar 1914 Schloffer's Urine Infusion Agar. 1945
. . .
Harvey's Placenta Blood Serum Sputum Agar...

Picker's Glycerol 1946
Agar 1915 Szaboky's Sputum Lung Infusion
Veillon's Serum Agar 1916 Agar 1947
Kutscher's Serum Placenta Agar.. 1917 Costa and Boyer's Gum Infusion
BezanQon's Serum Placenta Agar. . . 1918 Agar 1948
Shamine's Nucleic Acid Serum Agar. 1919 Tulloch's Ox Heart Infusion Pea
Robey, et al., Glucose Serum Agar. . 1920 Flower Agar 1949a
Noguchi's Ringer Solution Serum D2. Not containing additional materials of
Agar 1921 unknown chemical composition of animal
Todd's Lactose Serum Agar 1922 origin.
C3. Whether infusions or extracts were em- G2. At least one additional material or-
ployed not definitely specified. ganic.
Di. Containing tissues, cells or their Mandelbaum's Lactose Blood Agar. 1977
derivatives. Thompson's Glucose Plasma Agar. . 1978
El. Egg employed. Avery's Oleate Blood Agar (Stitt). 1979
Fi. Yolks only used. Esch's Ascitic Fluid Blood Agar 1980
Capaldi's Egg Yolk Agar 1949 Liston's Trypsinized Casein Blood
Nastiukoff's Egg Yolk Nutrient Agar 1981
Agar (Rechtsamer) 1950 F2.* Whole blood used.
Bezangon and Griffon's Glycerol Gi. Blood citrated or oxalated.
Egg Yolk Agar 1951 Bernstein's Basal Blood Agar 1982
Pergola's Tellurite Egg Yolk Agar. 1952 . Wordley's Oxalated Blood Agar 1983
F2. Whites of eggs only used. Wilson and Darling's Laked Blood
Lipschutz's Egg Albumin Agar 1953 Agar 1984
Krumwiede, Pratt and Grund's Egg Wilson and Darling's Lactose Blood
Albumin Agar 1954 Agar 1985
Oberstadt's Egg Albumin Agar 1955 Stitt's Glycerol Blood Agar (Choco-
F3. Whole egg (white and yolk) used. late Agar) 1986
Besredka and Jupille's Egg Agar Besson's Citrated Blood Agar 1987
(Besson) 1956 G2. Blood neither citrated nor oxalated.
Scales' Whole Egg Agar 1957 Hi. Additional constituents, if any, inor-
Stitt's Glycerol Egg Agar 1958 ganic, (exclusive of dyes).
Robertson's Alkaline Egg Agar Bezangon, Griffon and LeSourd's
(Park, Williams and Krumwiede) 1959 .
Blood Agar 1988
E2. Tissues or their derivatives other than Fleming's Brilliant Green Blood
eggs employed. Agar 1989
Fi. Tissue used. Hachla and Holobut's Alkaline
Cantani'juns' Sperm Agar 1960 Blood Agar 1990
Pettersson's Brain Ascitic Fluid Matthews' Trypsinized Blood Agar. 1991
Agar 1961 H2. At least one of the added constituents
Smith and Taylor's Fetus Agar 1962 organic.
Noguchi's Ascitic Fluid Tissue Agar . 1963 Bieling's Glucose Blood
Agar 1992
Gozony's Kidney Agar 1964 Mandelbaum and Heinemann's
Duval's Trypsinized Tissue Agar. . . 1965 Glycerol Blood Agar (Kolle and
F2. Tissue derivatives used. Wassermann) 1993
Gi. Containing gelatin.
Wassermann's Nutrose Blood Agar
Thoinot and Masselin's Gelatin Agar (Abel) 1994
. 1966
Fremlin's Phosphate Gelatin Agar. Savini and Savini-Castano's Bac-
. 1967
teriaBlood Agar 1995
G2. Not containing gelatin.
Bieling's Optochin Hydrochloride
Fat Agar
Vierling's 1968
Blood Agar 1996
Abe's Meat Water Infusion Agar. 1969 . .
F3. Hemoglobin used.

Esch's Hydrolyzed Meat Agar
Heim's Hemoglobin Agar 1997
(Kohlisch and Otto) 1970
Esch's Alkaline Hemoglobin Agar.. 1998
Wellman's Placenta Infusion Agar. . 1971
Kabeshima's Alkaline Hemoglobin
D2.* Containing animal fluids.
Agar 1999
El. Blood or its derivatives employed.
Besson's Glycerol Hemoglobin Agar
Fi. Defibrinated blood used. (Tanner) 2000
Gi. Additional materials, if any, inorganic, Kabeshima's Hemoglobin Extract
(exclusive of dyes). Agar 2001
Orcutt and Howe's Fat Blood Agar. 1972 E2 t Serum employed.
Dieudonne's Alkaline Blood Agar.. 1973 Finger, Ghon and Schlagenhaufer's
Pilon's Alkaline Blood Agar 1974 Dialyzed Serum Agar 2002
Filde.s' Pepsinized Blood Agar 1975 Miiller's Serum Agar 2003
Carpano's Hemolyzed Blood Agar. . 1976
* See F3.
See D3 and D4, page 488. See E3 and E4, page 488.
t
Joas' Alkaline Serum Agar (Klim- (3) Heat over a free flame until the ma-
mer) 2004 terials are completely dissolved.
Muhlen and Hoffman's Glucose (4) Make up to the original volume by
Serum Agar (Stitt) 2005 the addition of water.
Cantani's Glycerol Serum Agar (5) Add Na2C03 or sodium phosphate
(Besson) 2006 until the reaction is slightly alkaline.
Kodama's Fuchsin Sulphite Serum (6) Pour into a flask and steam until
Agar 2007 the albuminous material still to be
Wassermann's Nutrose Serum Agar. 2008 removed is completely separated
Kliglers' Nasal Secretion Serum (usually 2 hours if sodium phosphate
Agar 2009 is used and longer if Na2C03).
Meyers' Tuberculin Agar 2010 (7) Filter thru cotton, using compressed
Douglas' Tellurite Trypsinized Se- air to effect a fast filtration.
rum Agar 2011 (8) Distribute into smaller flasks.
Czaplewski's Alkaline Serum Glu- (9) Heat in streaming steam for 2 hours
cose Agar 2012 (if the glassware has been sterilized
Klein's AlkalineSerum Agar 2013 in the autoclave).

Frost, Charlton and Little's Milk (10) Finally distribute into sterile test
Serum Agar 2014 tubes that have been autoclaved in
Es. Ascitic fluid employed. the autoclave for 2^ hours. (The
Salomon's Basal Ascitic Fluid Agar. 2015 tubes are contained in an enamel
Rosenow's Glucose Ascitic Fluid container in the autoclave. The
Agar 2016 inner temperature reaching about
Ruediger's Inulin Ascitic Fluid Agar. 2017 150C.).
Klimenko's Glycerol Ascitic Fluid Sterilization: Not specified.
Agar 2018 Use: General culture medium.
Veilion's Ascitic Fluid Agar 2019 Variants: The following authors prepared
Herrold's Phosphate Ascitic Fluid similar media as indicated:
Agar 2020 (a) Schultz (1891).
E4. Bile employed. (1) Place 500.0 g. of the best quality
Loeffler's Dye Bile Agar (Abel) .... 2021 meat, without fat or tendons, in a
D3. Containing animal secretions or ex- glass container fitted with a lid.
cretions. (2) Pour 1300.0 cc. of distilled water
El. Milk or its derivatives employed. over the meat.
Hasting's Milk Agar 2022 (3) Store in a cool place until the
Valleti's Whey Agar 2023 next day.
Klimmer's Casein Agar 2024 (4) Filter thru 4 thicknesses of
E2. Milk or its derivatives not employed. cloth and press the remaining
Mayer's Mucin Agar 2025 meat to obtain as much fluid as
Fichtner's Sputum Agar 2026
Finger, Ghon and Schlagenhaufer's (5) Pour the filtrate into a kettle,
Urine Agar 2027 add 10.0 g. peptone (siccum),
D4. Containing materials of plant origin. 5.0 g, NaCl and the whites of two
Eberson's Yeast Infusion Agar 2028 eggs, beatenup in two or three
volumes of water.
1661. Jacobi's Meat Infusion Salt Agar
Boil under a gas flame for 15
(6)
Constituents minutes.
1. Meat infusion 1500.0 cc. (7) Adjust to faint alkalinity, using
2. Peptone (siccum) 15.0 g. phenolphthalein as an indicator,
3. NaCl 7.5 g. and the end point being a faint
4. Agar-agar 22.5 g. red color.
Preparation (8) Pour into an iron kettle, add
(1) Method of preparation of meat in- 100.0 cc. distilled water; boil
fusion not given. strongly for 5 minutes and filter.
(2) Add 2, 3 and 4 to (1). (9) Add 15.0 g. agar-agar to (8).
(10) Place in a steamer for 12-15 hours. (5) Dissolve 5.0 g. NaCl and 10.0 g.
(11) Filter thru a hot funnel. dried peptone in (4).
(12) The steaming for 12-15 hours is (6) Boil 15 minutes, preferably over
sufficient for sterilization. a free flame.
(b) Schultz (1891). (7) Add 10.0 to 20.0 g. agar to (6) and
Place 3 to 4 liters of distilled boil until dissolved.
(1)
water in an enamel container and (8) Neutralize if necessary and add
add 20.0 g. finely cut up agar- glycerol if desired.
agar to it. (9) Filter.
Boil two hours in a closed vessel. (10) Sterilization not specified,

(2)
(3) Place 500.0 g. of the best quality (d) Frothingham (1895).

meat, without fat or tendons in a (1) Add one pound of finely chopped
glass container fitted with a lid. lean meat to 1000.0 cc. of water
Pour 1300.0 cc. of distilled water and allow to stand from 12 to

(4)
over the meat. 24 hours in a cool place.
Store in a cool place until the (2) Strain thru a cheese cloth or a
(5)
ne.xt day. coarse towel and squeeze in a
(6) Filter thru 4 thicknesses of meat press or by twisting the

cloth and press the remaining ends of the cloth until 1000.0 cc.
meat to obtain as much fluid as of the meat juice is obtained.
possible. Make up to 1000.0 cc. by the addi-
Pour the filtrate into a kettle, tion of water if necessary.
(7)
add 10.0 g. peptone (siccum), (Three to 5.0 g. of Liebig's meat
5.0 g. NaCl and the whites two
of extract may be substituted for
eggs, beaten up in 2 or 3 volumes the meat infusion.)
of water. (3) Dissolve 5 g. NaCl and 10.0 g.
under a gas flame for 15 dried peptone in (2).

(8) Boil
minutes. (4) Boil 15 minutes either in steam
Adjust to faint alkalinity, using or over a flame.

(9)
phenolphthalein as an indicator, (5) Dissolve 10.0 to 20.0 g. of agar in
and the end point being a faint (5) by boiling.
(6) Neutralize, if necessary and add
red color.
glycerol if desired.
(10) Pour into an iron kettle, add
distilled water, boil (7) Boil from 45 minutes to an hour.
100.0 cc.
strongly for 5 minutes and filter. (8) Filter. The
filtrate should be
Add 1 liter of (10) to (2). clear. not add the yolks of

If
(11)
there remains only 2 eggs, mix rapidly and boil for a
(12) Boil until
1 liter of medium. quarter to a half hour. Filter.
(9) Sterilization not specified,
(13) Filter thru a hot funnel.
(e) Ravenel (1898-1900).
(1) Chop 500.0 g. meat and mix with
steam on 3 successive days for
500.0 cc. water.
one hour.
(2) Place in cool place over night
<c) Frothingham (1895). and then strain.
(1) Add one pound of finely chopped agar-agar and add to
(3) Chop
lean meat to 1000.0 cc. water, and 500.0 cc. of water.
let stand for about one hour.
(4) Put (3) in autoclave and run up
(2) Heat in the water bath to about to 2 atmospheres of pressure.
60C. for 3 hours. (5) Cool below 100 C. before opening.
(3) Boil 30 minutes. (6) Cool (5) to 75C.
(4) Filter and when cool make (7) Mix (2) and (6).
slightly alkaline (3.0 to 5.0 g. of (8) Add 1.0% dried peptone and
Liebig's meat extract may be sub- 0.5% salt.
stituted for the meat infusion). (9) Boil 3 to 5 minutes.
(10) Neutralize (indicator not speci- (6) Cool the autoclave at 100C. be-
fied). fore opening.
(11) Filter. (7) Cool to 75C.
(12) Sterilization not specified. (8) Mix (7) and (3).
(f) Jensen (1898) studied denitrification (9) Add 10.0 g. peptone and 5.0 g.
using the following medium: NaCl to (8).
(1) Prepare a meat infusion from (10) Boil for 3 to 5 minutes.
500.0 g. of meat and 1000.0 cc. (11) Neutralize (indicator not speci-
of water. fied).
(2) Dissolve 5.0 g. NaCl, 10.0 g. (12) Filter.

peptone and 15.0 g. agar in (1). (13) Sterilization not specified.
(3) Adjust to a slight alkalinity with (i) Thalmann (1900) used the following
soda. medium for the isolation of gono-
(4) Method of sterilization not given, cocci:
(g) Committee A. P. H. A. (1899). (1) Cut lean beef into small pieces in
(1) Macerate one part finely chopped , a meat cutting machine.

lean meat with 2 parts distilled (2) Add a double weight of distilled
water in the ice box for 18 to water to (1).
24 hours, stirring occasionally. (3) Boil for 15 minutes stirring con-
(2) Strain cold thru a fine cloth. tinually with a glass rod.
(3) Add 1.0% peptone and 0.5% NaCl (4) Make up the loss of water and
to the filtrate. Heat until solu- filter thru a filtering cloth.
tion is complete. (5) Add 1.0% peptone (siccum) and

(4) Add NaOH until the reaction is 0.5% NaCl.
slightly alkaline (practically neu- (6) Boil.
tral) to phenolphthalein. (7) Make up the volume of water

(5) Heat on a water bath for 30 min- lost.
utes and boil for 5 minutes over a (8) Cool (in a closed container) and
free flame. filter.
(6) Filter while hot thru paper or cot- (9) Distribute in 300 to 500.0 cc. por-
ton and cloth, and add 1.0 to tions in clean flasks with pat-
2.0% agar to filtrate. Dissolve ented sealers.
by boiling or autoclaving. (10) Sterilize in streaming steam for
(7) Add N/1 HCl to the filtrate to ob- one hour.
tain the desired reaction (+1.5). (11) Add 1.5% agar to (10).
(8) U the medium is clear distribute (12) Bring to a boil on a concentrated
in tubes or flasks. If not clear, salt solution bath and boil for
clarify by adding the white of

45 minutes shaking often.
one egg to the agar cooled to 50 (13) Take 30.0 cc. of (12) add a drop
of alcoholic phenolphthalein solu-
to 60C., and then boil vigor-
ously. Filter. tion and add N/1 sodium solution
until a red coloration is formed.
(9) Sterilize by the fractional
either
(14) Estimate the amount (12) and
or continuous method.
calculating from (13) add f the
(h) Ravenel (1899).
amount of sodium solution re-
(1) Mi.x 500.0 g. of freshly chopped
quired to give neutralization.
meat with 500.0 cc. of water.
(15) Keep in the autoclave or hot
(2) Allow to stand in a cool place
water for 15 minutes.
over night.
(16) Filter.
(3) Strain thru a towel. Tube.
(17)
(4) Chop agar into small pieces and (18) Sterilize (exact method not spec-
add to 500.0 cc. water. ified).
(5) Put in an autoclave and run the (j) Walbaum (1901).

pressure up to two atmospheres (1)Method of preparation of meat
(135.1). Turn out the flame. infusion not given.
(2) Add 10.0 g. of finely cut agar to (10) Cool to 90C. and allow to remain
1000.0 cc. of (1). at that temperature for several
<3) Allow to stand until the agar is hours.
completely soaked up. The time (11) Decant the clear agar and filter
required depends on the type of thru a folded filter paper. Filter

agar used. at 90C. The sediment may be
(4) Boil for about 45 minutes with filtered thru another filter paper.
constant stirring. (12) 1.0 to 2.0% glucose may be added.
(5) Prepare a solution of 5.0 g. NaCl (13) Distribute in tubes or flasks.
and 10.0 g. peptone by heating (14) Boil for one hour to sterilize.
at about 70C. (material in which (1) Thoinot and Masselin (1902).
the peptone and NaCl is to be (1) Macerate 500.0 g. of lean beef
dissolved not specified, whether with 1 liter of water for several
it be water or of some of the hours.
infusion). (2) Pass thru a linen cloth and ex-
(6) Add distilled water to (4) to make press the juice from the meat.
up the loss due to evaporation. (3) Add 10.0 g. peptone and 5.0 g.
(7) Add (5) to (6). NaCl.
(8) Neutralize and boil again for a (4) Boil and filter.
short time. (5) Make slightly alkaline by the

(9) Filter thru a single folded filter addition of soda.
paper, placing the funnel and agar (6) Add 15.0 g. of finely chopped agar
in an autoclave (requires 20-40 and heat until dissolved. Stir
minutes depending on the kind of constantly.
agar). (7) Pass thru a sieve.
(10) Sterilization not specified, (8) Cool to 55 C. and add the white
(k) Migula (1901). of an egg beaten up in 500.0 cc.
(1) Mix 500.0 g. of finely chopped of water. Mix well.
lean beef with one liter of water, (9) Heat to 120C. for 45 minutes.
and allow to stand in the ice box (10) Filter thru paper (hot filtration).
for 12 to 24 hours. (11) Distribute in tubes.
(2) Press the liquid thru a towel and (12) Heat at 115C. for 20 minutes,
make up the volume to one liter. (m) Frost (1903).
(3) Boil in the steam cooker for (1) Add 15.0 g. of agar to 500.0 cc. of
30 minutes. water and dissolve by heating to
(4) The infusion may be boiled for a 120C. in the autoclave, closing
hour before removing the meat off the gas and allowing to cool
and then filtered thru paper. or boil until the agar is dissolved
If the liquid is still red, boil (about 30 minutes) and make up
again for 15 minutes. the loss of water due to evap-
(5) Filter when cold to remove fat. oration.
(6) Dissolve 15 g. agar, 0.5% NaCl (2) Free 500.0 g. of lean beef from
and 1.0% Witte's peptone in (5) fat and connective tissue and
by boiling in a paraffin oven or an mince.
autoclave. (3) Add 500.0 g. of water to (2) and
(7) Neutralize carefully by the addi- set in the ice chest for 12 to
tion of concentrated Na2C03 solu- 24 hours.
tion until litmus paper is colored (4) Squeeze thru a cloth and add dis-
violet. tilled water to the filtrate to make
(8) Add soda solution as desired. 500.0 cc.

Generally 10.0 cc. of a 15.0% soda (5) Add 10.0 g. of peptone and 5.0 g.
solution is added per liter. NaCl.
(9) Place the agar in a tall narrow (6) Heat until solution is complete.
flask and boil in the steamer for (7) Neutralize to phenolphthalein.
one hour. (8) Cool to 60C.
(9) Add (1) to (8). (o) Smith (1905).

(10) Boil until albumin is coagulated (1) Add 1000.0 cc. distilled water to
and floats in the clear liquid. minced lean beef.
500.0 g. of finely
Restore to weight. (2) Infuse for 24 hours in the ice
(11) Readjust the reaction if neces- chest or heat for one hour in the
sary. water bath at 55C.
(12) Add 0.5% normal HCl.
of (3) Boil for 60 minutes in the steamer
(13) Filter thru cotton supported on or in a covered dish.
a coil of wire using a suction (4) Filter thru a clean cloth in a
pump to hasten filtration. meat press.
(14) Tube. (5) Cool and remove the fat by filter-
(15) Sterilize in steam for 15 minutes ing thru S and S filter paper.
on 3 successive days or in the (6) Make up the volume to 1000.0 cc.
autoclave for 20 minutes at by the addition of water.
120C. Slant some of the tubes (7) Add 1.0% Witte's peptone and
after final sterilization. 0.5% NaCl.
(n) Frost (1923). (8) Steam for one-half hour.
(1) Add agar to 500.0 cc. of
15.0 g. (9) Filter.
water and dissolve by heating to (10) Cool.
120C. in the autoclave, closing (11) Adjust the reaction.
off the gas, and allowing to cool (12) Steam again for 30 minutes.
or boil until the agar is dissolved (13) Filter.
(about 30 minutes) and make up (14) Add 10.0 g. powdered agar to the
the loss of water due to evap- filtrate.
oration. (15) Steam for 30 minutes and cool
(2) Free 500.0 g. of lean beef from to 58C.
fat and connective tissue and (16) Add the whites of 2 eggs (beaten
mince. thoroly and neutralized to litmus
(3) Add 500.0 cc. of distilled water. by dilute HCl).
(4) Place in a vessel for cooking and (17) Steam one hour and filter while
then cook for 30 minutes at 70 C. hot thru S & S filter paper which
(5) Filter thru paper and make up the has been thoroly warmed with
volume to 500.0 cc. boiling distilled water. The agar
(6) Add 10.0 g. of peptone and 5.0 g. may be filtered thru absorbent
of NaCl. cotton.
(7) Heat until solution is complete. (18) Tube.
(8) Neutralize to phenolphthalein. (19) Sterilize by heating in the auto-
(9) Cool to 60C. clave at 110C. for 10 minutes or
(10) Add (1) to (9). by short steamings in the steamer
(11) Boil until albumin is coagulated on each of 3 successive days.
and floats in the clear fluid. (p) Guillemard (1906) cultivated anae-
Restore the weight. robes on the following medium:
(12) Readjust the reaction if neces- (1) Mix 500.0 g. of chopped meat,,
sary. 10.0 g. peptone and 5.0 g. NaCl
(13) Add 0.5% of normal HCl. in 1000.0 cc. water.
(14) Filter thru cotton supported on a (2) Bring to a boil and boil 10 min-
coil of wire using a suction pump utes.
to hasten filtration. (3) Filter while hot.
(15) Tube. (4) Allow to cool for 12 hours.
(16) Sterilize in steam for 15 minutes (5) Filter while cold.
on 3 successive days or in the (6) The solution is clear and of an
autoclave for 20 minutes at 120C. acid reaction.
Slant some of the tubes after (7) Make up to a liter if necessary.
sterilization. (8) Add a piece of red litmus paper.
(9) Then add a 36B. soda solution (8) After the last washing allow the
drop by drop until the red litmus agar to drain for 10 minutes.
is turned blue. (9) Add 500.0 cc. of (4), 10.0 g. pep-
(10) Add 10.0 g. agar and dissolve by tone and 5.0 g. NaCl to the
heating. washed agar.
(11) Heat at 115 to 120 in the auto- (10) Autoclave at 115C. for 30 min-
clave. utes or in the steamer for 90 min-
(12) A precipitate forms which com- utes.
pletely clears the medium. (11) Make (10) up to a liter by the
(13) Filter and distribute in tubes. addition of (4).
(14) Sterilize at a temperature lower (12) Make slightly alkaline to litmus
than in (11). by the addition of KOH.
(q) Walbaum (1910). (13) Cool to 60C. and add the beaten
(1)Method of preparation of meat white of an egg.
infusion not given. (14) Heat in the autoclave for 45 min-
(2) Add 100.0 g. of finely cut agar to utes or in the steamer, for
1000.0 cc. of (1). 90 minutes.
(3) Place in the autoclave for 30 min- (15) Filter thru moistened filter paper,
utes at 0.5 atmosphere extra using a hot water funnel.
pressure. (16) Tube.
(4) Prepare a solution of 10.0 g. pep- (17) Steam for 30 minutes on each of
tone and 5.0 g. NaCl by heating 2 successive days.
at about 70C. (Material in (s) Abel (1912).
which the peptone and NaCl is (1) Chop 500.0 g. of fat free meat and
to be dissolved not specified, add to a water at 50C.
liter of
whether it be some of the infu- (2) Keep at 50C. for 30 minutes and
sion or water). then boil for 30 to 45 minutes.
(5) Place the agar, after autoclaving (3) Filter or strain the fluid from the
is completed, on a free flame, and meat.
add the distilled water to make (4) Make up the fluid to 1 liter.
up the loss in weight due to (5) Soak 1.5 or 2.0% agar in (4) for
evaporation. several hours.
(6) Add (4) to (5). (6) Add 1.0% Witte or Chapoteaut's
(7) Neutralize and boil again for a peptone and 0.5% NaCl.
short time. (7) Heat in the steamer until solution
(8) Filter thru a single or double is complete.
thickness of filter paper. (Re- Neutralize to litmus if necessary.
(8)
quires about 10 minutes.)
(9) Heat in the steamer for 15 to
30 minutes.
(r) Abel (1912).
(10) Filter thru cotton-wool to clarify.
(1) Chop 500.0 g. of fat free meat and
The agar may be allowed to
add to a liter water at 50C.
of
solidify in the steamer in straight
(2) Keep at 50C. for 30 minutes and
walled vessels and cut away the
then boil for 30 to 45 minutes.
bottom opaque layer.
heat. (11) Sterilize on each of 3 successive
Make up the fluid to 1 liter.
days in the steamer or autoclave
(4)
Place 20 agar in a 3 liter flask at 120 C. for 15 minutes.
(5) g.
and add 500.0 cc. of tap water and (t) Wilcox (1916) produced toxin by
2.5 cc. of glacial acetic acid. growing B. tetani on the follow-
(6) Allow (5) to soak 15 minutes and ing medium:
drain carefully. (1) Dissolve 5.0 g. agar, 10.0 g.
(7) Wash the agar thoroly with 4 lots Witte's peptone and 5.0 g. NaCl
of water. in 1000.0 cc. veal infusion.
(2) Adjust reaction so that it is (16) Bring the volume up to 1000.0 cc.
neutral to phenolphthalein. by the addition of hot water.
(3) Tube in 8 to 10.0 cc. quantities. (17) Estimate and adjust the reaction
(4) Autoclave at 15 pounds for J hour. to a definite pH value, or to
(5) To transfer cultures, a semi-solid faintly alkaline to litmus or
agar culture is melted and 1.0 cc. 1.0% acid to phenolphthalein.
portions transferred to fresh (18) Steam 30 minutes.
semi-solid melted agar tube. (19) Filter, while thru well-
hot,
(u) Meier (1918). wetted, thick paper or two
filter
(1) Boil 500.0 g. of fat and tendon paper or two layers of absorbent
free beef in 1 liter of water. cotton wool, by placing filter
(2) Filter. funnel, stand, and receptacle for
(3) Dissolve 15.0 g. agar, 10.0 g. filtrate in the steam sterilizer
Witte's peptone and 5.0 g. NaCl and steaming till filtration is
in the filtrate. complete.

(4) Neutralize by the addition of Note: If filtration thru paper
KOH. Add KOH until turmeric isnot sufficient to give a clear
paper is turned quite weakly medium, clarify with white of
brownish red. egg or other clearing agent.
(5) Sterilization not specified, (a) Beat up the white of one or two
(v) Harvey (1921-22). eggs along with the crushed
(1)Mince finely fat-free beef. shells in about 20.0 cc. water.
(2) Add 500.0 to 1000.0 cc. distilled Note: Raw meat juice, 15.0 cc.
water or clear tap water. per liter of medium may be sub-
(3) Heat the mixture 20 minutes stituted for white of egg.
over a free flame at a temperature (b) Add to the medium little by
not exceeding 50C. littlebefore filtration and at a
(4) Skim off fat floating on the sur- temperature not exceeding 60 C.
face. (c) Stir to mix.
(5) Raise the temperature to boiling (d) Steam 30 minutes.
point. (e) Remove from steamer and shake
(6) Boil 10 minutes. up well to mix.
(7) Pour the mixture onto a wet, (f) Steam again 15 minutes.
thick, clean cloth. (g) Filter in the steamer thru thick
(8) Collect the fluid which drains filter paper, or thru two layers
thru the cloth together with that of absorbent cotton,
obtained by squeezing the meat (h) Refilter, if necessary, the first
in the cloth. portion of the filtrate.
(9) Add 10 g. peptone 5.0g XaCl. (20) Distribute into flasks on test
(10) Mix fiber agar 1 part, glacial tubes,
acetic acid 0.05 parts, and water (w) Harvey (1921-22).
2 parts. (1)Mince finely fat-free beef.
(11) Allow the agar to soak in the (2) Add 500.0 g. to 1000.0 cc. dis-
acidified water 15 minutes. tilled water or clear tap water.
(12) Remove the fiber agar and wash
(3) Heat the mi.xture 20 minutes over
thoroly with water imtil quite
a free flame at a temperature not
free from any trace of acid reac-
exceeding 50C.
tion to litmus paper.
(4) Skim off fat floating on the sur-
(13) Squeeze in a cloth to get rid of
face.
excess of water.
(14) Add (13) to (9).
point.
(15) Steam gently 2^ hours to bring
the agar thoroly into solution. (6) Boil 10 minutes.
Note: Or heat 45 minutes at (7) Pour the mixture on to a wet,

118C. thick, clean cloth.
(8) Collect the fluid which drains (4) Raise the temperature to boiling
thru the cloth together with that point.
obtained by squeezing the meat (5) Boil 10 minutes.
in the cloth. (6) Pour the mixture on to a wet,
(9) Filter the fluid collected thru thick, clean cloth.
well-wetted, thick filter paper. (7) Add 2.5 g. XaCl to the filtrate.
(10) Add to the filtrate 10.0 g. pep- (8) Bring the volume up to 1000.0 cc.
tone, 5.0 g. sodium chloride. by the addition of water.
(11) Steam or boil 45 minutes. (9) Estimate and adjust the reaction.
(12) Bring the volume up to 1000.0 cc. (10) Steam 30 minutes.
by the addition of water. (11) Filter, while hot, thru well-
(13) Cut up 20.0 g. fiber agar into wetted, thick filter paper.
small pieces. (12) g. fiber agar in 500.0 cc.
Place 15.0
(14) Add to 1000.0 cc. hot bouillon tap water.
(12). (13) Wash the agar well by squeezing
(15) Bring the agar into solution with it thru the hands.
heat after having been allowed to (14) Decant and reject the dirty
soak 2 hours in the hot bouillon. water.
(16) Estimate and adjust the reaction (15) Replace with the same amount of
to a definite pH value or faintly clean tap water.
alkaline to litmus or 1.0% acid (16) Heat over a free flame with con-
to phenolphthalein. stant stirring to dissolve the agar.
(17) Steam 30 minutes. (17) Add peptone 5.0 g., sodium
(18) Clarify and filter. chloride 2.5 g.
(a) Beat up the white of one or two (18) Add to 500.0 cc. of meat extract
eggs along with the crushed (11), 10.0 g. egg albumin which
shells in about 20.0 cc. water. has been made into a paste or
Note: Raw meat juice, suspension with a little of the
15.0 cc. per liter of medium, meat infusion.
may be substituted for white of (19) Add the dissolved agar slowly to
egg. the meat extract thus prepared,
(b) Add to the medium little by with constant stirring.
before filtration and at a
little (20) Steam 60 minutes or heat 45 min-
temperature not exceeding 60C. utes at 120C.
(c) Stir to mi.x. (21) Bring the volume to 1000.0 cc.
(d) Steam 30 minutes. by the addition of water.
(e) Remove from steamer and shake (22) Estimate and adjust the reaction.
well to mix.
(23) Boil 15 minutes over a free flame.
(f) Steam again 15 minutes.
(24) Make up for any loss of volume
(g) Filter in the steamer thru thick
by addition of water.
filter paper, or thru two layers
(25) Filter, while hot, thru well-
of absorbent cotton wool.
necessary, the
wetted, thick filter paper by
(h) Refilter, if first
placing filter funnel, stand, and
portion of the filtrate.
receptacle for filtrate in the
steamer and steaming till filtra-
(20) Sterilize in the steamer or auto-
tionis completed.
clave.
Harvey (1921-22). (26) Distribute filtrate into flasks or
(x)
test tubes.
(1)Mince 250.0 g. fat-free beef with
500.0 cc. of water. (27) Sterilize in the steamer or auto-
(2) Heat the mixture 20 minutes over clave.
a free flame, at a temperature not (y) Harvey (1921-22).

exceeding 50C. (1) Soak 10.0 g. purified agar (see me-
(3) Skim off fat floating on the sur- dium 1401 for preparation) in
face. 500.0 cc. of Harvey's Infusion
Broth (see variant (bb) medium Add 20.0 g. peptone and 5.0 g.
(6)
779) for 2 hours. NaCl to 1000.0 cc. of the filtrate
(2) Bring the agar into solution by and dissolve by shaking.
heating.
(7) Filter thru a wetted filter paper.
(8) Make slightly alkaline to litmus
(z) Harvey (1921-22). by the addition of NaOH or
(1) Add 500.0 cc. of water to 500.0 cc. NaaCOs.
of the variant given by Harvey (9) Soak 20.0 g. of agar in (8) for
above, several hours.
(aa) Abbott (1921). (10) Boil until solution is complete,
(1) Add 500.0 g. chopped lean beef stirring constantly.
to 1 water and soak for
liter of
(11) Test, and readjust the reaction if
24 hours, kept at ice box tem- necessary.
perature. Cool to 60C.
(12)
(2) Strain thru a coarse towel and (13) Add the white of one egg beaten
press until a liter of fluid is up in 50 to 100.0 cc. of water.
obtained. Add
(14) (13) to (12).
(3) Dissolve 1.0% peptone, and 0.5% (15) Heat in the autoclave at 118"
NaCI in (2). for 15 minutes.
(4) Dissolve 1.0 to 1.5% agar in (3) by (16) Filter while hot thru paper.
boiling in a porcelain lined iron Tube.
(17)
vessel. Add 250.0 to 300.0 cc. (18) Sterilize at 115 for 20 minutes,
of water and boil until this vol- (cc) Pitfield (1922).
ume water has evaporated,
of
Cover 500.0
(1) g. of finely cut fat
leaving one liter volume.
free beef with 1000.0 cc. of water.
(5) Place the vessel in a large dish
(2) Shake well and place on ice over
of cold water.
night.
(6) Stir the agar constantly until
(3) Squeeze out the fluid by means
cooled to 68 to 70C.
of a cloth and make up the volume
(7) Add the white of one egg which to 1 liter.
has been beaten up in about
(4) Inoculate with a culture of the
50.0 cc. of water (a 10.0% dry colon bacillus.
albumin solution may be used).
(5) Allow to stand at room tempera-
(8) Mix (7) thoroly with (6).
ture over night.
(9) Allow to boil slowly for 30 min-
(6) Boil and add 10.0 g. Witte's pep-
utes. Do not allow the volume of tone and 5.0 g. NaCl.
the liquid to fall below the liter
(7) Weigh the saucepan and contents
mark.
and heat to 60C.
(10) Filter thru a heavy folded filter
(8) Make up the loss in weight by
paper.
(11) Sterilize by steam (method not
(9) Neutralize to litmus,
given).
(bb) Dopter and Sacquepee (dd) Klimmer (1923).
(1921).
(1) Add 1000.0 cc. of water to 500.0 g. (1) Preparation of meat water not
of finely chopped fat and tendon given.
free beef. (2) Soak the agar in water from 1 to

(2) Allow to stand in ice box for 12 hours.
12 hours, or heat at 50 to 55 for (3) Pour off the water and press
30 minutes. the agar free from liquid.
(3) Heat slowly to boiling. (4) Add (3) to (1) and boil until solu-
(4) Boil slowly for 10 minutes, stir- tion is complete (steamer or free
ring constantly. flame).
(5) Press the liquid thru a clean (5) ]\Iake up the loss in weight by the
cloth. addition of water.
(6) Add 10.0 g. peptone and 5.0 g. (gg) Park, Williams and Krumwiede
NaCl to (5). (1924).
(7) Neutralize. (1) Prepare double strength infusion
(8) Boil. broth, using double the amount of
(9) Readjust the reaction if neces- beef or veal. (See infusion broth
sary. Alkaline agar may be pre- medium 779, variant (kk) or (11)
pared by adding 7.0 cc. of normal for the method.)

soda solution to a liter of agar (2) Add double the amount of pep-
neutral to litmus. tone, (20.0 g. per liter) and NaCl
(10) Filter. (10.0 g. per liter) to (1).
(12) Distribute as desired. (3) Adjust the reaction.
(13) Sterilize by
steaming for 40 (4) To an equal quantity of water
minutes, on
one day, or for (1000.0 cc.) add a double quantity
15 minutes on each of 3 succes- (30.0 g.) of agar and dissolve.
sive days. (5) Cool the agar below 50C.
(ee) Park, Williams and Krumwiede (6) Mix equal parts of (3) (double
(1924). strength infusion broth) and (5)
(1) Dissolve 10.0 g. peptone and 5.0 g. (3.0% agar).
NaCl in 1000.0 cc. infusion broth
(7) Test the reaction and adjust if
(see variant (11) medium 779) by necessary.
heating to 50C. Remainder of preparation as
(2) Add 15.0 g. agar to (1) and dis- from step (5) variant (ee) above.
solve by heating in the autoclave References: Jacobi (1888 Schultz
p. 536),
at 10 to 15 pounds pressure for Frothingham
(1891 pp. 57, 60), (1895
30 minutes, or by boiling over the
p. 55), Ravenel (1898-1900 p. 89), Jensen
free flame.
(1898 p. 406), Committee A. P. H. A. (1899
(3) If boiled over a free flame, make p. 77), Ravenel (1899 p. 606), Thalmann
up the loss in weight by the addi- (1900 p. 829), Walbaum (1901 p. 796),
tion of water.
Migula (1901 p. 17), Thoinot and Mas-
(4) Adjust the reaction. selin (1902 Frost (1903 p.
p. 33), 16),
(5) Cool to 50C. and add one egg. Smith (1905 Guillemard (1906
p. 195),
(6) Heat in the autoclave at 10 to p. 157), Walbaum (1910 p. 796), Abel (1912
15 pounds for 30 minutes or Ar-
pp. 17, 18), Wilcox (1916 p. 333), Meier
nold sterilizer for 1 hour. P ilter. Harvey (1921-22 pp.
(1918 p. 435), 66, 70,
(7) Test the reaction and adjust if
71), Abbott (1921 p. 129), Dopter and
necessary.
Sacquepee (1921 p. 125), Pitfield (1922
(8) If more than 0.2% normal soda p. 117), Klimmer (1923 p. 193), Park,
is required per liter, heat again Williams and Krumwiede (1924 117).
p.
for 10 minutes.
(9) Filter thru cotton. 1662. Zettnow's Meat Infusion Agar
(11) Sterilize at 15 pounds pressure Constituents
for 30 minutes. 1. Water 5000.0 cc.
(ff) Park, Williams and Krumwiede 2. Meat (horse) 3000.0 g.
(1924). 3. Agar 11-5 g.
(1) Dissolve 10.0 peptone, 5.0 g.

g. 4. (NH4)2S04 1.0 g.
NaCl and 15.0 g. agar in infusion 5. Peptone 1.0 g.
broth. (See variant (11) me- 6. KNO3 1.0 g.
dium 779) by heating in the auto- Preparation
clave at 10 to 15 pounds pressure (1) Heat 3000.0 g. of horse (or other
for 30 minutes or by boiling over cheap fat-free meat) meat with
the free flame. 5000.0 cc. of tap water, or distilled
Remainder of preparation the water, to 60-65C. for 1 to 11 hours,
same as variant (ee) above. stirring occasionally.
(2) Heat then to a boil stirring con- if one wishes to remove the sugar,
stantly. 12 hours at 37C.
(3) Filter thru a filtering cloth, wash- (3) Place in an enamelled pot and bring
ing the residue with one liter of slowly to a boil.
water and squeeze out the residue. (4) Boil for 10 minutes.
(4) Distribute into glass flasks and (5) Throw on a thick cloth and press
sterilize twice before use (This the meat free from juice.
constitutes a stock meat infusion.) (6) Filter the juice thru moistened
(5) For the preparation of one liter of paper.
agar medium add 11.5 g. fibre agar (7) Add 10.0 g. of Chapoteaut's or
to 500.0 g. water. Allow the agar Defresne peptone, 5.0 g. XaCl and
to grow and swell in the water. about 1.0 g. of sodium phosphate.
(6) Add 8.0 g. ovi albumin (siccum) to (8) Boil stirring constantly until solu-
about 40.0 cc. of water. It dissolves tion is complete,
slowly. (9) Neutralize or make slightly alkaline
(7) Place 1000.0 g. of (4) (meat infusion) to litmus by the addition of soda
in a weighed kettle. solution.
(8) Add 250-300.0 cc. water to (7) and (10) Soak 20.0 g. of chopped thread agar
1.0 g. peptone. in cold water for several hours.
(9) Heat nearly to boiling and neutralize (11) Heat (10) and (9) at 100C. until
with NaOH until litmus paper be- the agar is dissolved.
comes strongly blue. (12) Readjust the reaction if necessary.
(10) Add (5) (the soaked agar) to (9) and (13) Allow to cool to 55 or 60C.
boil over a free flame to dissolve the (14) Beat the white of an egg in 100.0 cc.
agar. Do not over heat. of water and add to (13).
(11) Add 1.0 g. (NH4)2S04 and 1.0 g. (15) Mix well.
KNO3 to (10). (16) Autoclave at 120C, for one hour.
(12) Weigh and add distilled water until (17) Filter thru a moistened Chardin
the contents of the kettle weigh filter using a hot water funnel.
1000.0 g. (18) Tube.
(13) Cool to 50-55C. Sterilization:Sterilize at 115 for 20
(14) Add (6) (albumin solution) or the minutes.
whites of two eggs and place in a Use: General culture medium.
steamer for 50-60 minutes. Variants
(15) Filter thru a hot water funnel. (a) The agar may be treated with a 6 to
(May use a folded felty, so-called 100 solution ofHCl (500.0 cc. water
coffee filter paper.) to 30.0 cc.HCl for 24 hours and then
Sterilization: Method not given. with a 5.0% ammonia solution. Then
Use Cultivation of Spirillum undula majus
: thoroly wash the agar with water.
and other spirilla. (b) Besson also used 20.0 g. peptone in-
Reference: Zettnow (1895 p. 394). stead of 10.0 g.
1663. Besson's Phosphate Infusion Agar
1664. Smith's Infusion Agar
Constituents:
2. Beef 500.0 g. 1. Distilled water 2000.0 cc.
3. Peptone 10.0 g. 2. Beef 1000.0 g.
4. Agar 20.0 g. 3. Peptone (Merck, brown) . . 20.0 g.
5. Sodium phosphate 1.0 g. 4. Agar 20.0 g.
Preparation (1) Soak finely chopped fat free beefwith
(1) Remove all fat and tendons from 2000.0 cc. distilled water for some
beef and chop into small pieces. hours and then slowly raise to 65C.
(2) Allow 500.0 g. of (1) to macerate with on the water bath.
1000. cc. cold water for 6 hours, or (2) Finally steam and filter.
(3) Add 20.0 g. Merck's brown peptone (14) Boil 2 to 5 minutes over a free
and 20.0 g. agar. flame, stirring constantly.
(4) Steam (time not specified). (15) ]\Iake up
loss due to evaporation.
(5) Cool and add the whites of 10 eggs to (16) Filter thru absorbent cotton and
clarify. cotton flannel, passing the filtrate
(6) Resteam and filter. thru the filter until clear.
(7) Neutralize the egg albumin with HCI (17) Titrate and record the final
and then render the medium alkaline reaction.
with XaOH. (18) Tube in 7.0 cc. quantities.
Sterilization: Not specified. (19) Sterilize 15 minutes in the auto-
Use: General culture medium. Smith cul- clave at 110 or for 30 minutes in
tivated Pseudomonas campestris (Pam- streaming steam on 3 successive
mel) on the medium and reported that the days.
colonies developed moderately fast. (20) Store in the ice chest in a moist
They were circular or nearly so, thin and atmosphere, to prevent evapo-
well defined margin pale yellow wet and ration.
shining. Streaked cultures tended to (b) Committee A. P. H. A. (1905) recom-

spread. In plates or streaks crystals of mended the same method of prepara-
ammonium magnesium phosphate formed tion as in but sterilized the
1901,
after several days. medium by heating for 5 minutes at
(a) Committee A. P. H. A. (1901 recom- 120C. or for 30 minutes in stream-

ing steam on 3 successive days.
mended the following medium to be
used for bacterial counts in water (c) Committee A. P. H. A. (1905) pre-
analysis.
pared a basal medium as given under
Boil 15.0 g. thread agar in 500.0 cc. variant (b) above and added 10.0 g.
(1)
of glucose or lactose just before
water for half an hour and make
up weight to 500.0 g. or digest for
sterilization. The reaction is then
10 minutes in the autoclave at adjusted to neutral to phenolphthal-
ein. If medium is to be used in tubes
110C. Let this cool to about
60C. add sterilized azolitmin solution
Infuse 500.0 g. lean meat 24 hours (amount not specified) just before
(2)
with 500.0 cc. of distilled water in final sterilization. If medium is to
refrigerator. be used in Petri dishes, add sterilized

Make up any loss by evaporation.
azolitmin solution (amount not speci-
(3)
fied) just before the medium is to be
(4) Strain infusion thru cotton flannel.
Weigh filtered infusion.
poured into the plate.
(5)
(d) Committee A P. H. A. (1909) recom-
(6) Add 2.0% Witte's peptone.
(7) Warm on water bath, stirring till mended that the medium be adjusted
peptone is dissolved and not allow- to 4-1 5 instead of +1.0 as in variant
ing the temperature to rise above (a). Sterilize in the autoclave for
60C. one hour at 15 pounds pressure or in

streaming steam for 20 minutes on
(8) Neutralize.
3 successive days. To prepare lac-
(9) To 500 .0 g . of the meat infusion add
tose or glucose litmus agar, add 1.0%
500.0 cc. of the 3.0% agar, keeping
lactose or 1.0% glucose to the medium
the temperature below 60C.
just before sterilization. The reac-
(10) Heat over boiling water, (or
tion is adjusted to neutral to phenol-
steam) bath for 30 minutes.
phthalein. Add the sterilized azo-
(11) Restore loss by evaporation. litmin to the medium just before
(12) Titrate, after boiling one minute sterilization if the agar is to be used
to expel carbonic acid. in tubes. If to be used in plates,
(13) Adjust reaction to -|-1.0% by add- add the sterilized azolitmin solution
ing normal hydrochloric acid or to the Petri dishes when pouring
sodium hydrate as required. plates.
(e) Meier prepared a similar medium as (1) Remove spleen from a healthy rab-
follows: bit and cut into pieces 3 mm. in
(1) Boil 500.0 g. of fat and tendon free diameter under aseptic conditions.
beef in 1 liter of water. (2) Rub one piece of (1) on each agar
(2) Filter. slant of (a), (b) or (c) and allow
(3) Dissolve 15.0 g. agar, 10.0 g. pep- the spleen to remain on the surface
tone and 5.0, 20.0 or 40.0 g. of lac- of each slant just above the water
tose or glucose in the filtrate. of condensation.
(4) Neutralize by the addition of (e) Cystine agar. Add 0.02% cystine to
KOH. Add KOH until turmeric solid sterile 1.0% stock agar and
paper is turned quite weakly steam to melt agar and sterilize the
brownish red. cystine.
(5) Sterilization not specified. (f) Spleen agar. Rub pieces of spleen
References: Smith (1897 p. 480), Com- over 1.0% stock agar slants in the
mittee A. P. H. A. (1901 p. 384), (1905 manner as indicated under (d) (1)
p. 108), (1905 Sup. #1), (1909 p. 285), and (2) above.
Meier (1918 p. 436). (g) Serum glucose cystine agar.
(1) Add 0.1% cystine and 1.0% glucose
1665. Francis' Basal Infusion Agar
to solid sterile 1.0% stock agar.
Constituents: (2) Steam to melt the agar and to steri-
1. Infusion agar (1.5%) 1000.0 cc. lize the cystine and glucose.
2. Peptone (1.0%) 10.0 g. (3) Cool to 5bC.

Preparation (4) Add 5.0% horse serum.
(1) Prepare a stock beef infusion agar (h) Serum glucose cysteine hydrochloride
using fresh beef, 1.0% agar and 1.0% agar. Substitute cysteine hydro-
peptone. chloride for cystine in medium (g)
(2) Adjust (1) to pH-7.6. above.

(3) Add one of the added nutrients in the (i) Cystine hydrochloride agar. Add
manner indicated below. 0.1% cystine hydrochloride and 1.0%
(4) Incubate 24 to 48 hours to test glucose to sterile solid 1.0% stock
sterility. agar and heat to melt the agar and
Sterilization: Method not given. sterilize the cysteine hydrochloride
Use: Cultivation of Bacterium tularense. and glucose.

Variants Reference: Francis (1922 p. 102, 987, 988)
(a) The author used 1.0% agar in prepa- (1923 p. 1398).

ration of media as indicated under
1666. Deycke's Alkali Albuminate Agar
added nutrients.
(b) Francis adjusted the reaction of the Constituents:
stock agar to pH-7.3 when preparing 1. Water 1200.0 cc.
serum glucose cystine agar, serum 2. KOH (3.0%) 36.0 g.
glucose cysteine hydrochloride agar, 3. Veal 1000.0 g.
and cysteine hydrochloride agar. 4. NaCl
Added nutrients: Francis prepared the 5. Peptone
media listed below as indicated. "
6. Agar
(a) Serum glucose agar. Add 1.0% glu- Preparation
cose from a sterile 50.0% glucose solu- (1) Digest 1000.0 g. of finely ground fat
tion and 5.0% serum to the melted free veal in 1200.0 cc. of 3.0% KOH
agar cooled to 45C. solution for 2 days at 37C.
(b) Glucose blood agar. Substitute 5.0% (2) Heat (1) for one hour on a water bath
defibrinated blood for serum in (a). at 60 to 70C. until all the protein
(c) Blood agar. Same as (b) but add no has dissolved.
glucose. (3) Add HCl, carefully to precipitate
(d) Mediums (a), (b) and (c) plus spleen the albuminates.
tissue. (4) Collect on a filter.
(5) Suspend the precipitate in distilled 1668. Krasnow's et al. Meat Infusion Agar
water and add concentrated soda Constituents
solution until part of the precipitate
is brought into solution. Dissolve
Veal, Bacto
2. 75.0 g.
the remainder of the precipitate by
3. Peptone 10.0 g.
prolonged heating in steam. NaCl 5.0 g.
4.
(6) Correct the reaction to a weak
5. Agar 20.0 g.
alkalinity.
Preparation
(7) Dry 100.0 cc. of the solution and Bacto Veal
(1) Infuse 75.0 g. of in
determine the per cent of dry ma-
500.0 cc. of tap water in the Arnold
terial.
sterilizer at 100C. for two hours.
(8) Dilute (6) so that there is 2 to 3.0% Allow the coagulum thus formed to
(2)
solid materials present.
settle to the bottom of the container.
(9) Dissolve 1.0% peptone, 1.0% NaCl
Allow to cool very slowly.
and agar (amount not given) to (8), Strain thru a wire sieve.
(3)
method not given. Dissolve 3 and 4 in
(4) (3).
(10) Neutralize by Dahmen's method
(5) Adjust to pH 7.9.
with 0.33% soda.
(6) Steam in the Arnold for 15 minutes.
(7) Filter.
Use: Isolation of cholera bacilli from Add an equal quantity
(8) of a 4.0% agar
stools. Author reported that this me- solution.
dium eliminated practically all other
Sterilization : Sterilize in the autoclave for
organisms from stools.
Reference: Deycke (1893 p. 888).
Use: Cultivation of streptococci.
Reference: Krasnow, Rivkin and Rosen-
1667. Harvey's Basal Infusion Agar
berg (1926 p. 391).
Constituents
1. Infusion agar 1000.0 cc. 1669. Dopter's Liver Infusion Agar (White)
(1) Prepare infusion agar according to 1. Water 1000.0 cc.
variant (v) medium 1661. 2. Liver 500.0 g.
(2) Add one of the added nutrients to (1). 3. Agar (1.5%) 15.0 g.
Sterilization: Not specified. 4. Peptone (1.0%) 10.0 g.
Use Cultivation of B. acnes and anaerobes.
: (Witte, Chapoteau or Fair-
Also used as an enrichment medium for child)
the colon typhoid group. 5. NaCl (0.5%) 5.0 g.
Added nutrients: The author added one of Preparation
the following: (1) Mix 500.0 g. of well minced calf or
(a) Oleic acid 10.0 cc. beef liver with 1000.0 cc. of distilled
glycerol 20.0 cc. water.
(b) Glycerol 20.0 cc. (2) Stir well and infuse over night in the
oleic acid 1.0 cc. ice box.
(c) Glucose 20.0 g. (3) Boil 5 minutes.
sulphin digotate 10 g. (4) Filter thru paper.
(d) Glucose 20.0 g. (5) Make up to original volume.
(e) Sodium formate 4.0 g. (6) Add 1.0% Witte, Chapoteau or Fair-
Equal parts agar and 1.0% caffeine child's peptone, 0.5% NaCl and 1.5%
solution. agar.
The combinations (a) and (b) were (7) Boil 20 minutes and adjust to 4-0.2
used to cultivate B. acnes, (c) and (d) to phenolphthalein.
used for the cultivation of anaerobes. (8) Boil 5 minutes and filter.
Agar containing caffeine (e) was used Sterilization: Sterilize in the Arnold on
as enrichment medium for members three successive days.
of the colon typhoid group. Use: General culture medium. Also used
Reference: Harvey (1921-22 pp. 87, 92, 111). to cultivate and carry stock meningococci
cultures. Bronfenbrenner and Schles- (2) Cook (1) in flowing steam one
inger reported that the medium gave a hour.
large amount of water of condensation. (3) Strain thru cheese cloth and
To keep a pure culture on this medium cotton.
for several weeks, inoculate the water of (4) Add peptone (1.0%) and NaCl
condensation and tilt the tubes every day (0.5%).
or so. Other investigators cultivated a (5) Add 20.0 g. agar and heat in flow-
variety of organisms as B. welchii, B, ing steam until all is dissolved.

chauvoei, the gonococcus, etc., on sim- (6) Adjust to pH = 8.2.
ilarmedia. (7) Clarify with egg albumin.
Variants (8) Filter and tube.
(a) Bronfenbrenner and Schlesinger pre- (9) Autoclave at 15 pounds pressure
pared the mediima as follows: for 20 minutes.
(1) Prepare a liver infusion

using (d) Harvey prepared the medium as
1000.0 g. liver to 1000.0 cc. water. follows:
The extract is collected thru a (1) Prepare an infusion from 1000.0 g.
single layer of cheese cloth. Boil liver (or other organ, placenta,
and stir continually (Time not etc.)and 1000.0 cc. water in the
given). same manner as for ordinary infu-
Strain thru a single layer of cheese sion broth. (See variant (bb)
(2)
cloth and a thin layer of cotton. medium 779.)
(3) Do not adjust the reaction, and (2) Dissolve 3.0% agar, 2.0% peptone
sterilize at 10 pounds pressure for and 1.0% NaCl in water.
10 minutes. (3) Sterilize (2).
Prepare a 3.0% agar solution in (4) Mix equal parts sterile (1) and (3)
(4)
water (30.0 g. agar to 1000.0 cc. while hot under aseptic conditions.
water. (5) Tube in sterile tubes.
(5) Filter thru cotton. (e) Heller cultivated pathogenic anae-

(6) Add 2.0% (20.0 g.) peptone and robes in the following medium:
1.0% (10.0 g.)NaCl. (1) Infuse one part beef liver with
(7) Do not adjust the reaction. 4 parts distilled water in the
(8) Mix equal parts of sterile (3) and refrigerator over night.
sterile (7) while hot. (2) Bring to boil and strain thru cheese
(9) Tube in sterile tubes. cloth.
(b) Heller isolated B. Welchii, B. oede- (3) Add 15.0 g. Peptone (Difco), 5.0 g.
matiens and other soil anaerobes on salt and and dissolve
20.0 g. agar
the following medium: by heating.

(1) Grind 250.0 g. beef liver and infuse (4) Titrate and adjust to pH = 7.2.
with 4 parts water over night. (5) Cool to 60 and add white of egg
Boil and strain. and serum.
(2) Add 15.0 g. peptone, 5.0 g. NaCl (6) Autoclave for one hour.
and 2.0 g. Agar. (7) Titrate and adjust to pH = 7.2.
(3) pH 7.2 (faintly alkaline to litmus).

(8) Filter through cotton.
(4)Tube in deep tubes. Tube.
(9)
(5)Method of sterilization not given. autoclave (time not
(10) Sterilize in the
He reported that if large amount of
given).
H2O be produced, add KNO3 1.0%.
(f) Park, Williams and Krumwiede culti-
Use 3.0% agar if rapid growers out-
vated gonococci on the following
grow others.
medium:
(c) Goss etal. cultivated B. chauvoei and
Soak 5.0 pounds of finely chopped
other organisms on a medium pre- (1)
liver in 5000.0 cc. of tap water
pared as follows:
over night in an ice box or at room
(1) Grind 500.0 g. beef liver and add
1000.0 cc. water.
temperature.
(2) Weigh the kettle used in (1) and (9) Adjust to pH = 7.0.
its contents. (10) Add 1.0% dissolved egg albumin.
(3) Heat at 45C. for an hour. (11) Mix well and place in flowing
(4) Boil for 30 minutes. steam for 90 minutes.
(5) Make up the loss by weight by the (12) Decant the liquid from the clot.
addition of water. (13) Remove small clumps of albumin
(6) Strain thru cheese cloth. Squeeze by filtering thru a discarded pres-
by twisting the cloth or use the sure cooker. Place glass wool,
meat press. previously washed with dilute
(7) If used at once dissolve 1.0% pep- acid, in the barrel of the filter and
tone and NaCl in (6). a 30 mesh copper screen funnel for
(8) Dissolve 1.5% agar in (7) by heat- collecting large coagulated par-
ing in the autoclave at 10 to 15 ticles.
pounds pressure for 30 minutes or (14) Readjust to pH = 7.0 if necessary.
by boiling over the free flame. The reaction should fall to pH 6.6
(9) If boiled over a ree flame make up following sterilization.
the loss in weight by the addition (15) Sterilize at 15 pounds pressure for
of water. 30 minutes.
(10) Adjust the reaction to +0.2% to (16) For isolation work add sufficient
phenolphthalein quantity of a saturated aqueous
(11) Cool to 50C. and add one egg. solution of gentian violet so that
(12) Test the reaction and readjust if the concentration be 1:10,000.
necessary. References: White (1917 p. 49), Bronfen
(13) If more than 0.2% normal soda is brenner and Schlesinger (1918 p. 125)
required per liter, heat again for (1918-19 p. 219), Heller (1921 p. 460)
ten minutes. Goss, Barbarin and Haine (1921 p. 615)
(14) Filter thru cotton. Harvey (1921-22 p. 69), Heller (1922 p. 9)
(15) Distribute in tubes or flasks. Klimmer (1923 p. 201), Park, Williams
(16) Sterilize at 15 pounds pressure for and Krumwiede (1924 p. 131), Huddleson,
30 minutes. Hasley and Torrey (1927 p. 356).
(g) Stafseth (Huddleson, Hesley and
Torrey used the following medium 1670. Richardson's Mucosa Infusion Agar
for isolation and cultivation of Bac-
terium abortus (Bang): Constituents
(1) Grind fresh fat free beef liver in a 1. Distilled water 500.0 cc.
meat chopper to a plastic mass. 2. Mucosa of hog intestine 250.0 cc.
(2) Mix (1) with 500.0 cc. of tap water. 3. Peptone 5.0 g.
(3) Place in flowing steam for 20 4. NaCl 2.5 g.

minutes. 5. Agar 3.8 g.
(4) Remove the lid,and stir with a Preparation

glass rod in order to mix thoroly. (1) Take small intestine of a hog in
(5) Continue the heating in flowing and
fresh condition without cutting,
steam for Ij hours. wash thoroly with running water
(6) Remove and filter thru a wire until the water runs perfectly clear.
screen. (This infusion be may (2) Lay the intestine open and scrape
sterilized and stored until ready off the mucosa with a glass slide
for use, or used at once.) (150.0 g. of mucosa is easily obtained
(7) Mix 500.0 cc. of (6) with 500.0 cc. of from one hog).
tap water, 20.0 g. washed agar, (3) To 250.0 cc. of the mucosa add
10.0 g. Bacto peptone and 5.0 g. 500.0 cc. of distilled water. )
"^
NaCl, and place in a covered (4) Boil for 30 minutes. Cool.
container. (5) Boil again then neutralize and boil
(8) Place in flowing steam for 30 once more.
minutes. (6) Filter.
(7) Boil and add 5.0 g. peptone, 2.5 g. Sterilization: Not specified.
NaCl and neutralize again. Boil Use: Cultivation of saphrophytic organ-
for 20 minutes. isms. Author reported that diphtheria
(8) Filter. (The filtrate may be divided bacilli and streptococci grew on this me-
into two and using half of the
parts, dium better than on glycerol agar.
infusion as a liquid medium.) Saphrophytic organisms requiring sugar
(9) Add 1.5% agar to (8) and boil until in ordinary agar, grew luxuriantly on this
solution is complete. medium.
(10) Neutralize and cool to 68C. Reference: Lichtenstein (1915-16 p. 362).
(11) Add a well beaten egg to (10) and
boil until the egg is thoroly co- 1672. Stuart's Stomach-Liver Infusion Agar
agulated.
and tube. Constituents
(12) Filter
Sterilization: Sterilizein the Arnold on 1. Water 1000.0 cc.
each of three successive days for 30 min- 2. HCl 10.0 cc.

3. Liver 250.0 g.
utes, and 30 minutes just before inocu-
lation. 4. Stomach, pig 200.0 g.
Use: Study found in carcinoma-

of bodies 5. Peptone, Witte's (1.0%) .... 10.0 g.
6. Agar (1.2%) 12.0 g.
tous tissue. Author was unable to obtain
growth of carcinomatous bodies. Preparation
(1) Mix 250.0 g. of finely minced liver
Reference: Richardson (1900-1 p. 73).
and 200.0 g. of that portion of a pig's
1671. Lichtenstein's Blood Clot Infusion stomach lining containing the gastric
Agar glands, in a liter of water containing
Constituents 10.0 cc. ofcommercial HCl.

1. Distilled water 1000.0 cc. (2) Incubate the mixture for 18 hours
Blood clots 500.0 g. in a water bath at 50C.
2.
3. Peptone (3) Make Biuret and tryptophane tests
4. NaCl every six hours.
5. Agar (4) Stop digestion by heating in the
sterilizer in free flowing steam for
Preparation
Run the blood clots thru a meat 10 minutes.
(1)
grinding machine. (5) Allow to settle over night in the ice
Mix (1) with a double amount of box and decant.
(2)
distilled water. (6) Add 1.0% Witte's peptone and auto-
Place in an enamel kettle and boil clave into solution.
(3)
over a free flame for 10 minutes stir- (7) Add 1.2% agar.
ring constantly. (8) Adjust the reaction to pH = 7.0
(4) Add concentrated acetic acid and boil until the agar is dissolved.
(amount not given). (9) Adjust the reaction to pH = 7.2
Boil for five minutes. to 7.4.

(5)
Remove the flame and place the (10) Tube.
(6)
kettle in a slanted position so the Sterilization: Method not given.
precipitated material may settle Use: To study variation in Bacterium
out. typhosum.
(7) Collect the clear liquid after it has Reference: Stuart (1924 p. 591).
cooled.
(8) Transfer into glass flasks and add 1673. Bailey's Hormone Agar
the necessary amount (exact amount
not given) of peptone, NaCl and Constituents
agar.
Beef 500.0 g.
(9) When the agar has dissolved, neu- 2.
tralize and place in the autoclave 3. Peptone 100 g-
for 20 minutes. 4. NaCl 5.0 g.

5. Agar 15.0 g.
(10) Filter.
Preparation (b) Brom cresol purple 0.0005%

(1) Thoroly wash 15.0 g. of agar agar Cresol red 0.0005%
shreds in running water. (c) China blue 0.0025%
(2) Dissolve (1) in a liter of distilled (d) China blue 0.005%
water and cool to between 50 and Sodium rosolate 0.005%
60C. (e) China green 0.003%
(3) Add 500.0 g. of lean beef or beef (f China blue 0.0025 g.
heart, chopped to moderate fine- Phenol sulphonphthalein. . . 0.001 g.
ness. Keep the indicator in concentrated alco-
(4) Bring to boil and cook slowly for 15 hol stock solution of such a strength that a
to 20 minutes. definiteamount can be measured out per
(5) Filter thru a 16 mesh sieve (cullander medium, e.g., 1.0 cc. of 1.6 per cent
liter of
type) until clear. brom cresol purple per liter of medium.
(6) Add 10.0 g. peptone and 5.0 g. NaCl.
1676. Jordan and Victorson's Lead Acetate
Infusion Agar
(8) Adjust to desired reaction (pH 7.5).
(9) Allow to stand several minutes and Constituents
decant the supernatant fluid. 1. Water 1000.0 cc.
(10) Tube. 2. Peptone Witte's 30.0 g.
Sterilization: Sterilize by the fractional 3. Beef, lean 1.0 lb.
method or by autoclaving for about 4. Agar 15.0 g.
20 minutes at 5 pounds pressure. 5. Lead acetate (10.0% soln.).
Use: Hormone medium. Preparation
Reference: Bailey (1925 p. 341). (1) Dissolve 2 in fresh meat broth
(1 pound meat to 1 liter water, but
1674. Fasiani and Zironi's Veal Autolysate
exact procedure not given) by boiling.
Peptone Agar
(2) Filter.
Constituents (3) Dissolve agar in (2).
1. Autolysate of veal liver 1000.0 cc. (4) Adjust to 1.0% acid to phenol-
2. Peptone (1.0%) 10.0 g. phthalein.
3. NaCl (0.5%) 5.0 g. (5) Tube.
4. Agar (2.0%) 20.0 g. (6) Cool sterile (5) to 43C., add 2 drops
Preparation : 10.0% lead acetate solu-
(0.1 cc.) of a
(1) Dissolve 1.0% peptone 0.5% NaCl and tion prepared from recently sterilized
2.0% agar in an autolysate of veal water to each tube and mix well.
liver. Sterilization: Sterilize (5) method not
(2) Tube in 15 to 20.0 cc. lots. given.
Sterilization: Autoclave at 110C. for 30 Use : Differentiation of paratyphoid enteri-
minutes. tidis group. Inoculate by sliding the
Use: Isolation and cultivation of anae- needle in between the agar and wall of
robes. the tube. Authors reported that B. para-
Reference Fasiani and Zironi (1918
: p. 439). typhosus B and B. enteritidis produced
Taken from (1919 p. 147). H2S, blackening the medium, while
B. paratyphosus A, B. suipestiver did not ,
1675. Harvey's Basal Indicator Infusion

blacken the medium. Emile-Weil used
Agar
a similar medium to determine H2S pro-
See medium 1661 variant (v) for prepara- duction by Bacillus leprae.
tion of Infusion Agar. Add 1.0% of any Variants
desired carbohydrate, alcohol, etc., to the (a) Emile-Weil prepared the medium as
agar, adjust the reaction with brom thymol follows:
blue indicator to a pH value between 6.8 (1) Macerate 500.0 g. of finely divided
and 7.2. Add one of the following indi- beef with one liter distilled water
cators: for 12 hours.
(a) Brom cresol purple 0.001% (2) Boil slowly for 15 minutes.
(3) Allow to cool, skim and filter (10) Place (8) in a saturated salt solution
thru filter paper. bath and boil for 45 minutes, making
(4) Adjust the reaction so that the provision for loss of water by evap-
acidity is equal to 0.025 g. H2SO4 oration.
per liter (0.5%). (11) Add saturated Na2C03 solution until
(5) Add 5.0 g. of agar and 10.0 g. De- slightly alkaline to litmus (allow for
fresne peptone and heat for 15 min- a slight augmentation of the acid
utes at 118C. during the subsequent steps).
(6) Filter and tube in 6 to 8.0 cc. lots. (12) Cool to 55C. and add the white of
(7) Heat at 112 for 15 minutes. an egg and steam in Arnold sterilizer
(8) When ready for use add 0.1 cc. of a until clarification of the upper
1 to 10 sterile solution of lead sub- strata of the medium has occurred.
acetate in distilled water to each (13) Pipette off the clear supernatant
tube. fluid or filter.
(b) Tilley adjusted Jordan and Victor- (14) Transfer definite quantities of (13)
son's medium to pH = 7.2, sterilized to flasks.
the agar in the autoclave and added (15) To 100.0 cc. of hot sterilized distilled
3 drops of freshly prepared 10.0% lead water, add 10.0 g. of one of the added
acetate to each tube. nutrients, and expose the solution
(c) Harvey added 1.0 drop (0.05 cc. to to the action of live steam for 10 min-
each tube (4.0 cc.) of agar (see utes. (Use old Jena glassware to
variant (v) medium 1661) cooled to avoid alkaline production.)
45C. (16) Add to sterile (14) following the
References: Jordan and Victorson (1917 third sterilization, 1.5% of sterilized
p. 554), Emile-Weil (1917 p. 379), Tilley Kubel and Tiemann's litmus solu-
(1923 p. 115), Harvey (1921-22 p. 107). tion.
(17) Transfer to sterile tubes, slant and
1677. Elser and Huutoon's Basal Litmus
expose to incubator temperatures
Infusion Agar
for several days.
Constituents (18) The final reaction of the medium
1. Distilled water 1000.0 cc. should be very faintly alkaline to
2. Beef (lean) 500.0 g. litmus.
3. Peptone 10.0 g. Sterilization: Sterilize (14) by steaming for
4. NaCl 5.0 g. 15 minutes on each of three successve
5. Agar agar 16.0 g. days.
6. Litmus, Kubel and Tie- Use: Cultivation of meningococci, pseudo-
mann's 15.0 cc. meningococci and gonococci. The me-
Preparation dium is suited primarily for stock cul-
(1) Mix thoroughly 500.0 g. of chopped tures.
lean beef with 1000.0 cc. distilled Added nutrients: The author added 1.0%
water. of one of the following:
(2) Boil over a free flame for 15 minutes, glucose mannitol
stirring constantly. galactose dulcitol
(3) Filter. levulose inulin
(4) Add an emulsion of Bacterium coli. lactose dextrin
(5) Incubate at 37C. for 24 hours. maltose sucrose
(6) Sterilize for 10 minutes. Reference: Elser and Huntoon (1909
(7) Filter until clear. p. 406).
(8) Add 3, 4 and 5 that have previously
been soaked in water, to (7).
1678. Emile-Weil's Neutral Red Infusion
Agar
(9) A portion of the filtratereinocu- is
lated with Bacterium coli to estab- Constituents

lish the sugar free condition of the 1. Distilled water 1020.0 cc.
product. 2. Beef 500.0 g.
3. Peptone (Defresne) 10.0 g. (4) Boil 30 minutes.

4. Agar 5.0 g. (5) Add 7.0 cc. N/1 NaOH.
5. Neutral red 0.1 cc. (6) Neutralize to litmus with sodium
Preparation carbonate.
(1) Macerate 500.0 g. of finely divided (7) Add 5.0 cc. N/1 sodium carbonate.
beef with one liter distilled water for (8) Add nutrose to make 1% solution.
12 hours. (0.5% peptone may be used instead
(2) Boil slowly for 15 minutes. of nutrose).
(3) Allow to cool, skim and filter thru (9) Boil.
filter paper. (10) Distribute in 500.0 cc. flasks.
(4) Adjust the reaction to 1.5. (11) Sterilize several hours on each of two
(5) Add 5.0 g. of agar and heat for 15 successive days in streaming steam
minutes at 118C. (12) Let cool slowly in sterilizer.
(6) Filter. (13) Decant clear supernatant liquid.
(7) Prepare a solution of 0.1 cc. neutral (14) To each add 2-2.5 cc.
100.0 cc. of agar
red in 20.0 cc. distilled water. of a sterile 2.0% aqueous malachite
(8) Add 5.0 cc. of (7) to one liter of (6). green solution.
(9) Tube. (15) Pour 15.0 to 20.0 cc. in each petri
Sterilization: Sterilize at 112C for 15 dish.
minutes. (16)Leave dishes open until agar is cool
Use: Cultivation of Bacillus leprae. and hard.
Author reported that a change of color References: Lentz and Tietz (1903 p. 2139),
or fluorescence may be present or absent. Loefiler (1906p. 289).
Variants: Harvey added 5.0 cc. of a 1.0%
1680. Zielleczky's Phenolphthalein Infusion
solution of neutral red and 5.0 g. of any
desired carbohydrate, alcohol, etc., to
Agar
medium 1661, variant (v). Same as medium 783 but solidified by the
References: Emile-Weil (1917 p. 379), Har- addition of agar.
vey (1921-22 p. 89).
1681. Werbitzki's China Green Infusion
1679. Lentz and Tietz's Malachite Greea Agar
Infusion Agar
Constituents
1. Loeffler's 2.0% meat infusion peptone 2. Beef 500.0 g.
agar. 3. Peptone, Witte (1.0%) 10.0 g.
2. Malachite Green. 4. NaCl (0.5%) 5.0 g.
Preparation: 5. Agar (3.0%) 30.0 g.
(1) Add 1:1000, 1:2000, 1:4000, 1:6000, 6. China green (Boyer's)
1:8000 or 1:10,000 malachite green to Preparation
Loeffler's 2.0% meat infusion peptone (1) Add 500.0 g. of finely chopped bone
agar (method of preparation not and sinew free lean beef to one liter
given) of distilled water. Mix well.
(2) Pour into petri dishes. (2) Weigh (1).
Sterilization: Not specified. (3) Boil for 45 minutes stirring con-
Use: Isolation colon-typhoid bacteria from stantly.
feces. Author reported that coli grew (4) Weigh, and make up the loss in
weakly in 1:10,000 malachite green, weight by the addition of distilled
typhoid well in 1:4000 and para B 1:2000. water.
Variants: Loeffler gave the following (5) Filter thru a towel and measure the
method of preparation: filtrate.
(1) Prepare beef infusion 1 pound in two (6) Add 1.0% Witte's peptone and 0.5%
liters water. NaCl to (5).
(2) Add 30.0 g. agar. (7) Boil in the steamer.
(3) Add 7.5 cc. N/1 HCl. (8) Cool in a cold water bath.
(9) Filterthru filter paper. dye inhibited growth of B. paratyphoid B

(10) Dissolve 3.0% agar in (9) in the but did not hinder growth of B. paraty-
steamer. phoid A or B. enteritidis
(11) Adjust the reaction by the addition Reference: Teague (1918 p. 1).
of NaOH so that 1.3% NaOH will

1683. Drennan and Teague's Crystal Violet
be required to bring the reaction
Infusion Agar
to the neutral point of phenol-
phthalein. Constituents
(12) Heat. 1. Distilled water 1000.0 cc.
(13) Filter in the steamer. 2. Heart, beef 1.0 lb.
(14) Distribute in 100.0 cc. lots in Erlen- 3. Peptone 10.0 g.

meyer flasks. 4. NaCl 2.5 g.
(15) Prepare a 0.2% china green solution. 5. Agar 15.0 g.
(16) Add 1.4 to 1.5 cc. of (15) to each 6. N/1 NaOH 4.0 cc.
100.0 cc. of sterile melted and cooled 7. Crystal violet 1/70 g.
to 60-65C. (14). Mix well. Preparation:
(17) Pour into plates. (1) Pass one pound of beef heart thru a
Sterilization: Method of sterilization of meat chopping machine and soak in
(14) not given. one liter of distilled water over night
Use: Isolation of typhoid bacilli from inan ice box.
feces. Enrichment of typhoid bacilli. (2) Squeeze the fluid thru a cheese cloth,
Smear the plates with the stool or feces heat to boiling and filter thru filter
suspension. Incubate 20 hours at 37C. paper.
Wash the surface of the plates with 8 to (3) Add 1.0% peptone, 0.25% NaCl,
10.0 cc. physiological salt solution, and 1.5% agar-agar, 4.0 cc. of N/1 NaOH
smear 1 to 3 loops of the salt solution sus- and heat for 30 minutes in the auto-
pension on Drigalski-Conradi medium. clave at 15 pounds pressure.
Reference: Werbitzki (1909 p. 205). (4) Adjust the reaction to -fl (hot titra-
tion), clear with white of egg.
1682. league's Victoria Blue Infusion Agar Filter thru cotton.
(5)
Constituents (6) Distribute in flasks in 200.0 cc. lots.
1. Meat infusion 1000.0 cc. (7) Add to the melted sterile agar 1/700%
2. Peptone, Witte (1.0%) 10.0 g. of crystal violet.
3. NaCl (0.5%) 5.0 g. (8) Pour in plates.
4. Victoria blue 4R Sterilization: Sterilize in the autoclave for

Preparation : 20 minutes at 15 pounds pressure.
(1) Detailed method of
preparation or Use: Isolation of B. pestis from lesions.
composition of meat infusion agar Reference: Drennan and Teague (1919
not given except that it contains 1.0% pp. 521-529).
Witte's peptone and 0.5% NaCl, and
1684. Meyer and Batchelder's Sulphite
that it is to be prepared in the usual
Gentian Violet Infusion Agar
manner using the Arnold sterilizer.
(2) Clear (1) with egg white. Constituents

thru cotton. 1. Infusion agar 1000.0 cc.
(3) Filter
(4) Titrate to +1- 2. NaoSOa (10.0% soln.) 2.5 cc.
(5) Add stock solutions (method of 3. Gentian violet (1 : 1000) 25.0 cc.
preparation not given) of Victoria Preparation
blue 4R in varying amounts so that (1) Prepare heart infusion agar using
there is present 1/20, 1/30, 1/40 or 1/50 Berna peptone, according to Hun-
of dye, to sterile (4). toon's method. (See medium 1863
Sterilization: Sterilize by heating in Ar- for Huntoon's method.)
nold for 3 successive days. (2) Add 0.25% of a freshly prepared 10.0%
Use: Enrichment of B. paratijphosus A and solution) NasSOa and 1/400% (2.5 cc.
B. enteritidis. Author reported that the of a 1:1000 solution per 100.0 cc.) of
gentian violet (improved Coleman (2) The reaction is slightly alkaline.

Bell). (3) Filter in the steamer.
Sterilization: Sterilize in the autoclave. (4) on three successive days
Sterilize
Use: Isolation and cultivation of B. pestis. minutes in streaming steam.
for 30
Reference: Meyer and Batchelder (1926 (b) Muller dissolved 22.0 g. Ragit bouil-
p. 385). lon and 10.0 g. agar in 1000.0 cc. water
to cultivate milk bacteria. The reac-
1685. Miiller's Indicator Infusion Agar
tion was slightly alkaline.
Same as medium 784 but solidified by the References: Marx (1910 p. 361), Lohnia
addition of 18.0 g. of agar per liter. (1913 p. 17), Tausz and Peter (1919 p. 507),
Muller (1917 p. 390).
1687. Friedberger and Joachimoglus' Tellu-
rite Placenta Infusion Agar 1689. Bacto Nutrient Agar (Dehydrated)
1. Placenta infusion agar. 100.0 cc. 1. Distilled water
2. Potassium tellurite 2. Beef extract (Bacto) 3.0 g.
(1.0%) 1.0 to 2.0 cc.
Preparation 4. Agar (Bacto) 15.0 g.
(1) Prepare neutral placenta infusion Preparation
agar.
(1) Dissolve 23.0 g. of Bacto Nutrient
(2) Add from 1.0 to 2.0 cc. of a 1.0% po- Agar (Dehydrated) in 1000.0 cc. dis-
tassium tellurite solution to each tilled water by boiling or autoclaving.
100.0 cc. of (1).
(2) Restore loss if necessary.
(3) If sterilized 20 minutes at 15 pounds
Use; Cultivation exan-
of Bacillus typhi pressure pH = 6.6.
thematici {Bacillus Weil and
proteus,
Sterilization Sterilize in the usual manner.
:
Felix). This organism produces an in- Use: General culture medium. The me-
tense black color on this medium. dium conforms to "Standard Methods"
Reference: Friedberger and Joachimoglu formula.
(1918 p. 805). Reference: Digestive Ferments Co. (1925
p. 10).
1688. Ragit Agar
Constituents: 1690. Bacto Nutrient Phosphate Agar
1. Water 1000.0 cc.
(Dehydrated)
2. Ragit agar 42.0 g. Constituents
Preparation: (1) Dissolve 42.0 g. of Ragit 1. Distilled water
agar in 1000.0 cc. of water. Ragit agar is 2. Beef extract (Bacto) 3.0 g.
a trade name of a dried medium marketed 3. Peptone (Bacto) 10 g.
by Merck. It contains "Maggibouillon" 4. Agar (Bacto) 15.0 g.
Agar and Peptone in such amounts that 5. NaaHPOi 5.0 g.
42.0 g. of powder in 1 liter of water gives Preparation
a nutrient agar of the usual composition, (1) Dissolve 33.0 g. Bacto Nutrient
of
(i e. Beef infusion with 1.0% peptone Phosphate Agar (Dehydrated) in
and 2.0% agar.) 1000 cc. distilled water by boiling or
Sterilization: Not specified. autoclaving preferably by boiling.
Use: General culture medium. (2) If sterilized 20 minutes at 15 pounds
Variants pressure pH = 7.5 .
(a) Tausz and Peter cultivated Bac- (3) Cool to 50C. before adding albu-
terium aliphaticum, Bacterium ali- minous enrichment materials if these
phaiicum Uquefaciens and Paraffin materials are to be added.
bacteria, and prepared the medium Sterilization: Sterilize in the usual manner.
as follows: Use General culture medium.
:
(1) Boil 42.0 g. Ragit agar with 1 liter Reference: Digestive Ferments Co. (1925
of water for one hour. p. 11).
1691. Bacto Nutrient Agar 1.5% Sterilization: Do not heat over 75 C. when
(Dehydrated) sterilizing(method not given).
Constituents :
Use: To study oxalic acid formation by
acetic acid bacteria. Meier used a sim-
1. Distilled water
ilar medium to study the bacterial count
2. Beef extract (Bacto) 3.0 g.
of milk and whey.
3. Peptone (Bacto) 5.0 g,
4. Agar (Bacto) 15.0 g.
Added nutrients The author added one of
:
the following:
5. NaCl 8.0 g.
Preparation Methyl alcohol 10.0 g.
Dissolve 31.0 g Bacto Nutrient

of
Ethyl alcohol 30.0 g.
(1)
Agar 1.5% (Dehydrated) in 1000.0 cc. Propyl alcohol 20.0 g.
distilled water by boiling or auto-

Butyl alcohol 10.0 g.
claving.
Amyl alcohol 5.0 g.
Glycerol 10.0 g.
(2) Distribute as desired (Blood may be
added in suitable proportions for
Ethylene glycocol 10.0 g.
blood agar). Erythritol 10.0 g.
If sterilized at 15 pounds pressure for

Mannitol 10.0 g.
(3)
pH = Succinic acid 10.0 g.
20 minutes 7.0 .
Acetic acid 10.0 g.

Propionic acid 10.0 g.
Butyric acid 5.0 g.
Iso butyric acid 5.0 g.
p. 11).
Valearic acid 5.0 g.
1692. Bacto Nitrate Agar (Dehydrated) Glyoxalic acid 2.5 g.
Constituents: Lactic acid 10.0 g.
1. Distilled water Malonic acid 2.5 g.
2. Beef extract (Bacto) 3.0 g. Pyrotaric acid 2.5 g.
3. Peptone (Bacto) 5 g. Malic acid 10,0 g.
4. KKO3 (C. P.) 1.0 g. Salicylic acid 5.0 g.
5. Agar (Bacto) 12.0 g. Tartaric acid 10.0 g.
Preparation Potassium benzoate 5.0 g.
(1) Dissolve 21.0 g. Bacto Nitrate Agar GlycocoU 10.0 g.

(Dehydrated) in 1000.0 cc. of water Leucine 5.0 g.
by boiling or autoclaving, preferably TjTosine 2.5 g.
the latter. Potassium urate 5.0 g.
(2) Tube. Creatin 2.5 g.
(3) If sterilized at 15 pounds for 20 min- Creatinine 2.5 g.

utes pH = 6.8d=. Urea 10.0 g.
Sterilization: Sterilize in the usual manner. Hippuric acid 5.0 g.
Use: Study nitrate reduction. Sarcosine (amount not given)
Reference: Digestive Ferments Co. (1925 Glucose 20.0 g.
p. 13). Levulose 20.0 g.
Galactose 20.0 g.
1693. Banning's Basal Meat Extract Agar Maltose 20.0 g.
Constituents Sucrose 20.0 g.
1. Water 1000.0 cc. Lactose 20.0 g.
2. Peptone 10.0 g. Raffinose 10.0 g.
3. Meat extract 1.0 g. Rhamnose 10.0 g.
4. Agar 10.0 g. Arabinose 20.0 g.
Preparation Starch (wheat) 10.0 g.
(1) Dissolve 2, 3 and 4 in 1. Inulin 10.0 g.
(2) Do not adjust the reaction. Glycogen 10.0 g.
(3) Dissolve one of the added nutrients Dextrin 10.0 g.
in .(2). . Gum arabic 10.0 g.
Variants: Meier studied the bacterial (Blood serum may be used instead
counts of milk and whey, using the fol- of egg white.)
lowing medium: (8) Boil for 3 to 5 minutes and filter
(1) Dissolve 15.0 g. of agar, 10.0 g. thru paper.

Liebig's meat extract, 10.0 g. Witte's (9) Sterilization not specified.
peptone, 5.0 g. NaCl and 2.0% gly- (b) Frost (1903).
cerol, 2.0% glucose or 4.5% lactose in (1) Add 15,0 g. agar to 500.0 cc. of
1000.0 cc. water. water and dissolve by heating to
(2) Neutralize to litmus and then add 120C. in the autoclave, shutting
10.0 cc. N/1 soda solution per liter off the gas and allowing to cool,
medium, or boil until the agar
is dissolved
(3) Sterilization not specified. (about 30 minutes) and make up

References: Banning (1902 p. 395 and 426), the loss of water due to evapo-
Meier (1918 p. 438). ration.
(2) Weigh out 3.0 g. of beef extract
1694. Helm's Meat Extract Salt Agar such as Liebig's and add 500.0 cc.
Constituents of water.
1. Water 1000.0 cc. (3) Add 10.0 g. peptone and 5.0 g. NaCl
2. Agar 12.5 g. to (2).
3. Meat extract 10.0 g. (4) Heat until solution is complete.
4. Peptone 10.0 g. (5) Neutralize to phenolphthalein.
5. NaCl 5.0 g. (6) Cool to 60 and add one egg.
Preparation (7) Add (6) to (1).
(1) Dissolve 2, 3, 4 and 5 in 1. (8) Boil until albumin is coagulated

(2) Neutralize with NaOH to litmus. and floats in the clear liquid and
(3) Filter and distribute into culture restore the weight.
plates containing different amounts of (9) Readjust the reaction if necessary.
NaOH solution so that the final (10) Add 0.5% normal HCl.
volume be 10.0 cc. 0.5% alkaline (11) Filter thru cotton supported in a
gave the best results. coil of wire using a suction pump.
Sterilization: Sterilize in steam (exact (12) Tube.
method not given) and allow plates to (13) Sterilize in steam for 15 minutes
dry for 1 day with the cover removed. on days or in the auto-
3 successive
Use General culture medium. Heim used
: clave for 20 minutes at 120C.
the medium to study effect of alkalinity Slant some of the tubes after final
and acidity on growth of anthrax bacillus. sterilization.
Variants: The following authors prepared (c) Hesse (1908),
the medium as indicated: (1) Dissolve 10.0 g. Witte's peptone,
(a) Ravenel (1899). 5.0 g. Liebig's meat extract and 8.5
(1) Add 10.0 g. peptone, 5.0 g. NaCl g. NaCl in 500.0 cc. distilled water
and 5.0 g. Liebig's beef extract to by boiling.
500.0 cc. water. (2) Dissolve 5.0 g. agar in 500.0 cc. dis-
(2) Boil minutes and neutral-

(1) for 3 tilled water and boil.
ize (indicator not specified). (3) Filter (1) thru a filtering apparatus
(3) Chop 12.0 g. agar into small pieces and (2) thru paper.
and add to 500.0 cc. of water. (4) Mix the two filtrates.
(4) Place in the autoclave and run the (5) Make up to 1 liter and boil again.
pressure to two atmospheres (6) Distribute in 10.0 cc. lots in tubes.
(135.1C.). Turn out the flame. (7) Do not add alkali.
(5) Allow the autoclave to cool to (8) Sterilize in the autoclave under
100C. before opening. 2 atmospheres pressure for 20
(6) Cool (2) and (5) to 60C. and mix. minutes.
(7) Beat the whites of two eggs in (d) Hesse (1908).
50.0 cc. water and add to (6). (1) Dissolve 8.5 g. NaCl, 5.0 g. Liebig's
meat extract, 10.0 g. Witte's pep- (2) Neutralize to litmus and then add
tone and 5.0 g. agar in 1000.0 cc. 10.0 cc. normal soda solution pe?
water. liter of medium.
(2) Distribute in 10.0 cc. lots into test (3) Sterilization not specified,
tubes that will not give up alkali (i) Kligler and Defandorfer (1918).
by sterilization. (1) Dissolve 10.0 g. peptone, 3.0 g.
(3) Adjustment of reaction or steri- beef extract and 5.0 g. NaCl in

lization not specified. 1000.0 cc. water. (Meat infusion
(e) Stokes and Hachtel (1909). may be used instead of beef
(1) Dry agar at
105 for 30 minutes. extract.)
(2) Dissolve 4.5 g. of (1)

in 500.0 cc. (2) Add 15.0 g. agar to (1) and auto-
distilled water by boiling and make clave for 1 hour at 15 pounds
up loss in weight by adding dis- pressure.
tilled water. (3) Cool to 50C., an egg white is
(3) Dissolve 5.0 g. Liebig's beef ex- added and steamed in Arnold
tract, 10.0 g. Witte's peptone and 30 minutes.
8.5 g. NaCl in 500.0 cc. distilled (4) Adjust to pH = 7.4.
water. (5) Boil over free flame 6 or 7 minutes,
Mix (3) and (2) and boil 30 filter, flask and autoclave (length
(4)
minutes. of time not specified).
(5) Make up the loss in weight by the (j) Hesse (Ball) (1919).
addition of distilled water. (1) Digest 5.0 g. agar in 500.0 cc. of
(6) Filter.
water.
(7) Adjust the reaction to neutrality (2) Dissolve 10.0 g. peptone, 5.0 g.
(indicator not specified). beef extract and 8.5 g. NaCl in
(8) Tube in 9.0 cc. lots. 500.0 cc. water.
(9) Autoclave at 15 pounds pressure (3) Mix (1) and (2).
for 20 minutes. (4) Filter.
(f) Viehoever (1913) cultivated urea (5) Adjust the reaction to 1.0%.
splitting organisms on the follow- (6) Tube.
ing medium: (7) Sterilize in the autoclave,
(1) Dissolve 6.0 g. Witte's peptone, (k) Hesse (Tanner) (1919).
meat extract (amount not speci- (1) Dissolve 5.0 g. agar in 500.0 cc, dis-
fied) 1.0 g. NaCl and 10.0 g. agar tilled water.
in 500.0 cc. water. (2) Dissolve 10.0 g. peptone, 5.0 g.

(2) Add 0.25% NajCOa to (1). Liebig's beef extract, and 8.5 g.
(3) Tube in 5.0 cc. quantities. NaCl in 500.0 cc. distilled water.
(4) Sterilize (method not given). (3) Mix (1) and (2).
Bengis (1916). (4) Filter.

(g)
(1) Dissolve 30.0 g. powdered agar in (5) Tube.
1000.0 cc, distilled water. (6) Sterilization not specified.
(2) Add 10.0 g. Witte's peptone, 5.0 g. (1) Malm (Besson) (1920).
NaCl, and 5.0 g. meat extract (1) Dissolve 5.0 g. Liebig's meat ex-
to (1). tract (or Cibil's meat extract),
(3) Filter in incubating flasks. 10.0 g. Chapoteaut's peptone, and

(4) Sterilize in Bramhall-Dean auto- 5.0 g. NaCl in 1000.0 cc. water.
clave. (2) Soak 20.0 g. chopped thread

of
(h) Meier (1918) made bacterial counts of agar in coldwater for several
milk and whey using the following hours. Squeeze the water thru
medium a cloth.
(1) Dissolve 15.0 g. agar, 10.0 g. (3) Heat (2) and (1) at 100C. until the
Liebig's meat extract, 10.0 g. agar is dissolved.
Witte's peptone and 5.0 g. NaCl (4) Readjust the reaction if necessary.
in 1000.0 cc. distilled water. (5) Allow to cool to 55 or 60C.
(6) Beat the white of an egg in (9) Autoclave at 15 pounds pressure

100.0 cc. of water and add to (5). for 2 hours.
(7) Mix well. (10) Adjust the medium to the desired
(8) Autoclave at 120C. for one hour. reaction, by addition of normal
(9) Filter thru moistened Chardin NaOH or HCl.
filter using a hot water funnel. (11) Filter while boiling hot thru
(10) Tube. plaited filter paper.
(11) Sterilize at 115 for 20 minutes, (12) Distribute as desired.
(m) Giltner (1921). (13) Sterilize (method not given),
(1) Prepare an ordinary agar (method (p) Stitt (1923).
not given) from 5.0 g. NaCl, 5.0 g. (1) Place 3.0 g. Liebig'smeat extract,
Liebig's extract, 10.0 g. peptone 10.0 g. peptone, Agar 2.0 or 3.0%,
and 30.0 g. agar in sufficient water and 5.0 g. NaCl in 1000.0 cc. water
to make 1000.0 cc. in a rice cooker, or any other large
(2) Adjust the reaction to or +0.2%. straight sided granite-iron re-
(3) Store in 100 cc. quantities in ceptacle. The whites of one or
Erlenmeyer flasks. two eggs may or may not be dis-
(n) Wolf and Shunk (1921). solved in the water.
(1) Dissolve 10.0 or 20.0 g. agar, 3.0 g. (2) Place in the chamber of the
Liebig's beef extract, 10.0 g. Pep- dressing sterilizer and heat to be-
tone (Armour's) and 5.0 g. NaCI tween 5 and 10 pounds (110 to
in 1000.0 cc. water by heating in 115) for at least 45 minutes.
the autoclave. (3) Adjust the pH as desired.
(2) Flask. (4) Put back in the sterilizer for
(3) Sterilize at 15 pounds for 15 30 minutes at the same tempera-
minutes. ture as in (2). The greater the
(4) Coolto50C. pressure applied the darker the
(5) Pipette with sterile pipette, medium will be, so that a pressure
10.0 cc. quantities into sterile test not to exceed 3 pounds is used when
tubes and add appropriate quanti- a light medium is desired.
ties of strong acid or alkali to give (5) Leave the medium in the sterilizer
desired reaction. (Authors used over night with the steam turned
HCl sp. gr. 1.20 or 39.11% and off.
NaOH sp. gr. 1.226 or approxi- (6) In the morning dump out the
mately 20.0%.) jellymass and cut off and discard
(6) the tubes thoroly and cool.
Mix the bottom portion containing the
Do
not sterilize. sediment.
(o) Giltner (1921). (7) Melt the remainder of the agar.
(1) Place 500.0 g. of water in an agate (8) Distribute in flasks (or nursing
water pail and add 15.0 g. of agar. bottles).
(2) Wash the agar well, separating the (9) Sterilize (method not specified),
shreds and squeezing thru the (10) Store until ready for use.
hands. Wash until clean. (q) Stitt (1923).
(3) Make up to the original volume (1) Dissolve 15.0 g. of agar in 500.0 cc.
the addition of tap water. of water in the inner compartment
(4) Heat over a free flame until the of a rice cooker.
agar is dissolved, stirring con- (2) Cool to 55C.
stantly. (3) Dissolve the whites of one or two
(5) Add 3.0 g. of standard meat ex- eggs in 500.0 cc. of water.
tract (or use 500.0 cc. meat infu- (4) Prepare a paste from 3.0 g. Liebig's
sion) to 500.0 cc. of tap water. meat extract, 10.0 g. peptone and
(6) Add and 0.5% NaCl.
1,0%, peptone 5.0 g. NaCl by adding (3) little
(7) Cool (4) to 60C. and mix with (6). by little.
(8) Add 10.0 g. of albumin mixed with (5) Add the remainder of (3) to (4).
100.0 cc. water. (6) Heat (5) to 50 to 55C.
(7) Add (6) to (2). required per liter, heat again for
(8) Adjust to a desired pH value by 10 minutes.
the addition of normal acid or thru cotton.
(11) Filter
alkali. (12) Distribute in
tubes or flasks.
(9) Bring the agar to a boil in the inner (13) Sterilize at 15 pounds pressure for
compartment of a rice cooker. 30 minutes.
(10) Filter in an autoclave or Arnold References: Heim (1895 p. 193), Ravenel
sterilizer thru filter paper wetted (1899 p. 605), (1899-1900 p. 89), Frost
with boiling water. It may be (1903 p. 16), Hesse (1908 p. 441), (1908
filtered thru cotton or gauze if p. 89),Stokes and Hachtel (1909 p. 40),
clearness of the medium is not an Viehoever (1913 p. 214), Bengis (1916
essential. p. 392), Meier (1918 p. 435), Kligler and
(11) Sterilization not specified. Defandorfer (1918 p. 438), Ball (1919
(r) Stitt (1923). p. 80), Tanner (1919 p. 51), Besson (1921
(1) Prepare a paste of 3.0 g. meat ex- p. 43), Giltner (1921 p. 386), Wolf and
tract, 10.0 g. peptone, 5.0 g. NaCl, Shunk (1921 p. 325), Giltner (1921 p. 40),
15.0 g. powdered agar and the Heinemann (1922 p. 35), Stitt (1923
white of one egg by mixing with a pp. 36, 37), Park, Williams and Krum-
little water in a mortar. wiede (1924 p. 117).
(2) Add the remainder of the 1000.0 cc.

1695. Heinemann's Meat Extract Agar
of water and place in a rice cooker.
(3) Boil. Constituents
(4) Filter thru absorbent cotton 1. Water (tap) 1000.0 cc.
placed between two layers of 2. Agar (1.5%) 15.0 g.
gauze in a hot funnel. 3. Beef extract 3.0 g.
(5) Reaction is between +0.7 and 4. Peptone, Witte (1.0%) 10.0 g.

+0.8. Preparation
(6) Sterilization not specified. (1) Weigh accurately a sauce pan with-
(s) Park, Williams and Krumwiede out the lid.
(1924). (2) Measure 1000.0 cc. of tap water into
(1) Dissolve, 2.0 to 5.0 g. Beef extract the pan. Add 300.0 cc. of water to
(Liebig's or Armour's), 10.0 g. pep- allow for evaporation. Heat over
tone, and 5.0 g. NaCl in 1000.0 cc. the gas.
water by boiling over a fire. (3) Add 15.0 g. of shredded agar to (2)
(2) Boil 15 minutes. and boil slowly until solution is
(3) Determine the reaction and adjust, complete.

if necessary, by the addition of (4) Add 3.0 g. of beef extract to (3).
normal NaOH. (5) When dissolved remove from the
(4) Dissolve 1.5% (or 2.0%) agar in flame and slowly dust in 10.0 g. of
(3) by heating in the autoclave at Witte's peptone. Stir constantly
10 to 15 pounds for 30 minutes or until dissolved.
by boiling over the free flame. (6) Adjust the reaction to alkaline to
(5) Ifboiled over a free flame make up litmus or neutralize to phenolphthal-
the loss in weight by the addition ein and then add 0.5% normal HCl.
of water. (7) Allow the agar to cool to 60C.
(6) Adjust the reaction. (8) Dissolve the whites of two eggs in
(7) Cool to 50C. and add one egg. 75.0 cc. of water, and stir well
(8) Heat in the autoclave at 10 to into (7).

15 pounds for 30 minutes, or in an (9) Heat, without stirring, on a piece of
Arnold sterilizer for one hour. asbestos over the flame.
Filter. (10) Make up the weight due to the evap-
(9) Test the reaction and adjust if oration of water, or boil until the
necessary. proper weight is obtained if the
(10) If more than 0.2% normal soda is mixture weighs too much.
515
Cool to 45C. in a cold water bath,

(11) Filter thru paper or absorbent cot- (3)
vacuum pump. then warm to 65C. in the same

ton, using a
bath without stirring.
(12) Tube.
at 120C. for 5 (4) Make up the lost weight, titrate
and if the reaction is not already
minutes.
between +0.5 and +1.0 adjust
prepa- to +1.0.
Variants The following methods of
:
authors (5) Filter thru cloth and cotton until

ration have been described by
clear.
indicated:
H. A. (1916). (6) Distribute in 10.0 cc. lots in test
(a) Committee A. P.
tubes, or distribute in larger
Dissolve 3.0 g. Liebig's beef ex-
(1)
peptone in quantities if desired.
tract and 5.0 g.
the autoclave at
1000.0 cc. water by boiling. Make (7) Sterilize in
15 pounds (120C.) for 15 minutes
up the loss in weight due to
after the pressure reaches
evaporation.
15 pounds.
(2) Filter hot thru paper.
(d) Committee S. A. B. (1918).
(3) Add 12.0 g. of oven dried agar, or
market agar, and boil or (1) Prepare according to Committee
15.0 g. of
A. P. H. A. (1916-1917). May be
heat in the autoclave to dissolve.
clarified with white of egg.
(4) Restore the loss in weight due to
evaporation. (2) Adjust to pH = 6.6 to 7.4.
used the Sterilize.

If an autoclave is to be
(3)
(5)
peptone and beef extract may be (e) Tanner (1918).
Add 3.0 g. beef extract and 5.0 g.
added to about 300.0 g. of water (1)
peptone to 1000.0 cc. distilled
and the agar to 700 Auto- g.
water.
clave for 15 minutes at 15 pounds
pressure. Filter the hot broth (2) Heat agar to 105C. for 30 minutes.
Weigh out 12.0 g. of (2) and add
thru filter paper and mix the fil- (3)
to (1).
trate with the agar. Filter thru
(4) Boil over a water bath or cook in
absorbent cotton.
an autoclave until solution is
(6) Adjust the reaction between +0.5
complete.
and +1.0.
to boiling for Make up the loss due to evap-
(7) Cool to 45 and heat (5)
oration.
15 minutes.
Cool to 45 or 50C. and add 10.0 g.
(8) Filter thru paper or absorbent (6)
of desiccated egg albumin per liter.
cotton until the medium is clear.
Heat in the autoclave for 30
(9) Tube in 10.0 cc. quantities or dis- (7)
minutes.
tribute in flasks.
pressure for Make up the loss due to evapora-
(10) Sterilize at 15 pounds
(8)
30 minutes. The medium may be

tion.
Adjust the reaction to +1.0.
sterilized on 3 successive days for (9)
(10) Filter thru cotton or cloth until
20 minutes after the agar has
clear, or centrifuge in a Sharpies
melted.
centrifuge.
(b) Noyes (1916).
agar, 10.0 g. pep- (11) Tube.
(12) Sterilize in the autoclave at 120C.
tone and 5.0 g. Liebig's extract in
1000.0 cc. water. (f) Dawson (1919).
Dissolve 10.0 g, peptone, 10.0 g.
(c) Committee A. P. H. A. (1917).
(1)
meat extract and 20.0 g. agar in
Add 3.0 g. of beef extract, 5.0 g. of
(1)
1000.0 cc. water.
peptone and 12,0 g. of agar, dried
Neutralize (indicator not given).
for 30 minutes at 105C. before (2)
(3) Sterilization
not given.
weighing to 100.0 cc, of water.
Boil over a water bath until all the (g) Committee A. P. H. A. (1920).
(2)
(1) Same as for 1917,
but adjust the
agar is dissolved, and then make
reaction to a faint pink with
up the loss by evaporation.
phenol red, or adjust to a +1.0 (9) Make up

the loss in weight by the
to phenolphthalein if the reaction addition of hot distilled water.
is not between +0.5 and +1.0.
(10) Readjust the reaction if necessary.
(h) Cohen (1922). (11) Filter thru cotton or paper pulp in
(1) Dissolve 10.0 g. Difco peptone, a Buchner funnel, or run thru a
30 g. Liebig's beef extract and Sharpies centrifuge until clear.
20.0 g. of shredded agar in (12) Tube.
1000.0 cc. of water. (13) Sterilize in the autoclave for
(2) Adjust to pH = 7.0. 20 minutes after the pressure
(3) Method of sterilization not spec- reaches 15 pounds, or in streaming
ified. steam on 3 successive days for
(i) Committee A. P. H. A. (1923). Same 20 minutes, after the agar is com-
as for 1917 (see variant (c) above, pletely melted.
but adjust the reaction to a pH (1) Committee A. P. H. A. (1925).
= 6.2 to 7.0. (1) Add 3.0 g. beef extract, 5.0 g. pep-
(j) Park, Williams and Krumwiede tone and 15.0 g. agar (undried
(1924). market product as stored in the
(1) Soak 15.0 g. of agar (12.0 g. if dried ordinary laboratory cupboard) to
in the oven at 105 C. for 30 1000.0 cc. distilled water.
minutes) in water and wash. (2) Boil until all the agar is dissolved.
(2) Dissolve 3.0 g. beef extract (Lie- (3) Cool to 45C. in a cold water bath,
big's or equivalent) 5.0 g. Peptone then warm to 65 C. in the same
(Armour, Difco, Fairchild's or bath without stirring.
equivalent) and (1) in 1000.0 cc. (4) Make up the lost weight with hot
water. distilledwater and adjust the
(3) Adjust the reaction to between reaction so that the pH value,
+0.5 and +1.0 (phenolphthalein) after final sterilization, will be
or about pH 6.6 to 7.4, if necessary. between 6.2 and 7.0.
(4) Tube. (5) Bring to a boiling temperature,
(5) Sterilize at 15 pounds for 15 stirring frequently, restore the
minutes. lost weight with hot distilled water
(6) Cool rapidly. and clarify.
(k) Park, Williams and Krumwiede (6) Distribute in the desired con-
(1924). tainers.
(1) Dissolve 0.3% (3.0 g.) beef ex- (7) Sterilize in the autoclave at
tract and 0.5% (5.0 g.) peptone in 15 pounds (120C.) for 15 minutes
400.0 cc. distilled water by boiling after the pressure has reached
on the stove. 15 pounds.
(2) Adjust the reaction to between References: Heinemann (1905 p. 11), Com-
+0.5 and +1.0 (phenolphthalein) mittee A. P. H. A. (1916 p. 1316), Noyes
or about pH 6.6 to 7.4, if necessary. (1916 p. 93), Committee A. P. H. A. (1917
(3) Filter thru paper or paper pulp. p. 96), Committee S. A. B. (1918 p. 115),
(4) Soak 1.5% market agar (1.2% if Tanner (1918 p. 48), Ball (1919 p. 77),
oven dried) in water, and wash Dawson (1919 p. 142), Committee
under tap in a sieve. A. P. H. A. (1920 p. 96), Levine (1921
Add to (4) 600.0 cc. of distilled p. 108), Cohen (1922 p. 189), Committee
(5)
A. P. H. A. (1923 p. 4), Committee S. A. B.
water minus the water absorbed
(1923 p. 9), Park, Williams and Krum-
during the washing. Weigh.
wiede (1924 pp. 131, 132), Committee
(6) Mix (5) and (3).
A. P. H.A. (1925 p. 98).
(7) Heat on the stove until the agar is
completely melted, stirring con- 1696. Glaessner's Nahrstoff Heyden Extract
stantly. Agar
(8) Boil and stir constantly for Constituents
20 minutes.
1. Water 1000.0 cc.
2. Agar 15.0 g. 3. Peptone 10.0 g.
3. Nahrstoff Heyden 10.0 g. 4. NaCl 5.0 g.
4. NaCl 2.5 g. 5. Agar 10.0 g.
Meat extract 0-5 g. Preparation

5.
Preparation:
g. of agar in 500.0 cc. (2) Neutralize.
(1) Dissolve 15.0
water. Sterilization: Method not given.
Dissolve 3, 4 and 5 in 500.0 cc. water. Use: Show effect of pancreas extract on
(2)
Melt sterile (1) and cool to 44C. growth of organisms. Author reported
(3)
Mix equal parts of sterile (3) and that the organisms were inhibited by the
(4)
sterile (2) at 40C.
pancreas extract.
Pour in sterile plates. Reference: Kotlar (1895 p. 153).
(5)
1700. Bacto Andrade Maltose Agar
rately (method not given).
(Dehydrated)
Use: Cultivation of diphtheria bacilli.
Variants: The author used 1.0 g. meat Constituents
extract and 5.0 g. NaCl instead of 1. Water
amounts indicated. 2. Beef extract (Bacto) 3,0 g.
Reference: Glaessner (1900 p. 729). 3. Maltose (Bacto) 10.0 g.

1697. Guth's Selenicate Extract Agar
Same as medium 807, but solidified by the 6. Andrade Indicator (Difco) . . 0.0275 g.
addition of 30.0 g. of agar. 7. NaOH
Preparation
1698. Conn and Breed's Nitrate Extract Bacto Andrade
of
Agar
Maltose Agar (Dehydrated) in
Constituents: 1000.0 cc. of distilled water by boiling
1. Water 1000.0 cc. or autoclaving, preferably the latter.
2. Agar 150 g.
(2) Tube.
Peptone 10-0 S- 20 minutes at 15 pounds
3. (3) If sterilized
4. KNO3 10 g- pH = 7.7.
5. Beef extract 3.0 g. Sterilization: Sterilize in the usual manner,
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. avoiding excess heat.
Sterilization: Method not given. Use: General culture medium.
Use To determine the ability of bacteria to
: Reference: Digestive Ferments Co. (1925
reduce nitrates. p. 16).
Variants
(a) Committee S. A. B. prepared beef ex- 1701. Bacto Andrade Mannite Agar
tract agar according to the method of (Dehydrated)
Committee A. P. H. A. (1920 see Same as 1700, but substituting Bacto
variant (g) medium 1695 and adjusted Andrade Mannite Agar (Dehydrated) for
the reaction to pH = 6.6 to 7.4. Bacto Andrade Maltose Agar (Dehydrated)
(b) Percival sterilized the
medium on 3
successive days for 20 minutes each 1702. Bacto Andrade Dextrose Agar
day. (Dehydrated)
References: Conn and Breed (1919 p. 278),
Same as medium 1700, but using Bacto
Committee S. A. B. (1920 p. 128), Percival
S. A. B. (1923
Andrade Dextrose Agar (Dehydrated) in-
(1920 p. 163), Committee
stead of Bacto Maltose Agar (Dehydrated)
p. 10).
Pancreas Peptone Agar 1703. Bacto Andrade Lactose Agar

1699. Kotlar's
(Dehydrated)
Constituents
1. Water 1000.0 cc, Same as medium 1700, but using Bacto
2. Powdered pancreas 50.0 g. Andrade Lactose Agar (Dehydrated) in-
stead of Bacto Andrade Maltose Agar Preparation

(Dehydrated). (1) Prepare extract agar according to
Committee A. P. H. A. (1916), see
1704. Bacto Andrade Saccharose Agar
medium 1695.
(Dehydrated)
Same as medium
1700, but using Bacto (3) Tube in 5.0 cc. quantities.
Andrade Saccharose Agar (Dehydrated) (4) Dissolve 1.0 g. ofbasic lead acetate
instead of Bacto Andrade Maltose Agar in 1000 cc. water.
(Dehydrated). (5) Add 6.0 cc. of sterile (4) to each tube
of sterile (3).
1705. Percival's Basal Litmus Extract Agar
(6) Incubate to test sterility.
Constituents Sterilization: Sterilize (3) and (4) sepa-
1. Water 1000.0 cc. rately (method not given).
2. Meat extract, Lemco 5.0 g. Use: Detection of production of hydrogen
3. Peptone (Witte's) 10.0 g. sulfid.
4. Agar 15.0 g. Reference: Committee S. A. B. (1922
5. Litmus p. 528).
Preparation
Soak 15.0 g. agar in 500.0 cc. water 1707. Conradi's Brilliant Green Picric Acid
(1)
for 12 hours.
Extract Agar (Bezanfon)
(2) Dissolve 2 and 3 in 500.0 cc. water. Constituents:

(3) Mix (1) and (2) and steam in
boil or 1. Water 900.0 cc.
the sterilizer for 20 to 30 minutes until 2. Agar 30.0 g.
solution complete.
is 3. Meat extract, Liebig's 20.0 g.
(4) Neutralize to phenolphthalein and 4. Peptone, 10.0% soln 100.0 cc.
then add 10.0 cc. of normal HCl per 5. Brilliant green (1 1000) : 10.0 cc.
1000.0 cc. ofmedium. 6. Picric acid (1: 100) 10.0 cc.
(5) Cool to 40 to 50C. and add the white Preparation
of an egg beaten up in a little water. (1) Dissolve 2, 3 and 4 in 1. (Method not
(6) Heat in the steam sterilizer for given.)
90 minutes. (2) Make alkaline (indicator not spec-
(7) Filter hot thru a Chardin
while ified.
folded filter paper in a hot water (3) To 1.5 liter of agar add 10.0 cc. of 1
funnel. If not clear, repeat the to 1000 brilliant green and 10.0 cc. of
process. a 1 to 100 picric acid solution.
(8) Add one of the added nutrients and Sterilization: Not specified.
sufficient litmus solution to give the Use: Differentiation of colon-typhoid
desired color. (Chalk may be used group. Author reported that typhoid
instead of litmus.) colonies were green and transparent.
Sterilization Steam for 20 minutes on 3 suc-
: Paratyphoid colonies were large and the
cessive days. edges turned yellow; coli did not grow.
Use: General culture medium. Variants
Added nutrients: The author added 20.0 g. (a) Harvey used Lemco extract instead
glucose or lactose. of Liebig's extract.
Reference: Percival (1920 pp. 51, 57). (b) Stitt prepared the medium as follows:
(1) Dissolve 20.0 g. of Liebig's extract
1706. Committee A. B. Lead Acetate
S.
and 30.0 g. of agar in 1000.0 cc. of
Extract Agar
water.
Constituents (2) Adjust the reaction to +0.3 if
1. Water 1000.0 cc. necessary.

2. Beef extract 3.0 g. (3) Filter thru cotton.
3. Peptone 30.0 g. (4) Distribute in 150.0 cc. lots in
4. Agar 15.0 g. 250.0 cc. Erlenmeyer flasks.
5. Lead acetate, basic (5) Sterilize, method not given.
519
aqueous Add 3.0% HCl to used malachite

(6) Add 1.0 cc. of a 1 to 1000 (2)
of brilliant green and green agar slants or plates until the

solution
1.0 cc. of a 1.0% solution of picric HCl covers the agar. Stir.
(3) Allow the acid to react for one hour,
acid to each flask.
stirring continually.
(7) Pour into large petri dishes.
Pour the agar on a sieve.
References: Bezan<;on (1920 p. 345), Har- (4)
(5) Wash the pieces of agar with a stream
vey (1921-22 p. 91), Stitt (1923 p. 49).
of water until the wash water is
1708. Klinger's Malachite Green Extract clear.

Agar (6) Soak the agar in water for 24 hours,
changing the water often.
Constituents
2000.0 cc. (7) Place the agar on a sieve at the end
1. Water
20.0 g. of this time and allow to drip free
2. Meat extract (Liebig's)
20.0 g. from water.
3. Peptone (Witte)
10-Og- (8) Liquify the agar particles in stream-
4.NaCl
80 g- ing steam.
5. Agar
(9) To a liter of (8) add 8.0 to 10.0 cc. of
6. Malachite green (Hochst 120)
a 10 0% soda solution, 4.0 cc. of a
Preparation
and 5 to filtered and sterilized solution of
(1) Add 2, 3, 4 1.
hours in streaming 20,0%, peptone and 20.0% meat

(2) Heat for either 3
extract or meat equivalent.
steam, or 2 hours at 110 or 1 hour
(10) Sterilize for one hour in the steamer.
at 120C.
(11) The reaction of the agar determines
(3) Filter thru a thick layer of cotton.
the amount of malachite green to be
(4) Remove 50.0 cc. of the agar and
added. There is an optimum for
measure the rest.
solidify. each batch of agar, and must be
(5) Allow the agar to
determined in each case.
(6) After solidification add 0.05 g. Hochst
References: Klinger (1906 p. 52), Zipfel
120 malachite green to each 100.0 cc.
(1917-18 p. 479).
of agar.
(7) Liquefy the agar. 1709. Kohler's Basal Agar
(8) Determine the reaction. of the 50.0 cc.
Constituents
of agar removed in (4) and add
suffi-
1. Nutrient agar
1000.0 cc.
cient normal NaOH to melted mala-
Preparation: (1) Dissolve one of the added
chite green agar so that the alkalinity
normal nutrients in nutrient agar.
for 100.0 cc. of agar is 1.0 cc. of
using phenolphthalein as an Sterilization: Not specified.
NaOH
indicator.
Use: To study the effect of acids and other
If 0.9 normal cc. are re- materials on the growth of the typhoid
Example:
bacillus. Other investigators used sim-
quired to neutralize the 50,0 cc. agar,
ilar media for a variety of purposes.
then 1.8 cc. would be required for
100.0 cc. H one wishes to adjust the Added nutrients: The author added 0.05,
0.1, 0.15, 2, 0.25, 0.3, 0.35, 0.4, 45, 0.5,
reaction of 400.0 cc. of malachite
0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 95,
green agar add 4 X 0.8 = 3.2 cc.
1.0, 1.5 or 1.1% of one of the
following
normal NaOH.
materials:
(9) Pour into sterile plates.
Not specified. Lactic acid HNO3 (30%)
Sterilization:
Citric acid H2SO4 (97%)
Use: Isolation of typhoid bacilli. The NaOH
H3PO4 (2.0%) (33%)
author reported that other forms were
Tartaric acid KOH (33%)
inhibited.
Alum Methyl violet
Variants: Zipfel regenerated malachite
Carbolic acid Fuchsin
green agar in the following manner:
malachite green agar HCl (25%)
(1) Remove the
Variants
slants and plates from tubes and
(a) Thoinot added any desired amount of
Petri dishes.
sterile lactic, tartaric or hydrochloric given the following method of prepara-

acid to sterile tubes of agar. tion for similar media:
(b) Duval cultivated B. leprae in media (a) Smith (1902).
prepared as follows: (1) Grind 5.0 g. of agar and boil for one
(1) Method of preparation or composi- or two hours in 100.0 cc. of water in
position of 2.0% nutrient agar not a beaker. Add water from time
given. to time.
(2) Adjust to 1.5% alkaline to phenol- (2) Add 250.0 cc. of meat infusion
phthalein. (or extract) peptone solution.
(3) Distribute in 10.0 cc. lots. (3) Boil until the weight of the bouil-
(4) Method of sterilization not given. lon plus 5.0 g. of agar is obtained.
(5) Prepare 2 0% solutions of cystine, (4) Cool to 60C. or less and add one
(made from protein) leucine and half the white of an egg dissolved
tryptophane. in 25.0 cc. of boiled water.
(6) Sterilize (5) by passing thru a (5) Reboil for 10 or 15 minutes to
Berkefeld filter. coagulate the egg albumin.
(7) Add 5.0 cc. of one of (6) or in com- (6) Filter thru a folded and moistened
bination to melted (4). paper.
(8) Mix thoroughly and solidify in a (7) Make up to the weight of the
slanted position. bouillon plus 5.0 g. agar.
(c) Abel mixed one part serum, ascitic (8) If the agar is not clear, reheat
fluid or hydrocele fluid, warmed, to and refilter.
40 to 50C. with one or two parts agar. (9) Distribute.
(d) Tanner added 1.0% of any desired (10) Sterilize intermittently or in the
carbohydrate, alcohol, etc. to plain autoclave.
agar. (b) Roux and Rochaix (1911).
(e) Dopter and Sacquepee added 2.0% (1) Dissolve 15.0 agar in a liter of
g. of
of any desired carbohydrate, alco- peptone bouillon by heating at
hol, etc., to nutrient agar. 100C.
References: Kohler (1893 p. 76), Thoinot (2) Filter thru muslin.
and Masselin (1902 p. 35), Duval (1910 (3) Cool to 70-75C. and add the white
p. 655), Abel (1912 p. 26), Tanner (1919 of an egg. Mix well.
p. 48), Dopter and Sacquepee (1921 (4) Readjust the reaction if necessary.
p. 128). (5) Heat for 45 minutes.
(6) Filter while hot.
1710. Wurtz's Nutrient Agar (7) Distribute in tubes.
(8) Sterilize in the autoclave,
Constituents
(c) Roux and Rochaix (1911).
(1) Soak 25.0 g. of chopped agar for
2. Agar 12.0 g.
24 hours in 500.0 cc. of water,
Preparation
acidulated by the addition of 6.0%
(1) Dissolve 12.0 g. of agar in a liter of
HCI. Stir occasionally.
bouillon, slightly alkaline, by heating
(2) Wash thoroly with water.
in a salt water bath.
(3) Soak the agar for 24 hours in
(2) Cool to 50C. and add the whites of
500.0 cc. of a 5.0% ammonia
two eggs beaten up in a liter of
solution.
water.
(4) Wash thoroly.
(3) Heat minute at 125C.
for one
(5) Place in a liter of bouillon and
(4) Filter thru Chardin filter paper.
heat until dissolved.
(5) Distribute in tubes.
(6) Neutralize by the addition of a
Sterilization: Sterilize at 115C. for
saturated solution of NaHCOs.
15 minutes.
(7) Pass thru flannel and then filter
Use : General culture medium. using a hot water funnel.
Variants: The authors listed below have (8) Distribute into flasks or tubes.
MICROORGANISMS 521
CULTURE MEDIA FOR CULTIVATION OF
for Mix 10.0 cc. of (2) and 90.0 cc. of

Sterilize at 115 to 120C. (3)
(9)
water.
30 minutes,
Dissolve 1.5 g. agar in (3).
(d) Hoffmann (1912) made bacterial (4)
Sterilizeon three successive days
counts of the soil on a 0.1% peptone (5)
for 30 minutes in a steamer.
agar.
(6) Pour in sterile petri dishes.
<e) L6hnis(1913).
(7) Moisten the plant or material
(1) Cut agar into
small pieces.
containing the nematodes and
(2) Add 1.5% of (1) to bouillon.
place on the solidified agar.
(3) Allow to soak for one to two hours
(i) Stitt (1923).
or overnight in a cool place. powdered
Weigh 15.0 to 20.0 g. of
(4) Heat in the autoclave until solu- (1)
agar into a mortar.
tion is complete at 1.5 atmos-
(2) Make a paste by the addition of
pheres.
nutrient bouillon.
Filter thru cotton wool.
(5)
autoclave, (3) When a smooth even mixture is
made pour into the inner com-
(f) Roddy (1917).
partment of a rice cooker, and add
Add 1.0 to 3.0% agar to bouillon.
(1)
the remainder of the 1000.0 cc.
(Shredded agar is used, cut into
nutrient bouillon. Weigh the
small pieces before adding.)
agar is dis- mixture.
<2) Boil and stir until the
(4) Fill the outer compartment of the
solved.
rice cooker with 25.0% NaCl solu-
(3) Add water to make up the loss
of
tion, and boil until the solution
due to evaporation.
agar complete, (5 to 10 minutes
is
boiling). Do not stir.
(5) Filter in a warm
place.
(5) Filter the agar thru a pledget of
autoclave.
absorbent cotton, or cotton be-
<g) Bezangon (1920)
tween two layers of gauze, in a
Wash 15.0 g. of finely chopped agar
funnel that has been heated with
(1)
in water and soak for 12 hours.
boiling water. The filter-stand
Add (1) to bouillon.
with gauze cotton filter and flask
(2)
Place in the autoclave at 120C.
(3)
may be placed in an Arnold steri-
for 15 minutes.
lizer for 20 minutes and then
filter
Cool to 50C.
the agar. (1.5% agar may be
(4)
(5) Adjust the reaction to slightly
filtered thru paper.)
alkaline.
an egg in (6) Tube.
(6) Beat up the white of
autoclave or
50.0 cc. of water.
Arnold.
(7) Add (6) to (5).
Krumwiede
Park, Williams and
(8) Autoclave at 115C. for 20 mmutes. (j)
(1924).
cc.
Tube. (1) Dissolve 1.5% agar in 1000
(10)
the autoclave at
in bouillon by heating in the auto-
(11) Sterilize
115C. for 10 minutes. clave at 10 to 15 pounds for
(1917- 20 minutes, or by boiling over
a
(h) Hilgermann and Weissenberg
cultivated nematodes on the
18)
free flame.
medium. They reported (2) If boiled over a free flame make up
following
the loss in weight by the
addition
that nematodes appeared first after
of water.
4 days or possibly after 10 or 14 days.
Amoeba and bacteria grew at the (3) Adjust the reaction.
end of 24 hours. (4) Cool to 50 C. and add one egg.
composition of nutrient (5) Heat in the autoclave at 10 to
(1) Exact
15 pounds for 30 minutes or Arnold
bouillon not given.
alkaline. sterilizer for one hour. Filter.
(2) Adjust (1) so that it is
(6) Test the reaction and adjust if 1711. Warden's Salt Agar
necessary.
Constituents
(7) If more than 0.2% normal soda is
required per liter, heat again for
2. Bouillon 200.0
10 minutes.
3. Agar 2.5 g
Distribute in tubes or
4. Sodium bicarbonate 0.2 g
(9) flasks.
5. CaCla 0.25 g
(10) Sterilize at 15 pounds pressure for
30 minutes.
6. KCl 0.45 g
(k) Park,
7. NaCl 10.8
Williams and Krumwiede (1924) g,
Preparation
reported a very satisfactory medium
(1) Dissolve 3, 4, 5, 6 and 7 in 1 by aid of
can be made by simply using 0.5% or
less of agar instead of the usual 1.5%
heat until fluid shows a translucent
employed. This can be diluted by
ground glass appearance.
the addition of i of its bulk of an (2) Add bouillon (composition or method
of preparation not specified).
enrichment fluid. The finished
(3) Filter while hot, through gauze or
medium should just "set" suffi-
cotton into test tubes or flasks.
ciently for stab culture purposes.
Nutrose (1.0%) may also be added. (4) Adjustment of reaction not specified.
Sterilization: Sterilize once in autoclave
(1) Park, Williams and Krumwiede (1924)
(time not specified).
gave the following medium as Mus-
Use: Culture medium for gonococci.
grave and Clegg's Agar for the culti-
vation of amoeba:
Author reported that when cool medium
was semi-solid, and had a translucent
(1) Mix 90.0% tap water, 10.0% ordi-
silvery appearance. Nearly all strains
nary nutrient broth (preparation
of gonococci did not grow on this me-
not given) and 1.0% agar.
dium. If a few loopfuls of sterile human
(2) Dissolve.
blood be placed on this surface, all
(3) Reaction neutral to phenol-
strains grew.
phthalein.
Reference: Warden (1913 p. 94).
(4) Sterilize as usual (method not
given).
1712. Stroszner's Regenerated Agar
(m) Cunningham (1924).
(1) Add 1.5% agar to bouillon. Constituents
(2) Steam for 30 minutes to dissolve 1. Bouillon.
the agar. 2. Agar (used).
(3) Boil over an open flame for Preparation:
15 minutes, stirring constantly. (1) Place the used agar in an enamel con-
(4) Adjust the reaction to a slight tainer and melt in streaming steam
alkalinity using turmeric paper (add no water).
as an indicator (distinctly brown). (2) Measure the agar in a graduated
(5) Filter while hot thru a plug of cylinder.
cotton-wool in the bottom of an (3) To each liter of agar add 40.0 g. of
enamelled funnel. powdered charcoal and boil for 30 to
(6) Tube. 40 minutes in the steamer.
(7) Sterilize intermittently in steam. (4) Cool to 50C. and add 40.0 cc. defi-
References: Wurtz (1897 p. 29), Smith brinated blood per liter of agar.
(1902 p. 92), Roux and Rochaix (1911 (5) Boil in streaming steam once more
pp. 115, 116), Hoffmann (1912 p. 387), for 40 minutes.
Lohnis (1913 p. 16), Roddy (1917 p. 43), (6) Filter (method not given).
Bezanôn (1920 p. 112), Hilgermann and (7) Add meat infusion or
300.0 cc. 1 to
Weissenberg (1917-18 p. 470), Klimmer 1.5% Liebig's bouillon to each liter
(1923 p. 229), Stitt
(1923 p. 36), Park, of agar.
Williams and Krumwiede (1924 pp. 117, Sterilization: Method not given.
118, 134), Cunningham (1924 p. 15). Use: Regenerated agar.
MICROORGANISMS 523
223), 1715, Zipfel's Regenerated Drigalski's Agar

References: Stroszner (1917-18 p.
Zipfel (1917-18, p. 477). Constituents

1. Drigalski agar (used).
1713. Besson's Basal Litmus Agar
2. Peptone.
Constituents 3. Meat extract.
1. Nutrient agar
1000.0 cc 4. Kahlbaum's litmus solution.
2. Litmus Preparation
Preparation: (1) Soak used agar slants and plates in
(1) Dissolve
2.0 to 4.0% of one of the a 3.0% HCl solution for one hour.
added nutrients to plain agar. Stir constantly.
use, melt (1) and add a Wash the pieces of agar with water,
(2) Just before (2)
quantity of litmus to give
sufficient and soak in water for 24 hours.
a light blue color. (3) Place in a 1.0%, soda solution until

Sterilization: Not specified. the agar is completely blue.
Use: General culture medium. (4) Wash with water, and allow the agar
Added nutrients: The author added 2.0
to to drain.
4.0% of any desired carbon source. (5) Melt the agar and add to 1 liter,
Variants: Bezanôn gave the
following 8.0 to 10.0 cc. of a 10.0% soda solution,
method of preparation for a similar 40.0 cc. of a filtered sterile 20.0%

medium. peptone solution and 20.0% meat ex-
of any desired carbo- tract or meat equivalent.
(1) Dissolve 1.0 g.
cc. of Kahlbaum's
hydrate in 20.0 cc. of litmus solution. (6) Evaporate 250.0
(2) Heat for 20 minutes at 105C. litmus solution to 50.0 cc.
Melt agar and cool to 30 C. (7) Add 20.0 cc. of (6) to one liter of (5).
(3)
Decant (2) and add 1.5 cc. of the lit-
(8) The addition of lactose or other ma-
(4)
mus sugar solution to 5.0 cc. of agar. not specified.
terials
(5) Mix well. Sterilization:Method not specified.

Bezangon Use: Regenerated medium. This
medium
References: Besson (1920 p. 59),
prepared
(1920 p. 113). gave as good results as freshly
Drigalski agar.
1714. Mandelbaum's Basal Rosolic Acid p. 478).
Reference: Zipfel (1917-18
Agar
1716. Rothberger's Indicator

Agar
Constituents
1- Agar. ,
alcoholic solution).
. , .
Constituents
2. Rosolic acid (1.0%
1. Nutrient agar
Preparation: red, etc.)
2. Indicator (neutral
(1) Dissolve an
appropriate amount of
Preparation
one of the added nutrients in nutrient of preparation of nutrient
(1) Method
agar.
agar not specified.
To each 10.0 cc. of (2) add 0.3 cc. of a neu-
(2)
(2) Prepare a saturated solution of
1.0% solution of rosolic acid.
tral red.
Sterilization: Not specified. (Ij
(3) 10.0 cc. of melted sterile
To each
colon-
Use: To differentiate members of
3 or 4 drops of sterile (2).
sim- add
typhoid group. The author used a sterilization not
Sterilization: Method of
ilar medium to determine
both acid pro-
given.
duction and hemolysis. typhoid and
The author added an Use: Differentiation between
Added nutrients:
colon bacilli.
appropriate amount of any desired carbo-
Variants
hydrate, alcohol, etc. 3 or 4 drops of a
of (a) The author used
Variants: The author added 2.0 cc.
cc.
saturated watery solution per 10.0
human defibrinated blood to 5.0 cc. of
containing of agar of one of the following indi-
nutrient agar (cooled to 48C.)
glu- cators instead of neutral red.
small amounts of glycerol, lactose or
(a) Saffranin.
cose.
(b) Methylene blue.
Reference: Mandelbaum (1909 p. 2476).
524 CULTURE MEDIA FOR CULTIVATION OF
MICROORGANISMS
(c) Water blue.
diphtheriae and Bac. Pseudodiphtheriae.
(d) Berlin blue.
The author reported that coli organisms
(e) Gentian violet. changed the blue agar to yellow after
(f) Methyl violet.
15 to 20 hours. Typhoid organisms
(g) Crystal violet.
decolorized the agar after 3 or 4 days.
(h) Fuchsin. Bac. pseudodiphtheriae grew very
(i) Methyl green, luxu-
riantly with yellow colonies, and
after
(j) Malachite green, 2-3 days caused a decolorization
(k) Iodine green.
nearly
as intense as Bad. coli. Bac. diphtheriae
(1) Guignets-green.
grew less abundantly and did not cause
(m) Acid violet,
decolorization.
(n) Acid fuchsin.
Reference: Omelianski (1903 p. 4).
(o) Phlexin red.
(p) Magdali red. 1718. Burnet and Weissenbach's Lead
(q) Erythrosin.
Acetate Agar
(r) Benzoazurin.
(s) Benzopurpurin. Constituents :
(t) Indulin. 1. Nutrient agar.
(u) Nigrosin. 2. Lead acetate.
(v) Aniline blue, Preparation :
(w) Corallin. (1) Preparation of agar not given.

(x) Noggerath's color mixture. (2) Prepare a 1:10 solution of neutral
(y) Chrysoidin. lead acetate in water.
(z) Ruby S. (3) Add under aseptic conditions one
(aa) Kongo red. drop of freshly prepared sterile (2) to
(bb) Dahlia, each tube containing 4.0 cc. of melted
(cc) Victoria blue, sterile (1).
(dd) Indigo carmine, (4) Mix well.
(ee) Orseille-extract. (5) Slant.
(ff) Tcluidin, Sterilization: Sterilize (1) and sepa-
(2)
(gg) Magenta red. rately; method not given.
(hh) Bismarck Brown, Use: HoS production. Author reported
(b) Hunter added 0.1 to 0.5 cc. of
a con- that the medium is darkened if HoS is
centrated watery solution of neutral formed. Paratyphoid B and typhoid
red to nutrient agar. bacillus formed HoS. Paratyphoid A did
References Rothberger (1898 pp. 515, 517),
: not produce HoS.
(1899 p. 72), Hunter (1901 p. 614). Variants: Thompson prepared a similar
medium as follows:
1717. Omelianski's Indicator Agar (1) Exact composition of Standard 1.5%
Constituents : agar not specified but it is to be
1. Nutrient agar 1000.0 cc. prepared using "Difco" peptone.
2. Methylene blue (1.0% solution) (2) Adjust to pH 6.8 or 7.0.
Preparation: (1) Add 10 drops of a 1:100 (3) Distribute in 100.0 cc. lots in flasks.
solution of methylene blue to agar. Method of sterilization not given.
Sterilization: Not specified. (4) Prepare a 2.0% aqueous solution of
Use: Differentiation of Bad. coli and Bac. Merck's subacetate. Store in a
typhi. Author reported that coli gives tightly stoppered bottle. This solu-
decolorization after 6-7 hours. Typhoid tion may be kept several weeks under
colonies show no decolorization until these conditions.
later. (See variant.) Just before use melt
(5) (3) and add to
Variants: The author added 0.1 cc. of a each 100.0 cc. lot, 20.0 cc. of (4).
sterile 2.0% indigo-carmine solution Distribute into sterile tubes, inocu-
to (6)
10.0 cc. of sterile melted (not hot) agar. late and pour into sterile plates.
The medium was used for the differentia- References : Burnet and Weissenbach (1915
tion of Bad. coli and Bad. typhi, Bac.
p. 567), Thompson (1920-21 p. 384).
525
reported that cholera vibrio colonies

1719. Noeggerath's Indicator Agar
(Besson) were colored violet.
Reference: Krumwiede and Pratt (1913
Constituents: p. 562).
1. Agar
2. :Methylene blue (sat. soln.) ... 2.0 cc. 1722. Meier's Glucose Infusion Agar
3. Gentian violet (sat. soln.) .... 4,0 cc.
Constituents
Methyl green (sat. soln.) 1.0 cc. 1000.0 cc.
4. 1 Water
5. Chrysoidine (sat. soln.) 4.0 cc. 500.0 g.
2. Beef
6. Fuchsin (sat. soln.) 3.0 cc. 150
3. Agar S-
Preparation 4. Peptone 10.0 g.
(1) Prepare saturated
watery solutions 5.0 g.
5. Glucose
violet,
of methylene blue, gentian Preparation
methyl green, chrysoidine and fuchsin. (1) Boil 500.0 g. of fat and tendon free
(2) Mix 2.0 cc. of methylene blue, 4.0 cc. beef in 1 liter of water.
gentian violet, 1.0 cc. methyl green,
(2) Filter,
4.0 cc. of chrysoidine and 3.0 cc. of Dissolve 3, 4 and 5 in (2).
(3)
fuchsin.
(4) Neutralize by the addition of KOH.
(3) Add 200.0 cc. of distilled water to (2) Add KOH until turmeric paper is
and allow to stand several hours. turned quite weakly brownish red.
The color should be greenish blue. Sterilization: Not specified.
If not, obtain the original color
by
Use: Bacterial count of milk. Goss, Bar-
adding either blue, green or red dye. barin and Haines cultivated B. chauvoei
Add 7 to 10 drops of sterile (3) to a
(4) on a similarly prepared medium.
tube of sterile melted nutrient agar. Variants: Goss, Barbarin and Haines pre-
Sterilization: Sterilize (3) at 100C. pared a similar medium as follows:
Use General culture medium. infusion from 500.0 g.
(1) Prepare beef
:
Reference: Besson (1920 p. 60). beef and 1000.0 cc. water. (Exact
method not given.)
1720. Gasser's Fuchsin Agar NaCl and
(2) Add 10.0 g. peptone, 5.0 g.
20.0 g. Dextrose to (1).
Constituents
Nutrient agar. (3) Adjust to pH = 8.2.
1.
2. Fuchsin. (4) Add 2% agar.

(5) Heat in flowing steam until agar is
Preparation
drops of a saturated dissolved.
(1) Add twenty
Clarify with egg albumin.
aqueous solution of fuchsin to a tube (6)
(7) Filter and tube.
of nutrient agar.
petri dishes. (8) Autoclave at 15 pounds pressure for
(2) Pour sterile (1) in
Method not given. 20 minutes.
Sterilization:
References: Meier (1818 p. 436), Goss, Bar-
Use: Differentiation colon-typhoid
of
barin and Haines (1921 p. 615).
group. Author reported the typhoid cul-
ture was red after 24 hours at 37C. while 1723. Kitchens' Glucose Infusion Agar
B. coli communis decolorized the medium.
Constituents
Reference: Gasser (1890 p. 463).
1.
1721. Krumwiede and Pratt's Dahlia Agar 2. Beef, lean 1000.0 g.
3. Peptone 40.0 g.
Constituents:
1. Agar. 4. KNO3 4.0 g.
5. Glucose 4.0 g.
2. Dahlia.
Agar 60.0 g.
Preparation: (1) dahlia in the ratio of
Add 6.
dahlia per Preparation

1 to 100,000 (0.5 cc. of 1.0%
Stir 1000.0 g. of lean beef into
500.0 cc. agar) to nutrient agar. (1)
1000.0 g. of water.
of cholera vibrio. Author (2) Incubate at 37C. for 48 hours.
Use: Detection
(3) Strain and heat in water bath to (5) After warming this mixture in a
boiling and strain again. double boiler and stirring it for a
(4) Dissolve 3 and 4 in (3). few minutes to dissolve ingredients,
(5) Adjust to pH = 7.5. titrate with N/20 sodium hydrate,
(6) Filter and autoclave at 15 pounds using phenolphthalein as an indicator,
pressure for 30 minutes. and neutralize with normal sodium
(7) Dry agar thoroly, weigh and wash in hydrate.
running water over night. (6) Boil vigorously for 30 minutes in a
(8) Dissolve 60.0 g. (7) in 1000.0 cc. of double boiler, and 5 minutes over a
water in the autoclave. free flame with constant stirring to
(9) Adjust to pH = 7.5 and clear by prevent the caramelization of the
straining through cotton and gauze. dextrose.
(10) Add glucose to (6) just before addi- (7) Make up any loss in weight by evapo-
tion of agar solution. ration and filter thru cotton flannel
(11) In making the mixtures the amount and filter paper.
of hot fluid, 6.0% agar, necessary to (8) Tube.
obtain the various percentages, is Sterilization: Sterilize in an autoclave for
diluted with hot distilled water to 15 minutes.
a volume equal to that of the double Use: Cultivation of B. sporogenes and
strength broth (9) also hot, and other bacteria. Medium also used for
mixed. presumptive test for B. coli in water
(12) Check reaction (pH = 7.5). analysis.
Sterilization: Sterilize in autoclave at Variants: Harvey solidified medium 833
15 pounds for 20 minutes. variant (a) by the addition of agar. He
Use: To study growth at different agar reported that when 1.0 acid, B. bifidus
concentrations. The author recom- grew on medium. When 4.0% acid
this
mended a 0.1% agar concentration for B. acidophilus grew on this medium.
growth of aerobic and anaerobic bacteria. References Jackson and Muer (1911 p. 290),
:
Reference: Kitchens (1921 p. 391). (1911 p. 929), Harvey (1921-22 p. 110).
1724. Jackson and Muer's Liver Infusion 1725. Hall's Testicular Infusion Agar
Agar
Constituents
1. Water 1000.0 cc. 2. Testicles, beef 500.0 g.
2. Liver, beef 500.0 g. 3. Peptone (Witte or Difco) . . 20.0 g.
3. Agar 5.0 g. 4. Agar 30.O g.
4. Peptone (Witte's) 10.0 g, 5. Glucose 5.0 g.
5. Glucose 10 g 6. NaH2P04 3 0g.
Preparation Soak over night, 500.0
(1) g. of ground
(1) Chop 500.0 g. beef liver into small beef testicles from which the tunica
pieces, add 500.0 cc. distilled water vaginalis has been stripped, in
and boil slowly for two hours, stir- 1000.0 water
cc. distilled at room
ring occasionally. temperature.
(2) Add 3
(dried at 105C. for 30 minutes) (2) Heat to 50C. Keep warm in 37
to 500.0 cc. distilled water and digest incubator for one hour.
for 30 minutes in an autoclave at Boil strain, and
(3) restore to 1000.0 cc.
120C. (15 pounds). If in excessdo not boil to reduce
(3) After making up the loss by evapora-
volume, overheating is injurious.
tion, strain the liver infusion thru a Add 3, 4, 5 and 6 to (3).
(4)
wire strainer, add 500.0 cc. filtrate to (5) Soak at least an hour to soften the
the agar solution. agar.
(4) To the filtrate add 4 and 5, then (2). (6) Melt in autoclave at 10 pounds for
Weigh the infusion and container. 30 minutes.
527
Mince finely.
(7) Titrate with N/1 NaOH to neutral (2)
(3) Allow to macerate in water over-
point with phenolphthalein.
(8) Check the titre by repetition using ^l^^^' . ^ , ,. .^,
Heat in a water bath with con-
N/20 NaOH-5 cc. should require (4)
stant stirring until the proteins
1 to^2 cc. to display color while
, have been coagulated.
,
(5) Filter thru coarse cloth.
^
autoclave at (6) Make up to volume, 750.0 cc, with
hot distilled water^
10 pounds for 30 minutes.
Cultivation of gonococci. The (7) Add to the fluid, while hot,
Use:
20.0 g. peptone and 3i) g. di-
author reported that media with 1.0%,
tried hydrogen sodium phosphate,
2 0%, 3.0% or 4,0% agar were also
Allow the temperature to fall to
with and without glucose and varying (8)
NaOH. The concentrations 40C.

amounts of , j. , ,,
be the most satis- (9) Estimate and adjust the reaction
given were found to
to pH 7.6.
^
the medium as (10) Add 30.0 g. agar Previously dis-
(a) Clark prepared
solved in 250.0 cc. distilled water
follows-
discard tunica vagi- and finally 5.0 g. glucose,
(1) Remove and
nalis from fresh beef testicles and (H) Mix.
rinse in running water and grind. (12) Distribute in test tubes.
Mix ground testicles (500.0 g.) (13) Sterilize at 120C. _

(2)
with equal weight of distilled (14) Rotate the tubes to mix before
allowing to solidify
water (500.0 cc.) at room tempera-
References Hall (1916 p. 351), Clark (1920
ture, and infuse over night. :
In the morning heat to 50C. for p. 100), Harvey (1921-22 p. 98).

(3)
one hour. Heat in steam kettle or
1726. Kligler's Lead Acetate Infusion Agar
double boiler, stirring, until the
proteins are coagulated and tend constituents:
to collect in large flocculi. Do not
infusion agar (agar
^^ ^^^^^
heat over free flame. ^^ ^^ jq q ^ -^ 1000.0 cc.
(4) Strain thru coarse cloth and, if
^ Glucose 0-1%
necessary, add distilled water to ^- .'.'.'.'."
0.05%
^^^^ acetate!
750 cc
Dissolv; 20^0 g. Peptone (Parke
(5)
^^^^''^f^^^^^ ^^^, i,,^,,i,, ,g,, i, the
Davis) and 3.0 g. NaH.PO. H.O + ^J^^ ^^^_ ^^^ ^ ^^ ^^ ôor, ^^^^^
m warm (4, (40 C. however. Details of method not
(6) Adjust while warm to pH = 7.4 to
7.8. Heat to boiling and adjust be between

The^'Veaction should
more precisely.
dH = 7 2 to 7 6^
(7) Soak 25 to 30.0 g. agar in 250.0 cc. ^^.^^^^^^ ^^^^^^ ^^^
^^^ 05^^
distilled water, and autoclave long
^^^^ ^^^^^^^ .^ ^^^^^ separately.
u f(7)
agar to 60 and
.c^
(S)
T.T^
Add melted m Tto TfiT"
(6).
(4) Cool sterile melted
^^^ ^^^ ^^^^.j^ ^^^^^-^^ ^f gl^.^se
(9) Add 5.0 g- glucose- to the agar under
^ .^ .
^
mto and lead acetate
(10) Mix thoroly and distribute
conditions,
desired containers
^^^^^^
(11) Autoclave at lo pounds for
[gj ^^.^âte to test sterility.

(12) RoTaTe tubes to mix before
rately-method not given
slanting.
(b) Harvev gave the following method of Use: Differentiation of typhoid and para-
preparation: typhoid group. Author reported that
(1) Remove the tunica vaginalis of the stab cultures showed the following
bulls testicles. reactions:
B. typhi browned the medium along the Preparation

line of growth. Add
(1) 2, 3, 4 and 5 to 1.
B. paratyphi B produced browning and (2) Boil well (time not given).
gas. (3) Filter and neutralize.
B. paratyphi A gave gas but no browning. (4) Add 10.0 cc. of 10 0% soda solution to
B. dysenteriae gave neither gas nor make alkaline.
browning. (5) Add 10.0 g. of chemically pure lac-
Variants: Harvey added 4 drops of sterile tose and 5.0 cc. of a filtered alcoholic
30% glucose solution at 45C. and 2 drops fuchsin solution (strength not given).
of a sterile 5.0% lead subacetate at 45C. Add 10,0 cc. of a 10.0% sodium sulfite
(6)
to 10.0 cc. of melted sterile infusion agar solution. This nearly decolorizes the
(see variant (v) 1661). red medium.
The sulfite solution
Reference: Kligler (1917 p. 1043), Harvey must be kept in a well stoppered
(1921-22 p. 107). bottle or prepared fresh.
1727. Gage and Phelps' Neutral Red (7) Distribute in 15.0 cc. lots,
Infusion Agar (8) Store sterile (7) in a dark place until
ready for use.
Constituents
minutes in a
steaming apparatus.
2. Agar 10.0 g.
Use: Diagnosis of typhoid fevers. Author
reported that typhoid colonies were
4. Glucose 3.0 g.
colorless; coli colonies red, and after
5. Neutral red (1.0% watery
24 hours a green shiny fuchsin crystal-
solution) 10.0 cc.
like upper surface appeared on them.
Preparation
Variants: The following authors have pre-
(1) Prepare 1000.0 cc. of meat infusion.
pared the medium as indicated below:
(2) Dissolve 2, 3 and 4 in (1).
(a) Reitz.
(3) Add 10.0 cc. of a 1.0% watery solution
(1) Boil 500.0 g. of finely chopped beef
of neutral red to (2).
with 1 liter water, 10.0 g. Witte's
siccum peptone and 30.0 g. agar.
Use: To study the neutral red reaction by
members (2) Filter and neutralize.
of the colon group. Color
changes from red to yellow (3) Make alkalineby adding 10.0 cc.
if the reaction
of a 10.0% soda solution.
is positive.
Reference: Gage and Phelps (1902 (4) Add 10.0 g. of c. p. lactose and
p. 408).
5.0 cc. of a filtered alcoholic fuch-
1728. Harvey's Malachite Green Infusion sin solution.
Agar (5) Decolorize the red medium by the
Add 10.0 g. glucose and from 16 to 25.0 cc.
addition of 25.0 cc. of a freshly
of a 1-1000 malachite green solution to prepared 10,0% Na2S03 solution.
1000.0 cc. infusion agar, (see variant v (6) Sterilization not specified.
medium 1661) with a reaction of (b) Stroszner prepared a regenerated
0.3% acid
to phenolphthalein. Endo agar as follows:
(1) Place the Endo agar in enamel
1729. Endo's Fuchsin Sulphite Infusion containers and melt in streaming
Agar steam.
Constituents (2) Measure the melted agar in a
1. Water 1000.0 cc. graduated cylinder.
2. Beef 500.0 g. (3) To a liter of (2) add 50.0 g. of
3. Peptone 10.0 g, powdered charcoal and boil for
4. NaCl 5.0 g. 30 to 40 minutes in a steamer
5. Agar 30.0 g. (Stir).
6. Lactose (C. P.) 10.0 g. (4) Cool to 50C. and add 50.0 cc.
7. Fuchsin (alcoholic solution) 5.0 cc. defibrinated blood per liter of
8. Sodium sulfite (10.0% soln.). 25.0 cc. agar at room temperature. Add
blood slowly and stir to avoid the used directly in the preparation of
formation of clumps. agar after neutralization and fil-
(5) Boil in the steamer for 40 minutes. tration by adding 2 or 3% peptone
(6) Filter (method not given). per liter.
(7) Sterilize (method not given). (14) To prepare plain agar from used
(The agar is light red. Endo agar take the slants and
(8) To a liter of agar add 300.0 cc. of plates and treat with a 0.5% NaOH
infusion broth or 1.0 to 1.5% solution after washing (5) until the
Liebig's extract broth, 4.0 g. lac- red color disappears. Wash in
tose, 4,0 cc. alcoholic concentrated water until the agar reacts neutral.
fuchsin solution and 2.0 cc. of a Add peptone and meat extract as
10% NazSOs solution. (Alkali in (9). To completely remove the
need not be added.) pink color add 1.0 cc. of a 1.0%
(c) Zipfel prepared regenerated Endo bismark brown solution per liter
agar as follows: of agar,
(1) Remove the Endo agar slants and (d) Levine gave the following method of
plates from tubes and petri dishes. preparation as Kendal's modification
(2) Add 3 0% HCl to used Endo agar (1) Prepare plain, sugar-free nutrient
slants or plates until the HCl agar, using 15 grams of agar per
covers the agar. Stir. liter.
(3) Allow the acid to react for one (2) Adjust the reaction to a point just
hour, stirring continuously. alkaline to litmus.
(4) Pour the red agar on a sieve. The (3) Flask the agar, 100.0 cc. to a flask,
HCl solution may be saved and and sterilize in the autoclave.
utilized (see end). (4) Prepare a 10.0% solution of basic
(5) Wash agar with a
the pieces of fuchsin in 96.0% alcohol. This
stream of water until the wash solution is fairly stable if kept
water is clear. away from light.
(6) Soak the agar in water for 24 hours, (5) Prepare a 10.0% aqueous solution
changing the water frequently. of chemically pure anhydrous
(7) Place the agar on a sieve at the sodium sulphite (1.0 g. in 10.0 cc.
end of this time and allow to drip water). This solution does not
free from water. keep.
(8) Liquify agar particles in
the (6) Add 1.0 cc. of (4) to 10.0 cc. of (5)
streaming steam. and heat in the Arnold sterilizer
(9) To a liter of (8) add 8 to 10.0 cc. for 20 minutes. The color of the
of a 10% soda solution, 4.0 cc. of fuchsin is nearly discharged if the
a filtered and sterilized solution of solutions are of proper strength.
20.0% peptone and 20.0% meat This solution must be prepared
extract or meat equivalent.
each day it does not keep.
(10) Sterilize for one hour in the (7) Add 1.0 g. of C. P. lactose (free
steamer. from glucose) to 100.0 cc. of agar
(11) To prepare Endo agar add 3.0 cc. and place in the autoclave until
of a concentrated fuchsin solution, melted and the lactose is thoroly
25.0 cc. of a 10.0% NajSOs solution dissolved.
freshly prepared and 8.0 g lactose (8) Add a sufficient volume of (6)
dissolved in a little water, per (about 1.0 cc.) to impart a faint
liter of (10). pink color to the medium.
(12) Pour into sterile plates. (9) Pour into sterile Petri dishes and
(13) To utilize the HCl washings from allow to harden in a dark place
(4), boil and then neutralize with with the covers partly removed.
soda, (charcoal may be
filter When cool the medium should be
added) and concentrate by evap- colorless when viewed from above
oration. The washings may be and a very faint pink when viewed
from the edge. The medium must p. 722), Abel (1912 p. 131), Stroszner
be kept in a dark place because the (1917-18 p. 224), Zipfel (1917-18 p. 477),
color is restored by the action of Levine (1921 p. 114), Harvey (1921-22
daylight. pp. 92, 93).
(e) Harvey.
(1) Add 3.7 cc. of a 10% anhydrous 1730. Wurtz's Litmus Lactose Agar
sodium carbonate solution to
Constituents:
1000.0 cc. of infusion agar (see
1. Infusion Agar 1000.0 cc.
variant (v) medium 1661).
2. Lactose (2.0%) 20.0 g.
(2) Dissolve 2.0 g. lactose in 25.0 cc.
3. Litmus
of distilled water.
Preparation
(3) Dissolve 0.5 g. anhydrous NaoSOs,
(1) Add 2.0% lactose to infusion agar
and saturated alcoholic
1.0 cc. of a
with a slightly alkaline (0.5% re-
basic fuchsin solution in 10.0 cc.
action).
distilled water.
(2) Tint sterile (1) by the addition of a
(4) Mix (2) and (3).
sufficient quantity of sterile litmus
(5) Add (4) to (1).
solution.
(6) Pour in plates.
and the litmus
(7) Dry the surface of the medium
solution separately, method not given.
for 15 minutes in the incubator.
Use: Determine fermentation of lactose.
(f) Harvey.
Add 10.0 cc. of a 10.0% crystalline Variants
(1)
(a) Smith (1905) gave the following
sodium bicarbonate solution to
1000.0 cc. of infusion agar (see method of preparation.
variant (v) medium 1661) neutral (1) Add 10.0 g. of c. p. lactose to

1000.0 cc. of sugar free meat infu-
to litmus.
Prepare an alcoholic fuchsin solu- sion agar.
(2)
Add 20.0 cc. of a saturated watery
tion by adding 1.0 g. basic fuchsin (2)
solution of lime-free blue litmus
to 20.0 cc. absolute alcohol, allow-
ing it to stand 24 hours, and centri- to (1).
Sterilization not specified.
(3)
fuge. (A filtered saturated alco-
same or half (b) Heinemann prepared a similar me-
holic fuchsin in the
the quantity may be used.) dium as follows:
and Add 1.0% lactose to sugar free
(3) Add 10.0 g. c. p. lactose 5.0 cc. (1)
infusion agar.
of (2) to (1).
Add 10.0%, freshly prepared crys- (2) Tube in 8.0 cc. quantities.
(4)
(3) Add 1.0 cc. of 1.0% sterile litmus
talline sodium sulphite 25.
Distribute into test tubes or flasks. solution to each tube before using,
(5)
Sterilize at 115C. (c) Committee A. P. H. A. (1913).
(6)
(7) Keep in the dark until used, (1) Boil 10.0 or 15.0 g. of thread agar
Harvey. in 500.0 cc. of water for half an
(g)
(1) Add 10.0 g. lactose to infusion hour and make up weight to
agar variant (v) medium

(see 500.0 g. or digest for 15 minutes.
1661) with a reaction of 0.5% (2) Infuse 500.0 g. lean meat for
alkaline to phenolphthalein. 24 hours with 500.0 cc. of distilled
(2) Filter (1). wâter in a refrigerator.
(3) Add 5.0 cc. of alcoholic fuchsin (3) Make up lost weight.
solution prepared as indicated in (4) Strain thru cotton flannel.

step (2) variant (d) above. (5) Weigh.
(4) Mix. (6) Add 2.0% Witte's peptone and
(5) Add 10.0% freshly prepared so- warm on the water bath until solu-
dium sulphite 25. tion is complete. Do not heat
(6) Sterilize. above 60C.

References: Endo (1905 p. 109), Reitz (1906 (7) Mix 500.0 cc. (6) and 500.0 cc. of
(1), keeping the temperature below References: Smith (1902 p. 94), (1905,
60C. p. 196), Heineman (1905 p. 127), Com-
(8) Titrate and adjust the reaction to mittee A. P. H. A. (1913 p. 129), Giltner
neutral to phenolphthalein adding (1921 p. 379, 380).
normal HCl or NaOH.
1731. Bitter's China Blue Malachite Green
(9) Heat on a water bath for 40 min-
Agar
utes.
(10) Make up lost weight. Constituents
(11) Readjust to neutrality if neces- 1. Infusion agar (containing
sary and boil 5 minutes. 2.0 or 3.0% peptone and
(12) Restore lost weight. NaCl) 1000.0 cc.
(13) Filter thru absorbent cotton and 2. Lactose 20.0 g.
cotton flannel. 3. China blue (sat. aqueous
(14) Titrate and record final reaction. solution (Hochster)) 90 drops
(15) Add 1.0% lactose and sufficient 4. Malachite green (0.1%
azolitmin solution. solution, crystalline extra
(16) Tube in 10.0 cc. quantities. Hochst.) 25.0 cc.
(17) Sterilize minutes in the
for 15 Preparation
autoclave at 120C. or for 30 min- (1) Prepare meat infusion agar with
utes on each 3 successive days. 2.0 or3.0% peptone and NaCl.
(d) Giltner gave the following methods (2) Neutralize (1) with N/1 NaOH.
of preparation. The medium was (3) Add 2.0% lactose.
used in water analysis. (4) Boil a few minutes.
I. (1) Prepare infusion agar using (5) To each add 9 drops
100.0 cc. of (4)
equal parts meat infusion and of a saturated (about 10%) watery
water, with 1.0% peptone and china blue solution (Hochster Farb-
1.5% agar. werks 10 g. 2M) by means of a 1.0 cc.
(2) Adjust the reaction to -f-1.0. pipette.
(3) Add 1.0% lactose and 2.0% (6) Then add to each 100.0 cc. of (5)
azolitmin just before tubing. 2,5 cc. of a 0.1% malachite green
(4) Tube. (cryst extra Hochst, ) solution.
(5) Sterilize for 30 minutes on 3 (7) Pour sterile (6) in thin layers in
successive days. sterile plates.
II. (1) Preparation of meat infusion Sterilization: Sterilize 10 minutes in the
not given. autoclave.
(2) Strain (1) thru a piece of clean Use: Diagnosis of typhoid fever. Author
cheese cloth. reported that all acid forming organisms
(3) Place 2.0% washed agar in (coli, etc.) gave bright blue colonies.
distilled water. All non-lactose fermenting organisms
(4) Weigh (3). (typhoid) or alkali forming organisms
(5) Digest over a free flame. gave a colorless or yellowish colony.
(6) Add distilled water to make Reference: Bitter (1911 p. 474).
up the loss in weight.
Add lactose 2.0% and peptone 1732. Guth's Alizarine Lactose Agar
(7)
and mix (Klimmer)
2.0% to (6) until solu-
tion is complete. Constituents
(8) Add (2) to (7). 1. Meat infusion (beef or
(9) Adjust the reaction to 0. horse) 1000.0 cc.
(10) Add 2.0% azolitmin solution. 2. Agar 30.0 g.
(11) Boil over a free flame. 3. Peptone 10.0 g.
(12) Distribute in 100.0 cc. lots in 4. NaCl 5.0 g.
250.0 cc. Florence flasks. 5. Lactose 10.0 g.
(13) Sterilization not specified. 6. Alizarin 0.8 g.
Preparation 3. Water blue (6B extra P)

(1) Preparation of horse or beef infusion (1.0%) 175.0 cc.
not given. 4. Lactose 100.0 g.
(2) Dissolve 10.0 g. peptone, 5.0 g. NaCl Preparation
and 30.0 g. agar in 1. (1) Prepare meat infusion agar (yeast
(3) Clarify. may be used instead of meat).

(4) Neutralize to litmus. (2) Neutralize to litmus.
(5) Add 50.0 to 70.0 cc. of tenth normal (3) Prepare a 2.0% solution of meta-
NaOH and 10.0 g. lactose dissolved in chrome yellow (II RD) in water.
20.0 or 30.0 cc. water. (4) Boil two minutes.
(6) Dissolve 0.6 g. NaOH and 0.8 g. ali- (5) Prepare a 1.0% water blue (6B extra
zarine in 100.0 cc. distilled water and P) in solution in water and dissolve
boil several minutes. 100.0 g. lactose in 175.0 cc. of the
(7) Add (6) to (5). solution.
(8) Thoroly mix sterile (7) and pour into (6) Boil (5) 10 minutes.
sterile plates. (7) Add 125.0 cc. of (4) and 175.0 cc. of
Sterilization: Sterilize by steaming for (6) to 2000.0 cc. (2).
30 minutes. Sterilization: Method not given.
Use Detection of typhoid and paratyphoid
: Use: Detection dysentery or typhoid
of
bacteria. Author reported that B. coli bacilli. The author reported that Bad.
lightened the medium coloring it yellow, coli gave a deep blue medium. Typhoid
typhoid bacilli, paratyphoid and enteri- and dysentery gave a yellowish medium.
tidis bacilli left the medium opaque and Medium was green. This medium inhib-
formed greyish blue colonies. The addi- ited neither the typhoid, dysentery nor
tion of malachite green completely in- coli organisms.
hibited the colon bacteria; however, 40 Reference: Gassner (1917-18 p. 221).
to 48 hours were required for the develop-
ment of the typhoid colonies. 1735. Torrey's Brom Cresol Purple Lactose
Variants: Klimmer added 1.7 cc. of a 0.1% Agar
malachite green solution to each 100.0 cc. Constituents
ofmedium. 1. Water 1000.0 cc.
Reference: Klimmer (1923 p. 215). 2. Beef heart 500.0 g.
3. Peptone 10.0 g.
1733. Harvey's Starch Agar 4. Agar 15.0 g.
5. Lactose 1.0 g.
Constituents:
6. Brom Cresol Purple (Sat.
1. Infusion agar 1000.0 cc.
ale. soln.) 0.75 cc.
2. Starch (water soluble) 2.0 g.
Preparation
Preparation
(1) Boil 500.0 g. finely chopped beef
(1) See medium 1661, Variant (v) for the
heart (other meat material may be
preparation of infusion agar.
used) in one liter of water for 15
(2) Add 2.0 g. water soluble starch to
minutes over a free flame
1000.0 cc. of (1).
(2) Strain through canton flannel and
absorbent cotton.
(3) Make up the loss in filtrate and add
10.0 g. peptone and 15.0 g. flaked
agar.
1734. Gassner's Metachrome Yellow Water
(4) Heat in the Arnold until solution is
Blue Infusion Agar
complete.
Constituents: (5) Adjust to pH = 7.4 and place in the
1. Infusion agar 2000.0 cc. Arnold for 30 minutes.
2. Metachrome Yellow (II RD) (6) Readjust the reaction if necessary.
(2.0%) 125.0 cc. (7) Filter thru cotton and add 1.0 g.
lactose and a sufficient quantity of a infusion agar (see variant (v) medium
saturated alcoholic solution of brom 1661). The medium was used to
cresol purple (generally about 0.75 cultivate V. cholerae.
CO.) to give the desired depth of (b) Harvey added 50.0 cc. of glycerol to
color. 1000.0 cc. of infusion agar (see
(8)Flask in 200.0 cc. quantities. variant (v) medium 1661).
Sterilization: Autoclave at 15 pounds pres- Reference: Abbott (1921 p. 132), Harvey
sure for 10 minutes. (1921-22 p. 87).
Use: Analysis of fecal flora.
1737. Kowalski's Glycerol Lung Infusion
Agar
1736. Abbott's Glycerol Infusion Agar
Constituents
1. Water 1000.0 cc. 2. Lung, calf 1000.0 g.
2. Beef 500.0 g. 3. NaCl 18.0 g.
3. Peptone (1.0%) 10.0 g. 4. Potassium phos-
4. NaCl (0.5%) 5.0 g. phate 9.0 g.
5. Agar (1.0 to 1.5%) . . 10.0 to 15.0 g. 5. (NH4)2S04 9.0g.
6. Glycerol (5.0 to 7.0%). 50.0 to 70.0 g. 6. Na2S04 25.0 g.
Preparation: 7. Sug.ar 90.0 g.
(1) Add 500.0 g. of chopped lean beef to 8. Peptone 25.0 g.
1 water and soak for 24 hours,
liter of 9. Agar 40.0 g.
kept at ice box temperature. 10. Glycerol 20.0 to 25.0 g.
(2) Strain thru a coarse towel and press Preparation
until a liter of fluid is obtained. (1) Remove the lung of a calf imme-
(3) Dissolve 10.0 g. (1.0%) peptone, and diately after death. Grind it in a
5.0 g. (0.5%) NaCl. meat grinding machine, keeping as
(4) Dissolve 1.0 to 1.5% agar in (3) by clean as possible during the
boiling in a porcelain lined iron procedure.
vessel. Add 250 to 300.0 cc. water (2) Add two liters of water to 1000.0 g.
and boil until this volume water
of ground (1 )
of finely Place in a glass
.
has evaporated, leaving one liter container and boil.

volume. (3) After a half hour, strain thru a sieve.
(5) Place the vessel in a large dish of Press out the residue, and refilter
cold water. the liquid obtained. It is then added
(6) Stir the agar constantly until cooled to the original filtrate.
to 68 to 70C. (4) Add to the quite clear fluid, 18.0 g.
(7) Add the white of one egg which has NaCl, potassium phosphate,
9.0 g.
been beaten up in about 50.0 cc. of 9.0 g. (NH4)2S04, 25.0 g. Na2S04,
water, (a 10.0% dry albumin solution 90.0 g. sugar, 25.0 g. peptone.
may be used). (5) After complete solution of the mate-
(8) Mix (7) thoroly with (6). rials, add 2.0% agar which has been
(9) Allow to boil slowly for 30 minutes. previously soaked for 24 hours in
Do not allow the volume of the water.
liquid to fall below the liter mark. (6) Boil to dissolve the agar, stirring
(10) Filter thru a heavy folded filter continually.
paper. (7) Neutralize very carefully with equal
(11) Add 5.0 to 7.0% glycerol to (10). portions of KOH
and NaOH.
Sterilization: Sterilize by steam (method (8) Make up to 2500.0 cc. and cool to
not given). 58C.
Use: General culture medium. (9) Add the whites of four hen eggs,
Variants beaten to a foam.
(a) Harvey added 10.0 cc. glycerol, 3.0 (10) Boil a minute, and filter thru a hot
cc. normal NaOH to 87.0 cc. of water funnel.
To the clear, straw colored filtrate, of water and alcohol. Dilute with 20
(11)
whose reaction should be neutral, or parts water.
at the most slightly alkaline, add 8 (2) Compositionof usual infusion broth
to 10.0% glycerol with continual not given. (A 0.5% meat extract

shaking. may be substituted for meat in-
(12) Distribute in test tubes or flasks. fusion.)
Sterilization: After 3 appropriate steriliza- (3) Dissolve 2 and 3 in (2).
tions (exact details not given), the (4) Add200.0cc.of (l)to(3).
medium may be slanted or remain Sterilization: Method not given.
upright. Use: To study decomposition of formic
Use: Cultivation of influenza bacilli, tu- acid. Author reported that after 24
bercle bacilli and other pathogenic hours the agar was colored red near the
organisms. colonies. After several days the whole
Reference: Kowalski (1890, p. 245). plate had turned red.
Reference: Omelianski (1903-04 p. 186).
1738. Henssen's Glycerol Kidney Agar
1741. Harvey's Salicylate Infusion Agar
Constituents
500 1. Infusion agar 1000.0 cc.
2. Kidney g.
3. Agar 15.0 g. 2. Sodium salicylate (1.0<%) 10-0 g-

5. Glycerol 30.0 g. (1) Add 1.0% sodium salicylate to
6. NaCl 5.0 g. Harvey's Infusion Agar (see variant
Preparation (v) of medium 1661).
(1) Prepare kidney infusion in the
same Sterilization: Not specified.
manner as beef infusion (exact Use: Cultivation of molds and torulae.
method not given). Bacterial growth is inhibited.
(2) Dissolve 2, 3, 4, 5 and 6 in (1). Reference: Harvey (1921-22 p. 93).

1742. de Gasperi and Savini's Glucose
Use: Cultivation of pathogenic organisms. Lactose Agar
Reference: Henssen (1895 p. 406).
Constituents
1739. Harvey's Mannitol Infusion Agar 1. Water 1000.0 cc.
2. Beef 500.0 g.
Constituents:
1000.0 cc. 3. Agar 12.0 g.
1. Infusion agar
2. Mannitol 10.0 g.
(v) medium 1661 for 6. Glucose 4.0 g.

(1) See variant
7. Lactose 4.0 g.
(2) Add 1.0 g. mannitol to 100.0 cc. of (1). Preparation
Sterilization: Not specified. (1) Mix 500.0 g. finely chopped lean beef
Use: General culture medium. with 1000.0 cc. of water, and allow to
Reference: Harvey (1921-22 p. 111). stand for 24 hours at room tempera-
ture.
1740. Omelianski's Formate Agar Boil for 15 minutes over a free flame.
(2)
Constituents: (3) Filter thru a double chardin filter
1. Meat infusion 1000.0 cc. paper and press out the residue.
20.0 g. Add chopped agar,
12.0 g. of finely
2. Agar (4)
10.0 g. peptone and 5.0 g.
10.0 g. of Witte's
3. Sodium formate
4. Phenolphthalein solution . . 200.0 cc. of NaCl, and boil over a free flame,
Preparation or at 110C. in the autoclave, until
(1) Prepare a phenolphthalein solution

the agar is dissolved.
by dissolving 0.5 g. phenolphthalein (5) Cool to about 55C. and add 4.0 g. of
in 100.0 cc. of a mixture of equal parts glucose and 4.0 g. lactose and a little
egg albumin stirred up well in a little (5) Adjust to +1 with 2X, NaOH.
water. (6) Heat in Arnold sterilizer for ^ hour.
(6) Add soda solution until the reaction (7) Adjust again to -|-1.
is distinctly alkaline and heat in the (8) Cool to 55C., clear with egg white
autoclave at 120C. for 20 minutes. and filter thru cotton.
(7) When taken from the autoclave pour (9) Flask in 100.0 cc. or 200.0 cc. lots.
immediately upon a Chardin filter (10) When ready for use melt sterile (9),
paper. add 1.0% lactose and 1.0% saccha-
(8) Distribute in about 10-12 cm. layer in rose. (1.0 g. to 100.0 cc. medium of
mm. by 22 cm. test tubes.
a 20 to 22 each sugar.)
Sterilization: Sterilize at 112 to 115C. for (11) Prepare a 3.0% stock solution of
15 to 20 minutes. yellowish eosin in distilled water.
Use: Cultivation of anaerobes. Meier (12) Prepare a 1.0% stock solution of
made bacterial counts from milk and brilliant green in 50.0% alcohol, a
whey in a similar medium. g% solution in distilled water.
Variants : Meier prepared a similar medium (13) Add1.0 cc. of (11) and 1.0 cc. of (12)
as follows: to each 50.0 cc. of medium.
(1) Boil 500.0 g. of fat and tendon-free (14) Shake well.
beef in one liter of water. (15) Pour into sterile Petri dishes.
(2) Filter. Sterilization: Sterilize (9) by heating in the
(3) Dissolve 2.5 g. lactose, 2.5 g. glucose, autoclave at 120C. for 20 minutes.
5.0 g. Witte's peptone, 5.0 g. NaCl Use: Isolation of typhoid bacilli from
and 5.0 g. agar in 1. stools. Author reported that typhoid
(4) Neutralize by the addition of KOH. colonies after 18 hours were grayish in
AddKOH until turmeric paper is color by reflected light. B. coli colonies
turned quite weakly brownish red. were red. In transmitted light typhoid
(5) Sterilization not specified. were colorless and transparent. Liebig's
References: de Gasperi and Savini (1911 p. meat extract cannot be substituted for
248), Meier (1918 p. 454). beef infusion.
References: Teague and Clurman (1916 p.
1743. Teague and Clurman's Eosin Brilliant
651), Tanner (1919 p. 54).
Green Agar
Constituents:
1744. Kan-Ichiro Morishima's Lead
Acetate China Blue Agar
2. Beef, chopped 500.0 g. Constituents
3. Peptone (Witte) 10.0 g. 1. Meat infusion agar (compo-
4. XaCl(C. P.) 5.0 g. sition not given) 2000.0 cc.
5. Agar 15.0 g. 2. Neutral 2.0% solution lead
6. Lactose 10.0 g. acetate in distilled water. . . 50.0 cc.
7. Saccharose 10.0 g. 3. 1.0% solution China blue
8. 3.0% 3'ellowish eosin 4. N sodium hydrate solution
solution 20.0 cc. 5. Lactose 10.0 g.
9. 1.0% brilliant green solu- 6. Glucose 1-0 g.
tion 20.0 cc. Preparation
Preparation (1) Preparation or composition of meat
(1) Soak beef in water in the ice chest infusion agar not given except that
over night. it is to be prepared in the usual way.
(2) Squeeze infusion thru cheese cloth, (2) Clear with egg white.
heat in Arnold sterilizer and pass (3) Titrate while hot to -0.2 to -0.4
thru filter paper. with phenolphthalein.
(3) Add 3, 4 and 5 to warm (2). Rub (4) Prepare 2.0% neutral lead acetate
peptone into paste before adding. in sterile distilled water. Heat for
(4) Heat in autoclave for 30 minutes at 5hour at 100C. in water bath.
120C. (5) Melt (3), and add 5.0 cc. of (4) to
every 100.0 cc. agar. Cool agar to (7) Steam 15 minutes. The mixture
60C. or precipitate will be formed. becomes dark brown and very
(6) Transfer to small test tubes, to a cloudy.
depth of 1.5 cc. (8) Add 50.0 cc. of sterile (2), 50.0 cc.
(7) Stock solution of 1.0% china blue in of sterile (3), 2.5 cc. sterile (4) and
distilled water is prepared. 25.0 cc. of sterile (5) to hot (7).
(8) Add 0.4 cc. N sodium hydrate to (9) Place the flasks in a sloping position
10.0 cc. of (7). Heat on water bath to allow the precipitate formed to
for 10 minutes at 100C. (Color settle.
changes from blue to brown.) (10) Pour plates carefully, avoiding as
(9) added to 100.0 cc. of
1.2 cc. of (8) is far as possible the transference of
(3) reaction -0.2 to -0.4. settled precipitate.
(10) Add 1.0% lactose and 0.1% glucose (11) Place the plates to dry in the in-
to (9) and heat in water bath at cubator with their agar surface
100C. for 10 minutes. downwards.
(11) Cool to 60C. (12) Preserve in the dark for three days
(12) Add to (6) making a second layer in before use.
the tubes. Sterilization: Sterilize (1), (2), (3) and (5)
(13) Incubate over night make stab at 100C.
inoculation. Use : Isolation of V. cholerae.
Use: Differentiation of B. paratyphosus
A, B. paratyphotus B, B. enteritidis and
1746. v. Szaboky's Glycerol Lung Agar
B. coll.
Variants: The author prepared a similar Constituents
medium but used 1.0% inositol and 0.1% 1. Water 2000.0 cc.
arabinose instead of 1.0% glucose. 2. Lung 1000.0 g.
Reference: Kan-Ichiro Morishima (1918 3. Agar 10.0 g.
p. 19). 4. Peptone (Witte) 20.0 g.
5. Glycerol 100.0 g.
1745. Aronson's Fuchsin Sulphite Agar
6. Glucose 10.0 g.
(Harvey)
Preparation
Constituents (1) Boil 1 kilogram of lung with 2 kilo-
1. Infusion agar 1000.0 cc. (Time not specified.)
grams of water.
2. NasCOa (10.0% anhydrous (2) Filter and dissolve 3, 4, 5 and 6 in the
soln.) 60.0 cc.
filtrate.
3. Sucrose (20.0% soln.) 50.0 cc.
(3) Neutralize to litmus.
4. De.xtrin (20.0% soln.) 50.0 cc.
(4) Heat again.
5. Fuchsin (sat. alcoholic Distribute in 20.0 cc. lots into suitable
(5)
soln.) 2.5 cc. wide sterile test tubes.
6. Na.SOa (10.0% soln.) 25.0 cc.
manner
Preparation
by short heating in streaming steam (time
(1) Prepare a 10.0% anhydrous NaaCOs
or number of days not specified).
solution.
Use: Cultivation of tubercle bacilli. The
(2) Prepare a 20.0% sucrose solution.
author reported that colonies developed
(3) Prepare a 20.0% dextrin solution.
in one day when reaction was 0.5% alka-
(4) Prepare a saturated alcoholic fuch-
line to 0.5% acid.
sin solution.
Variants Tubercular lung may be used as
(5) Prepare a 10.0% NaaSOs solution :
(must be freshly prepared). well as a normal lung. This tubercular
Mix 60.0 cc. sterile (1), heated to material must be sterilized on 5 days for
(6)
2 hours each.
45C. and 1000.0 cc. melted sterile
infusion agar (see variant (v) Reference: v. Szaboky (1907 p. 652), Kolle
1661 for preparation), cooled to 45 C. and Wassermann (1912 p. 413).
1747. Hulton-Frankel's Inositol Infusion 4. NaCl 5.0 g.

Agar 5. KH2PO4 5.0 g.
Constituents 6. Sodium citrate 2.0 g.
1. Infusion agar (3.0%) 1000.0 cc,
7. MgS04 2'og'
2. Dextrin 10.0 g.
8. Glucose 5.0 g.
3. Inositol 10.0 g.
9. Glycerol 60.0 g.
4. Litmus (Griibler's) Preparation
Preparation (1) Prepare 1000.0 cc. of beef infusion.
(1) Method of preparation
exact or (2) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in (1).
composition of 3.0% infusion agar not (3) Adjust faintly alkaline to litmus.
given. Sterilization: Sterilize at 12 pounds for 15
(2) Adjust reaction to about +0.7 acid. minutes.

(3) Sterilize in the autoclave (time or Use: General culture medium. The author
pressure not given). reported that 2.0% opsine gave better
(4) Readjust reaction to +0.7 acid. generalmedium than using 1.0%.
(5) Add 2 and 3. (Inositol prepared Medium supported the growth of some
according to Nelson (Jour. Am. pathogenic forms.
Chem. Soc. Variants: The authors used only 1.0%
37: 1552, 1915) and
dextrin, Merck's C. P.) opsine instead of 2.0%.
(6) Add sterile solution of litmus Reference: Robinson and Rettger (1918 p.
(Griibler's) till a light violet color is 214).
obtained.
(7) Tube and slant after final steriliza- 1749. Lubenau's Lactose Caffeine Agar
tion. Constituents
Sterilization: Partial sterilization given in 1. Distilled water 1000.0 cc.
step (3) above. Sterilize (7) once in the 2. Beef 5OO.O g.
Arnold sterilizer as further heating tends 3. Agar (2.0 to 3.0%) . . . 20.0 to 30.0 g.
to break up the carbohydrates. Incubate 4. Peptone 3.0 g.
24 hours and discard tubes showing 5. NaCl (0.5%) 5.0 g.
growth. 6. Lactose (0.5%) 5.0 g.
Use To differentiate between typhoid and
:
7. Caffeine
paratyphoid A and B bacilli. Author 8. Litmus
reported that typhoid bacillus fermented Preparation
dextrin-inositol with acid formation. Boil 500.0 g. of finely chopped lean
(1)
Para-typhoid bacillus B fermented dex- beef with one liter distilled water for
trin-inositol with acid and gas formation. 30 minutes.
Para-typhoid bacillus A did not ferment Filter, and make up to one liter.
(2)
dextrin-inositol. Shiga-Kruse and Hiss- Add
(3) 3, 4, and 5 to (2) and dissolve by
Russel dysentery types did not ferment boiling in a salt water bath.
dextrin-inositol. Flexner Rosen dysen- (4) Neutralize to litmus.
tery types fermented dextrin-inositol.
B. typhi murium and B. pullorum did not
(6) Add 60.0 cc. of sterile litmus solution
ferment dextrin-inositol. B. aerogenes (preparation not given) to hot ster-
fermented dextrin-inositol with gas ile (5).
formation. Mix thoroughly, and allow to cool.
(7)
Reference: Hulton-Frankel (1918 p. 380). Add 110.0 cc. of a sterile 6.0% caffeine
(8)
1748. Robinson and Rettger's Opsine solution to (7). Mix well.
Infusion Agar (9) Pour in plates.

Sterilization: Sterilize (5), method not
Constituents given.
1. Beef infusion 1000.0 cc. Use Detection of typhoid bacteria.
:
2- Agar 15.0 g. Variants: Harvey added 5.0 g. lactose,

3. Opsine (2.0%) 20.0 g. 110.0 cc. of 6.0% caffeine, 60.0 cc. litmus
solution containing 6.0% peptone. The (5) Add 7.0 cc. of normal soda solution
lactose, litmus and caffeine were added to or 10.0 cc. of a 10.0% soda solution.
the hot sterile nutrient agar. (6) Boil.
References: Lubenau (1907 p. 249), (7) Filter.
Harvey (1921-22 p. 92). (8) Add 10.0 g. c.p. lactose, 5.0 cc. of a
concentrated filtered alcoholic
1750. Harvey's Caffeine Endo Agar fuchsin solution and 25.0 cc. of a
Add 33.0cc. of 10.0% caffeine to 1000.0 cc. 10.0% freshly prepared sodium
of Harvey's modification of Endo agar (see sulphite solution.
variant (e) medium 1729). (9) Add 0.33% crystalline caffeine to
(8).
1751. Gathgen's Caffeine Fuchsin Sulphite Klimmer reported that typhoid,
Agar (Bezanson) paratyphoid and cholera colonies
Constituents were blue; colon colonies were red.
1. Infusion agar (3.0%) 1000.0 cc. References: Bezangon (1920 p. 345),
2. Lactose (10.0% solution). . . 120.0 cc. Harvey (1921-22 pp. 92, 93), Klimmer
3. Fuchsin (1923 p. 210).
4. Na.SOa (10.0% soln.) 25.0 cc.
5. Caffeine 3.3 g. 1752. Viehoever's Basal Glucose Extract
Preparation: Agar
(1) Add 120.0 cc. of a 10.0% soda solution
Constituents
to a liter of neutral 3.0% infusion
1. Water 500.0 cc.
agar.
(2) Add 120.0 cc. of a 10.0% lactose
3. Meat extract (Liebig's) 4.0 g.
solution to (1).
4. NaCl 1.0 g.
(3) Add 10.0 g. of crystalline fuchsin to
5. Glucose 50 g.
100.0 cc. of 96.0% alcohol and allow to
6. Agar 10.0 g.
stand for 2 hours.
Preparation
(4) Decant (3) and add 2.5 cc. of the
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
supernatant fluid to sterile (2).
(5) Prepare a 10.0% solution of NazSOa-
in (1) and adjust the reaction as
(6) Add 25.0 cc. of sterile (5) to (4).
indicated.
(7) Add 0.33 g. of pure caffeine to each
100.0 cc. quantity.
(8) Pour in plates.
Use: To study spore production by urea
Sterilization: Sterilize (2) at 100C. for one
splitting organisms, Bac. probatus.
hour. Sterilize (5) by heating at 80C.
Added nutrients: The author prepared one
for one hour.
colon typhoid of the mediums as indicated:
Use: Differentiation of
group.
(a) Neutral basal medium + 0.25%
NasCOs.
Variants
(a) Harvey added 33.0 cc. of a 10.0% (b) Neutral basal medium + 1.0%
caffeine solution to 1000.0 cc. of CaCOa.
Harvey's modification of Endo agar (c) Neutral basal medium + 2.0% urea
(see variant (e) 1729).
+ 0.3% (NH4)2C03.
(b) Klimmer prepared a similar medium Variants: The author gave the following
as follows: variants:
Soak 30.0 to 40.0 g. agar in meat (a) Basal medium with 0.3, 0.2 or 0.1
(1)
water or a 1.0% meat extract solu- concentration of nutrients 0.1% +
tion for several hours. NasCOa.
(2) Boil for 3 hours. (b) Basal medium with 0.3 concentration
(3) Dissolve 10.0 g. peptone and 5.0 g.

of nutrients + 0.05, 0.25 or 1.0%
NaCl in (2).
Na.COs.
(4) Neutralize to litmus. (c) Basal medium with 0.3 concentration
of nutrients + 2.0% or 0.2% urea + (2) Neutralize with concentrated

0.1%NaoCO3. NaaCOs solution.
(d) Basal medium with 0.3 concentration (3) Heat for a time in a sterilizer.
of nutrients + 0.2% (NH4)2C03 + (4) Filter.
0.1% NaaCOs. (5) Soak 16.0 g. of agar in 500.0 cc.

Reference: Viehoever (1913 p. 214). water for about three hours.
(6) Mix (4) and (5).
1753. Bacto Dextrose Agar (Dehydrated) (7) Allow to stand for 3 hours at 100C.
(8) Neutralize once more with Na2C03
Constituents: and heat for a short time.
1. Distilled water
(9) Filter.
Add 10.0 g. glucose and
(10) sterilize
on three successive days.
4. Glucose (Bacto) 10.0 g.
(b) Bredeman cultivated Bac. amylo-
bacter and prepared the medium as
Preparation follows:
(1) Dissolve 33.0 g. of Bacto Dextrose Wash 215.0 g. of agar in running
(1)
Agar (Dehydrated) in 1000.0 cc. tap water for three or four hours.
distilled water by Ijoiling or prefer-
(2) Add water to (1) until the total
ably autoclaving for 10 minutes at 15
weight is 10 kilograms. Dissolve
pounds. the agar in the water.
(2) If sterilized 10 minutes at 15 pounds Clarify with egg white and filter.
(3)
pH = 7.4. (4) Dissolve 7.2 g. Witte's peptone,
6.0 g. dextrose, 4.8 g. Liebig's meat
Use: General culture medium. Add 8.0 g. extract and 1.2 g. NaCl in 200.0
NaCl per liter to prevent hemolysis when cc. of water.
using as a base for blood agar.
(5) Adjust the reaction so that it is
Reference: Digestive Ferments Co., (1925
slightly alkaline.
p. 11).
(7) Liquify400.0g. of (3).

1754. Henneberg's Glucose Extract Agar
(8) Mix and (6).
(7)
Constituents: (9) Sterilize on three successive days
1. Water 1000.0 cc. in streaming steam. (The author
2. Glucose 20.0 to 50.0 g. also preparedthe medium by
3. Meat extract 10.0 g. dissolving the constituents in the
4. Peptone 10.0 g. water and adjusting the reaction
5. NaCl 2.0 g. to a slight alkalinity to litmus.)
6. Agar 10.0 g. (c) Zikes used medium containing 1.0%
Preparation agar, 1.0% peptone, 1.0% meat
(1) Dissolve 2,3, 4, 5 and 6 in 1. extract, 0.5% NaCl and 0.25% glu-
Sterilization: Not specified. cose and cultivated apiculatus yeast,
Use: Cultivation of acetic acid bacteria, Torula alba, Torula Molischiana.
B. pasteurianutn, B. oxijdans, B. aceti, Mycoderma cervisiae. Blastoderma
B. kutzingianum. Other investigators salmonicolor.
cultivated a large variety of organisms on (d) Bachmann cultivated obligate ana-
similar media. erobes using Gottheil's medium, but
Variants used 1.0% peptone instead of 1.2%.
(a) Gottheil cultivated organisms found e) Burckhardt and Enriquez cultivated
in the soil and on the roots and diphtheria bacilli on a medium pre-
rhizomes of plants: pared as follows:
(1) Dissolve 2.0 NaCl, 8.0 g. meat
g. (1) Dissolve 10.0 g. agar, 10.0 g.
extract, and 12.0 g.
Liebig's, peptone, 10.0 g. meat extract, 5.0
Witte's peptone in 500.0 cc. of g. NaCl in 1000.0 cc. of water.
water. (2) Neutralize to litmus.
(3) Add 15.0 g. glucose. Preparation

(4) Add 12.5 cc. of N/1 soda solution. (1) Dissolve 2, 3 and 4 in 1.
(5) Boil for 30 minutes. (2) Add soda solution until a weak alka-
(6) Allow to slowly solidify. line reaction is obtained.
(7) Remove from the container in a (3) Boil one hour and filter.
solid state and cut away the (4) Adjust the reaction once more and
bottom part containing the sedi- add 0.3% agar to (3).
ment. (5) Dissolve the agar by boiling in the
(8) Melt the clear top portion in a autoclave for one hour.
steamer and pour onto a filter. (6) Filter while hot.
(9) Distribute into small test tubes (7) Add 1.0 or 2.0 cc. of a concentrated
(10 mm. in diameter and 12 cm. neutral red solution (exact con-
high). centration not given).
(10) Sterilize two times (method not (8) Dissolve 0.15% glucose in (7).
specified). (9) Distribute into 5.0 cc. lots in test
(f) Robinson and Rettger used a medium tubes.
containing 2.0% opsine, 0.5% NaCl, Sterilization : Sterilize for 15 minutes to two
1.5% agar, 10.0 g. glucose, and 5.0% hours in the autoclave (pressure not
Liebig'smeat extract. specified.)
(g) Dawson studied the variation of B.
Use: Differentiation between coli and ty-
on a medium composed of
coll 2.5 g.
phoid organisms. Author reported that
peptone, 10.0 g. meat extract, 10.0 g. coli types produced decolorization and
glucose and 20.0 g. agar (10.0 g. fluorescens after 24 hours. Typhoid
glycerol may be added). colonies caused no change in color of the
(h) Stitt prepared the medium as follows: medium.
(1) Mix 3.0 g. meat extract, 10.0 g.
References: Oldekop (1904p. 123), Klimmer
peptone, 5.0 g. NaCl, 10.0 g. glu-
(1923 p. 210).
cose and 15.0 g. agar with 1000.0
cc. water containing the whites of
1756. Bacto Purple Lactose Agar
one or two eggs.
(Dehydrated)
(2) Boil in a rice cooker until solution
is complete. Constituents:
(3) Filter. 1. Distilled water
(4) Sterilization not specified. 2. Beef extract (Bacto) 3.0 g.
References: Henneberg (1898 p. 18), Gott- 3. Peptone (Bacto) 5.0 g.
heil (1901 p. 432), Bredeman (1909 p. 4. Agar (Bacto) 10.0 g.
409), Zikes (1911 p. 148), Bachmann 5. Lactose (Bacto) 10.0 g.
(1912-13 p. 7), Burckhardt and Enriquez 6. Dibromcresolsulphonephth-
(1917-18 p. 16), Robinson and Rettger alein 0.025 g.
(1918 p. 202), Dawson (1919 p. 142), Preparation
Stitt (1924 p. 38). Dissolve 29.0 g. of Bacto Purple
(1)
Lactose Agar (Dehydrated) in 1000.0
1755. Oldekop's Neutral Red Glucose
cc. of distilled water by boiling or
Extract Agar
autoclaving.
Constituents necessary.
(2) Restore the loss if
2. Meat extract (Lie-
20 minutes, pH = 6.5.
big's) 5.0 g.
3. NaCl 2.5 g.
4. Peptone (Witte sic- Use: To determine lactose fermentation.
cum) 10.0 g. Acid colonies are yellow, alkali producers
5. Agar 1.5 g. are purple. Author reported that this
6. Neutral red, concen- medium is superior to 1757.
trated solution 1.0 or 2.0 cc. Reference: Digestive Ferments Co. (1925
7. Glucose (0.15%) 0.75 g. p. 12).
1757. Bacto Litmus Lactose Agar Sterilization: Sterilize (6) in the autoclave
(Dehydrated) at 15 pounds pressure (120C.) for 15
minutes.
Constituents:
Use: To determine the fermentation of
1. Distilled water
lactose.
Variants: The following investigators pre-
pared similar media as indicated below:
(a) Meyer.
5. Lactose (Bacto) 10.0 g.
(1) Dissolve 10.0 g. peptone, 10.0 g.
6. Azolitmin 10 g.
meat extract and 30.0 g. agar (3.0%)
Preparation
in 1000.0 cc. of water.
(1) Dissolve 29.0 g. of Bacto Litmus
(2) Distribute in 400.0 cc. portions.
Lactose Agar (Dehydrated) in 1000 not given.
(3) Sterilization
cc. of distilled water by boiling or add 16.0 g.
(4) To each 400.0 cc. lot
autoclaving.
of a sterile 25.0% lactose solution
(2) Restore the loss if necessary.
and 12.0 cc. of a sterile 8.0% litmus
(3) If sterilized at 15 pounds for 20
solution.
minutes pH = 7.0.
(5) Pour into sterile plates and allow to
Sterilization : Sterilize in the usual manner.
harden in open air.
Use: To determine fermentation of lactose.
(b) Bendick (Tanner).
Reference: Digestive Ferments Co. (1925 Dissolve 15.0 g. agar, 5.0 g. beef
(1)
p. 12). extract, 10.0 g. NaCl and 10.0 g.
peptone in 1000.0 cc. of water.
1758. A.P.H.A, Litmus Lactose Extract
(2) Clarify with egg and filter.
Agar (1917) not
(3) Adjustment of reaction is
Constituents: necessary.
1. Distilled water 1000.0 cc. (4) Distribute in 250.0 cc. lots in 500.0
2. Beef extract 3.0 g. cc. flasks.
3. Peptone 5.0 g. (5) Add 1.0 g. CaCOa to each flask.
4. Agar 12.0 g. (6) Sterilize method not given.

5. Lactose (1.0%) 10.0 g. (7) Add 25.0 cc. of Kahlbaum's aqueous
6. Litmus 100.0 cc. litmus solution and 5.0 g. lactose to
Preparation each flask.
peptone Tube by pouring directly into the

(1) Add 3.0 g. beef extract, 5.0 g. (8)
and 12.0 g. agar, dried for 30 minutes tube from the flask, keeping the
at 105C. before weighing, to 1000.0 contents mixed well.
cc. distilled water. (c) Stitt.
Prepare nutrient agar by dissolving
(2) Boil over a water bath until solution (1)
is complete. Make up lost weight. 5.0 g. Liebig's extract, 5.0 g. NaCl,

bath and 10.0 g. peptone and 30.0 g. agar in
(3) Cool to 45C. in a cold water
warm to 65C. in the same bath 1000.0 cc. water.
(2) Adjust the reaction to or +0.2
without stirring.
Restore lost weight, titrate and to phenolphthalein.
(4)
to neutral to Keep this agar base in 100.0 cc.
adjust the reaction (3)
phenolphthalein. quantities in Erlenmeyer flasks.

thru cloth and cotton until (4) Color 100.0 cc. of neutral agar base
(5) Filter
clear. to a lilac color with azolitmin.
Add 5.0 cc. of a hot freshly prepared
(6) Add 1.0% lactose. (5)
20.0% lactose solution in distilled
Add 1.0 cc. of sterilized litmus or water.
(8)
Tube in 10.0 cc. quantities or dis-
azolitmin solution to each 10.0 cc. of (6)
sterile agar just before it is poured tribute in 50.0 or 100.0 cc. Erlen-
into the Petri dish. The mixture meyer flasks.

Sterilize in the autoclave at 10
may occur in the plate itself if desired. (7)
pounds for 15 minutes, or in the (12) Distribute in 200-400 cc. lots and
Arnold, store in the dark.
(d) Conradi-Drigalski (Stitt). (13) Sterilization not specified.
(I) Add 1.0 cc. of a 1 to 100 solution of (b) KastleandElvove.
crystal violet in distilled water to (1) Dissolve 10.0 g. Liebig's extract,
100.0 cc. of the medium as prepared 10.0 g. peptone and 5.0 g. NaCl in
by Stitt above (variant (c)). 1000.0 cc. distilled water by heat.
References: Committee A.P.H.A. (1917 (2) Cool and add 40.0 g. powdered
p. 97), Meyer (1917 p. 238), Tanner (1919 agar. Allow agar to settle.
p. 48), Committee A.P.H.A. (1920 p. 97), (3) Place in Arnold and cook 3 hours.
Stitt (1923 p. 49). (4) Neutralize to litmus with NaoCOs.
(5) Filter thru cotton on a Buchner
1759. Endo's Fuchsin Sulphite Agar
filteror allow to settle, rejecting
(Heinemann)
turbid bottom part.
Constituents Add 10.0 of a sterile
(6) cc. 10.0%
1. Extract agar (3.0%) 1000.0 cc. Na2C03 solution to^(5) after
2. Lactose 10.0 g. filtering.
3. Fuchsin (ale. soln.) 5.0 cc. (7) This medium may be storedTin
4. NaaSOa (10.0% soln.) 25.0 cc. 100-200 or 400.0 cc. flasks until
5. NaOH (10.0% soln.) 10.0 cc. desired for use.
Preparation (8) When desired for use melt (7) and
(1) Prepare a medium from 2, 3 and 4 to each liter add c.p. 10 g. lactose
using 3.0% extract agar as a base. and 5.0 cc. of a 10.0% alcoholic
(2) Add 10.0 cc. of a 10.0% NaOH fuchsin solution (freshly | pre-
solution. pared).
Sterilization: Not specified. (9) Prepare a 10.0% alcoholic fuchsin
Use: Isolation of typhoid bacilli. Coli solutionby shaking 10.0 g fuchsin
colonies large and red, typhoid colonies (not acid fuchsin) with 100.0 cc.
small, colorless and bluish. of 96% alcohol, allow to stand 24
Variants: The following authors have pre- hours. Decant supernatant fluid
pared similar media as indicated: and filter this fluid each time
(a) Klinger. immediately before use.
(1) Add 20.0 g. Liebig's meat extract, (10) After fuchsin and sulphite have
20.0 g. Witte's peptone, 10.0 g. been added to (7), shake vigor-
NaCl and 80.0 g. agar to 1000.0 cc. ously and place unstoppered in
of water. sterilizer for 5 to 10 minutes to
(2) Heat for either 3 hours in stream- allow foam to settle.
ing steam, or 2 hours at 110, or (11) Add 25.0 cc. of a freshly prepared
one hour at 120C. sterile 5.0% anhydrous Na.SO,
(3) Filter thru a thick layer of cbtton. solution to (10) and mix by rotat-
(4) Neutralize to litmus. ing flask.
(5) Add 20.0 cc. of a sterile 10.0% soda (12) Sterilize in Arnold for a few minutes
solution to (4). and pour in Petri dishes while
(6) Add 20.0 g. of chemical pure lac- steaming hot. Solidified medium
tose to (5). nearly colorless to transmitted
(7) Add 10.0 g. of crystalline fuchsin to light and rose or flesh to reflected
100.0 cc. of 96% alcohol and allow light.
to stand for 20 hours. (c) Kendall and Day.
(8) Pour off the saturated solution. (1) "Dust" 15.0 g. powdered agar
(9) Add 10.0 cc. of (8) to (6). upon the surface of cold tap water.
(10) Add 50.0 cc. of a freshly prepared Allow to settle into the medium
10.0% NasSOs solution to (9). before heat is applied or other
(II) INIix well. ingredients added.
peptone and Cut the clear agar in small pieces,

(2) Add 10.0 g. Witte's (8)
3.0 Liebig's meat extract to replace in the beakers and melt

g.
again.
(1) and cook in a double boiler for
one hour. (9) Cool to 55C.
Make faintly alkaline to litmus by (10) Place 18 lots of 10.0 cc. each in an
(3)
cautious addition of NaOH. equal number of sterile test tubes
Cook 15 minutes to set the and divide the tubes in two series.
(4)
reaction. (11) To one series add 0.01, 0.03, 0.05,
0.07, 0.09 and 0.1 cc. of normal
(5) Filter thru absorbent cotton.
Distribute in 100.0 cc. lots in HCl.
(6)
flasks and sterilize in the auto- (12) To the other series add 0.0, 0.01,
clave. 0.03, 0.05, 0.07, 0.09, 0.1, 0.3, 0.5,
Prepare a 10.0% solution of 0.7and 0.9 cc. of 2.5% solution of

(7)
fuchsin in 96. 0% alcohol. NasCOs.
of sodium (13) To each tube of (U) and (12) add
(8) Prepare a 10.0% solution
sulphite in water. 1.0 cc. of a 10.0% solution of
crystalline lactose, 1.0 cc. of a
(9) Add 1.0 cc. of (7) to 10.0 cc. of (8)
and heat in the Arnold sterilizer 2.5% solution of NazSOs, freshly
for 20 minutes.
prepared, and 1.0 cc. of a half
must be saturated alcoholic solution of
(10) Add 1.0%, lactose (which
chemically pure) to (6).
basic fuchsin.
Heat in Arnold sterilizer until the (14) Pour each tube into sterile petri
(11)
agar is melted and the lactose dish and allow to harden.
thoroly distributed in it. (15) Inoculate one-half the surface of

each plate with a 24 hour broth
(12) Add (9) (decolorized fuchsin solu-
tion) in the proportion of 1.0 cc. culture of typhoid bacilli, and the
to each 100.0 cc. of the medium. other half of each plate with a
Mix thoroly and pour into sterile similar coli culture.
(13)
plates. (16) Incubate for 24 hours.
Allow to harden (with covers (17) The plate showing most typical
(14)
removed) in the incubator for 30 luxuriant growth of both the coli
minutes. The plates are now and typhoid is chosen as the
ready for inoculation. proper amount of acid or alkali
Kinyoun and Deiter. to add to the remainder of the
(d)
Mix 80.0 g. Witte's peptone, 40.0 medium.
(1)
powdered (18) Add the necessary amount of acid
g. NaCl and 160.0 g.
agar with sufficient water to make or alkali to the entire lot of agar
a smooth paste. (9).
Add suflBcient water to make 8000.0 (19) Distribute in 100.0 cc. lots m
(2)
cc.
(20) Sterilize in the Arnold for an hour
(3) Add 80.0 g. Liebig's meat extract
(not necessary to mix). on two successive days.
Place in the Arnold sterilizer and (21) Store until ready for use.
(4)
steam until solution is complete. (22) When ready for use add 1.0 g.
crystalline lactose to each flask of
Usually requires about an hour.
Cool to 55C. and titrate to +1 melted agar.
(5)
using phenolphthalein as an (23) When the lactose has dissolved
indicator. add 5.0 cc. of a 5.0% solution of
Place in tall beakers, steam an anhydrous NaaSOs, freshly pre-
(6)
hour and allow to solidify. (Allow pared and still hot to each flask.
Add 1.0 cc. of a half saturated
the beakers to stay in the steamer (24)
alcoholic solution of basic fuchsin
over night.)
Remove the solid agar from the to each flask.
(7)
beaker and cut away all the agar (25) Mix well.
containing sediment.
(e) Committee A.P.H.A. (1913). (4) Adjust the reaction. Best results
(1) Add 30.0 g. of agar to one liter of are obtained when the medium is
cold water by sifting successive just slightly alkaline to litmus.
small portions upon the surface (5) Add exactly 3.0 cc. of a saturated
and allowing them to settle. alcoholic fuchsin solution and 30.0
(2) Add 10.0 g. Witte's peptone, 5.0 cc. of a 10.0% solution of anhy-
g. meat extract to (1) and boil drous Na2S03 (or 3.0 cc. of a satu-
until solution is complete. rated aqueous solution of Na2S03
(3) Neutralize to litmus by adding may be added).
NasCOs. (6) Tube.
(4) Distribute in 100.0 cc. quantities. (7) Sterilize for 20 minutes on each of
(5) Prepare a 10.0% solution of 3 successive days in an Arnold
Na2S03 in water. sterilizer.
(6) Prepare a 10.0% basic fuchsin (h) Committee A.P.H.A. (1917-1920).
solution in 96% alcohol. (1) Add beef extract, 10.0 g.
5.0 g.
(7) Add 2.0 cc. of (6) to 10.0 cc. of (5) peptone and 30.0 g. agar, dried at
and steam for a few minutes in 105C. for one hour before weigh-
the Arnold steamer. ing to 1000.0 cc. of distilled water.
(8) Add 1.0 g. chemically pure lactose (2) Boil on a water bath until all the
to each 100.0 cc. of sterile (4). agar is dissolved and then make
(9) Melt (8) and add 0.5 cc. of (7). up the water lost by evaporation.
(10) Pour in plates. (3) Cool to 45C. in a cold bath, then
(11) Sterilize (4) for 2 hours in stream- warm to 65 C. in the same bath
ing steam. without stirring.
(f Robinson and Rettger. (4) Make up lost weight.
(1)Dissolve 25.0 g. powdered agar, (5) Titrate and if the reaction is not
10.0 g. Peptone (American brand), already between neutral and +1,
and 5.0 g. Liebig's meat extract in adjust to neutral.
1000.0 cc. water. (6) Filter thru cloth and cotton until
(2) Make neutral to litmus. clear.
(3) Steam in the autoclave at 12 to 15 (7) Distribute in 100.0 cc. (or larger)
pounds extra pressure for 35 to 40 quantities in flasks.
minutes. (8) Sterilize in the autoclave at 15
(4) Filter thru absorbent cotton and pounds pressure (120C.) for 15
cheese cloth. minutes after the pressure reaches
(5) Add NajCOs and heat for 10 15 pounds.
minutes on boiling water bath. (9) Prepare a 10.0% solution of basic
Reaction 0.1 to phenolphthalein. fuchsin in 95.0% alcohol, allow to
(6) Add 10.0 g. lactose and 5.0 cc. of a stand 20 hours, decant and filter
saturated alcoholic solution of the supernatant fluid. This is a
fuchsin to hot liquid. The me- stock solution.
dium is now brilliant red. (10) When ready for use prepare a
(7) Add 10.0 cc. of a 10.0% anhydrous 10.0% anhydrous sodium sulphite.
sodium bisulphite solution. (11) To each 10.0 cc. of (8) add 2.0 cc.
(8) Distribute into large test tubes in of (7) and steam for 5 minutes in
20.0 cc. lots. the Arnold or water bath.
5 to 7 minutes at 10
(9) Sterilize for (12) To each 100.0 cc. of agar, add 1.0
pounds extra pressure. g. of lactose, and dissolve in
(g) Roddy. streaming steam or on a water
(1) Prepare a 3.0% extract agar. bath and 0.5 cc. of (9).
(2) Liquify (1) in the Arnold steam (13) Pour into Petri dishes and allow
sterilizer. to harden thoroly in the incubator
(3) Dissolve 10.0 g. lactose in (2). before use.
(i) Krumwiede, Kohn, Kuttner and (3) Distribute in 100.0 cc. quantities
Schumm. in flasks.
(1) Dissolve 25.0 g. agar, 10.0 g. pep- (4) Prepare a 10.0% basic fuchsin
tone and 5.0 g. meat extract in solution in 96% alcohol.
1000.0 cc. water by heating over a (5) Prepare a 10.0% solution of chem-
gas stove. ically pure anhydrous sodium
Adjust reaction neutral to litmus sulphite (1.0 g. in 10.0 cc. water).
(2)
using 10.0% Na2C03 solution. (6) Add 1.0 cc. of (4) to 10.0 cc. of (5),
Add 10.0 cc. of 10.0% NajCOa. freshly prepared.

(3)
(4) Cool to 45C. and add one egg. (7) Heat in the Arnold sterilizer for 20
Autoclave for 30 minutes at 15 minutes.

(5)
pounds pressure. (8) Add 1.0 g. lactose to 100.0 cc. of
and add 10.0 lactose (3)and place in the autoclave until

(6) Filter g.
(boil to dissolve). melted and the lactose is dissolved.

(7) Add 10.0 cc. of 10.0% Na2C03 (9) Add about 1.0 cc. of (7) to each
solution, giving a reaction of 100.0 cc. flask.
-0.3 to phenolphthalein (hot (10) This should give a pink color.

titration). (11) Pour into sterile Petri dishes.
Distribute in bottles in 100.0 cc. (1) Bezangon.

(8)
lots. (1) Add 120.0 cc. of a 10.0% soda
Autoclave for 10 minutes at 10 solution to a liter of neutral 3.0%
(9)
pounds pressure. extract agar.
(10) To melted (9) just before use add (2) Add 120.0 cc. of a 10.0% lactose
0.5 a saturated alcoholic
cc. of solution to (1).
solution of fuchsin and 1.0 cc. of a (3) Sterilize at 100 for one hour.
10.0% solution of amorphous so- (4) Add 10.0 g. of crystalline fuchsin
dium bisulphite (per 100.0 cc.?). to 100.0 cc. of 96% alcohol and
Tanner (Hygienic Laboratory). allow to stand for 2 hours.
(j)
Add 3.7 cc. of a 10.0% anhydrous (5) Decant (4) and add 2.5 cc. of the
(1)
Na2C03 solution to 1000.0 cc. of a supernatant fluid to (3).
3.0% extract agar with a reaction (6) Sterilize a 10.0% solution of
of +0.5 to phenolphthalein. Na2SO3at80for one hour.

(2) Flask in 200.0 cc. quantities, (7) Add25.0cc.of (6)to(5).
Dissolve 2.0 g. of chemically pure (8) Pour in plates,
(3)
lactose in 25.0 to 30.0 cc. of dis- (m) Giltner.
tilled water with the aid of gentle (1) Prepare an ordinary agar from
heat. 5.0 g. Liebig's meat extract, 5.0 g.
Dissolve 0.5 g. anhydrous sodium NaCl, 10.0 g. peptone, 30.0 g. agar

(4)
sulphite in 10 to 15.0 cc. distilled and 1000.0 cc. water.
water. (2) Adjust the reaction to or +0.2%.

(3) Store in 100.0 cc. quantities in
(5) Add a saturated solution
1.0 cc. of
Erlenmeyer flasks.
of basic fuchsin in 95.0% alcohol
(4) Melt a flask of (3), and add 6 drops
to (4).
of a saturated alcoholic solution
(6) Add (5) to (3).
of basic fuchsin (4.0 g. fuchsin to
(7) Add (6) to 200.0 cc. of melted agar.
100.0 cc. of 95% alcohol).
(5) Mix well.
(9) When hard, dry for 15 minutes in (6) Add about 18 to 20 drops of a
the incubator. freshly prepared 10.0% solution of
(k) Kendall (Tanner). NazSOa.
(1) Prepare plain sugar free extract (7) Add 5.0 cc. of a freshly hot 20.0%
agar using 15.0 g. agar per liter. aqueous solution of lactose to the
(2) Adjust to slightly alkaline to hot agar.
litmus. (8) Pour into plates.
(n) Savage. (4) The medium must be stored in the

(1) Peptone 10.0 g., Liebig's extract of dark, since light gradually turns
beef, 10.0 g., sodium chlorid 5.0 g., it red. When solidified it is almost
are boiled up in an enamalled dish free from color,
with one liter of distilled water. (o) U. S. A. Med. Dept.
The mixture is then poured into a (1) Into a container put 1 liter of tap
flask, 30.0 powdered agar
g. of water, marking the level of the
added, and the whole heated in the fluid. Add 30.0 g. thread agar,
autoclave at 115C. for one hour. 10.0 g. peptone, 5.0 g. NaCl, 5.0
The flask is removed, and after g. beef extract. Cook until dis-
cooling to about GO'^C, the white solvedit is best to autoclave 30
of one egg mixed with a little minutes at 15 pounds; filter thru
distilled water is added. The sterile gauze or cotton. If neces-
contents are coagulated by heating sary clear with egg. For this
in current steam in the usual way, purpose, for each liter beat up the
filtered, and the filtrate made up to white of one egg with 10.0 cc. of
1 liter. The mixture is made warm water until the egg is well
neutral, litmus paper being used mi.xed. Add this to agar cooled to
as the indicator. Then 19.0 cc. 55C., mix thoroly, heat for 30
of normal sodium carbonate solu- minutes or autoclave and filter
tion and 10.0 g. c.p. lactose are thru cotton.
added. The flask is replaced for (2) This stock agar is kept on hand in
30 minutes in the steam sterilizer. quarter-liter flasks or bottles.
Almost invariably there is a con- Agar is standardized just before
siderable precipitate and the mix- use and reaction adjusted to 0.2%
ture has to be again filtered. acid to phenolphthalein. Before
(2) Seven cc. of the fuchsin solution use, fuchsin and sodium sulphite
(see below) are added, followed by are added. A filtered, saturated
25.0 cc. of a quite freshly prepared solution of basic fuchsin in 95%
10,0% sodium sulphite solution. alcohol kept on hand. A
is 10.0%
The mixture becomes much less solution of dry sodium sulphite
red, but is not immediately crystals in sterile water is freshly
decolorized. It is then distributed made.
conveniently into small flasks, (3) Teague has shown that a 10.0%
each containing 50.0 to 60.0 cc. of solution of crystalline sodium
media, and sterilized in current sulphite can be heated for 20
steam for 2 days, 30 minutes each minutes at 15 pounds pressure with
day. practically no change, and that the
(3) The fuchsin solution is made as 10.0% sodium sulphite solution
follows: 3.0 g.powdered crystal- covered with a layer of liquid
line fuchsin are placed in a dry petroleum about one cm. thick
flask, and 60.0 cc. of absolute and sterilized in the autoclave
alcohol are added. The contents can be kept at room temperature
are thoroly mixed, and the flask for 3 weeks and probably much
tightly stoppered, allowed to stand longer with but very slight change.
for exactly 24 hours at 20 to 22C. One and
(4) eight-tenths cc. of fuch-
The alcoholic extract is then sin solution is added per liter to
decanted and preserved in a clean the agar. After this has been
glass-stoppered bottle. Made in done the sodium sulphite solution
this way a uniform fuchsin extract is added gradually until the hot
is obtained which keeps well, agar is almost decolorized usually
and the same quantity of fuchsin about 25.0 cc. to the liter. A pale
is added each time a fresh batch of
rose color should be present in the
medium is prepared; a matter of hot agar which fades to a very
much importance. faint pink on cooling; 10.0 g. of
lactose is dissolved in a little (7) Mixthoroly.

water, filtered and added to each (8) Pour into plates.
liter. [t) Kligler and Defandorfer (Park, Wil-

Various fuchsin solutions may- liams and Krumwiede).
differand the absolute quantities (1) Prepare a beef extract agar. The
given above may not be exactly reaction should be from pH = 7.6
the proper balance in separate to 7.8.
lots. These are aproximate, (2) Sterilize (1) in 100.0 cc. quantities
however, and the proper balance (method not given).
can easily be attained by a little (3) When desired for use add 1.0 g. of
preliminary testing in which so- lactose to each bottle.

dium sulphite solutionis added to (4) Melt the agar. This sterilizes the
small quantities of fuchsin solu- lactose.
tion in a test tube. (5) Add 1.0 cc. of a saturated alco-
The finished product is poured holic solution of fuchsin to 10.0
into large sterile Petri dishes. cc. of a freshly prepared solution

The cover is left off until the agar of sodium bisulphite.
is hard. Smears are made on these (6) Heat in the Arnold sterilizer for
plates. 30 minutes.
It is helpful to lay a piece of (7) Add 0.5 cc. of (6) to each bottle of
filter paper into the lid of the melted agar.

petri plate in order to absorb (8) Pour in plates and allow to harden
liquid evaporating from the agar without the covers.
in the incubator. If there is (9) Dry in the incubator for 30
not enough filter paper for this, minutes, protecting the plates
the plate should be placed upside from dust.
down in the incubator, (s) Committee A.P.H.A. (1925).
(p) Committee A.P.H.A.
(1923) Same (1) Add 5.0 g. beef extract, 10.0 g.
as for 1917-1920 (variant (h) above) peptone and 30.0 g. agar (undried
but adjust the reaction to an alka- market product) to 1000.0 cc. dis-
linity slightly higher than required tilled water.
instead of to +1.0. (2) Boil until the agar is dissolved.
(q) Stitt. This may
be done over a free
(1) Prepare nutrient agar using 5.0 g. flame with constant stirring or
Liebig's meat extract, 5.0 g. NaCl, place the autoclave, in a
in
10.0 g. peptone and 30.0 g. agar walled container, and
straight
per 1000.0 cc. water. (Method autoclave at 15 pounds pressure
not given.) for 15 minutes. Shut off the steam
Adjust the reaction to or +0.2
(2) and allow the agar to stand in the
to phenolphthalein. autoclave until it has solidified.
(3) Keep this agar base in 100.0 cc. Then dump the solidified agar on a
quantities inErlenmeyer flasks. clean paper and cut away the
(4) When ready for use melt the agar, sediment. Cut the agar into small
and while liquid add 6 drops satu- pieces and melt. If the agar is
rated alcoholic solution of basic dissolved over a free flame, it is
fuchsin to each flask. necessary to filter thru cotton or
(5) Then add about 20 drops of a cloth.
freshly prepared 10.0% solution
(3) Adjust the reaction so that the
of sodium sulphite. This de- pH is between 7.8 and 8.2.
colorizes the intense red color to a in
(4) Distribute in 100.0 cc. lots
light rose pink.
flasks.
(6) Add 5.0 cc. of a freshly prepared
(5) Sterilize in the autoclave at 15
hot aqueous 20.0% solution of
pounds pressure for 15 minutes.
chemically pure lactose to each
flask. (6) Prepare a 10.0% solution of basic
fuchsin in 95% alcohol and allow (6) Mix (5) thoroly.

to stand 24 hours. (7) Pour into sterile plates, about 8.0 cc.
(7) Decant and filter the supernatant per plate, and allow to solidify with
liquid. cover removed.
(8) Add to each 100.0 cc, of sterile Sterilization : Not specified.
melted (5), 1.0% of chemically Use: Diagnosis of cholera. Authors re-
pure lactose in sterile solution 0.5 ported that by transmitted and reflected
cc. of (7), and 0.125 g. anhydrous light after 10 hours the cholera colonies
Na2S03, dissolved in a small were blue, and about 1.5 mm. in diameter.
amount of hot distilled water. The blue color of cholera colonies was
Mix thoroly. much darker than that of typhoid colo-
(9) Pour in plates. nies. After 20 hours the colonies were
References: Heinemann (1905 p. 130), about 2 mm. in diameter. By reflected
Klinger (1906 p. 52), Kastle and Elvove light they appeared milk blue, and by
(1909 p. 622), Kendall and Day (1911-12 transmitted light a deep i'ky blue. The
p. 96), Kinyoun and Deiter (1912 p. 979), entire plate remained intensively blue
Committee A.P.H.A. (1913 p. 133), (1917 with a pure culture. Bad. colt colonies,
p. 97), (1920 p. 97), (1923 p. 96), (1925 p. both by transmitted and reflected light,
98), Robinson and Rettger (1916 p. 367), were red.
Roddy (1917 p. 43), Krumwiede, Kohn, Reference: Hirschbruch and Schwer (1903
Kuttner and Schumm (1918 p. 286), p. 587).
Tanner (1919 p. 53), Ball (1919 p. 82),
1761. Chesney's Indicator Lactose Agar
Bezangon (1920 p. 342), Giltner (1921 pp.
384, 385, 387), Levine (1921 pp. 112, 113, Constituents :
115), Abbott (1921 p. 524), Pitfield (1922 p. 1. Beef extract agar

120), Klimmer (1923 p. 209), Stitt (1923 p. (3.0%) 1000.0 cc.
48), Park, Williams and Krumwiede 2. Lactose 10.0 g.
(1924 pp. 127, 131). 3. Brom-cresol purple
(0.04%) 50.0 to 80.0 cc.
1760. Hirschbruck and Schwer's Crystal Preparation :
Violet Litmus Lactose Agar E.xact method of preparation or

(1)
Constituents :
composition of 3.0% beef extract agar
1. Water 1000.0 cc. not given.
2. Agar 20.0 g. (2) Adjust (1) to pH of 7.2 to 7.4.
3. Meat extract, Liebig's 10.0 g. (3) Add 1.0% lactose from a sterile 20.0%
4. Peptone 10.0 g. solution to sterile (2).
5. NaCl 5.0 g. (4) Add from 5.0 to 8.0 cc. of a sterile
6. Litmus solution, Kubel and 0.05% aqueous solution of brom cresol

Tiemann 130.0 cc. purple, to every 100.0 cc. of sterile
7. Crystal violet B (Hochst) (3).
0.1% 10.0 cc. (5) Pour into sterile Petri dishes, about
Preparation :
20.0 cc. to each plate.
(1) Boil 2, 3, 4 and 5 in 1 for 1.5 hour. Sterilization: Method of sterilization of
(2) Filter and boil again for 30 minutes. agar or lactose solution not given.
(3) Add 15.0 g. lactose to (2) and boil Sterilize the 0.04% brom cresol purple
for 15 minutes in the autoclave.
(4) Add a sterile soda solution (any Use: Isolation of typhoid and dysentery
desired strength, but not too strong) bacilli from stools. Author reported that
until red litmus paper is turned blue. medium was clear and deep blue in color.
(5) Add 130-0 Kubel-Tiemann's
cc. of Lactose fermenters produced greenish
litmus solution, and 10.0 cc. of a yellow colonies with a yellow zone. Non-
crystal violet solution that has been lactose fermenters produced bluish colo-
prepared by dissolving 0.1 g. Hochst nies. Typhoid colonies have typical
crystal violet in hot sterile distilled woolly appearance. In thickly seeded
water to (4) plates, typhoid colonies were bluish
green, while colon colonies were brilliant (4) Readjust the reaction if necessary.
yellow. The addition of brilliant green (5) Allow to cool to 55 or 60C.
did not interfere with the color produc- (6) Beat the white of an egg in 100.0 cc.
tion of the non-lactose fermenters. of water and add to (5)
Variants: The author prepared a similar (7) Mix well.
medium as follows: (8) Autoclave at 120C. for one hour.
(1) Prepare a 3.0% beef extract agar and (9) Add 5.0 g. of sucrose to (8).
adjust to pH = 7.6 to 7.8. (10) Tube.
(2) Clear (1) with white of an egg. Sterilization: Sterilize at 115C. for 20
(3) Sterilization not mentioned. minutes.
(4) Add 1.0% lactose from a sterile 20.0% Use: General culture medium.
solution. Reference: Besson (1920 p. 43).
(5) Add for every 100.0 cc. of the medium,
10.0 cc. of an 0.04% aqueous solution 1763. Hesse's Starch Extract Agar (Stokes
of phenol red. This solution may be and Hachtel)
sterilized by autoclaving.
Constituents:
(6) Pour into sterile Petri dishes, about
20.0 cc. of agar to the plate.
2. Agar 5.5 g.
The author reported that the medium was
3. Beef extract (Liebig's) 5.0 g.
originally salmon pink or old rose. Lac-
4. Peptone (Witte's) 10.0 g.
tose fermenters produced vivid greenish
5. Starch 10.0 g.
yellow colonies with a surrounding zone
6. NaCl 8.5 g.
of green. The typhoid bacillus, the
7. Azolitmin solution (Kahl-
paratyphoid bacillus (A and B) and the
baum's)
dysentery bacilli (Shiga, Flexner and
Hiss Y) all produced pink colonies. In Preparation
very thickly seeded plates the colon (1) Dry agar at 105C. for 30 minutes.
(2) Dissolve 5.5 g. of (1) in 500.0 cc.
colonies assumed a bright green or yellow
distilled water.
green color and are opaque, whereas the
typhoid colonies were more translucent (3) Add 5.0 g. beef extract to 500.0 cc.
and possessed a bluish green color. of distilled water and dissolve by

heat.
Typhoid colonies had a corrugated,
(4) Filter into a sterile flask and
(3)
woolly appearance. The addition of
brilliant green did not interfere with the
inoculate with the colon bacillus
color production of the non-lactose
and incubate at 37C. for 24 hours.
(5) Filter the sugar free bouillon and
fermenters.
dissolve 10.0 g. Witte's peptone,
Reference: Chesney (1922 pp. 183, 184).
10.0 g. starch and 8.5 g. NaCl in it.
1762. Salomonsen's Sucrose Extract Agar (6) Make up the loss due to evaporation
(Besson) by the addition of distilled water.
Constituents: (7) Mix (6) and (2) and boil for 30
minutes, making up the loss in
1. Water 1000.0 cc.
2. Meat extract (Liebig's) .... 5
weight by the addition of distilled
g.
water.
3. Peptone 30.0 g.
(8) Filter.
4. NaCl 5.0 g.
5. Agar 20.0 g. (9) Adjust the reaction to neutral.
(10) Color with Kahlbaum's azolitmin
6. Sucrose 5.0 g.
solution.
Preparation:
Dissolve 4 in 1.
(1) 2, 3,
chopped thread agar (12) Store in the ice box until ready for
(2) Soak 20.0 g. of
use.
in water for several hours.
cold
Squeeze water thru a cloth. Sterilization: Autoclave at 16 pounds pres-
(3) Heat (2) in (1) at 100C. until the sure for 20 minutes.
agar is dissolved. Use: Differentiation of typhoid bacillus.
dysentery bacillus, cholera spirillum and Preparation

other intestinal forms. (1) Dissolve 2, 3 and 4 in 1 by heating on
Reference: Stokes and Hachtel (1913 p. the salt water bath.

(2) Neutralize to phenolphthalein using
10.0% soda.
1764. Committee S.A.B. Starch Extract Add 18.0 to 20.0 g. of agar to boiling
(3)
Agar
(2).
Constituents (4) Clarify (Method not given).

1. Distilled water 1000.0 cc. (5) Tube in about 10.0 cc. lots.
2. Water sol. starch 2.0 g. (6) Add by means of a sterile pipette
3. Beef extract 3.0 g. graduated in tenths, 0.5 cc. of a 10.0%
4. Peptone 5.0 g. sterile citric acid solution to each
5. Agar 15.0 g. tube of melted sterile (5) cooled to

Preparation 50C.
(1) Prepare beef extract agar according (7) Mix thoroly and slant.
to Committee A.P.H.A. (1916-1917). Sterilization: Method not specified.
(See extract agar 1694). May be Use: Isolation of molds and fungi, Mucor,
clarified with white of egg. Rhizopus, Penicillium, Aspergillus and
(2) Adjust to pH = 6.6 to 7.4. Scelerotinia. Authors reported that it
(3) Add starch. may be necessary to add 8 or even 10.0 g.
Method not given.
Sterilization: of citric acid to keep down bacterial
Use: Standard culture medium. growth.
References: Committee S. A. B. (1918 p. Reference: Piettre and de Souza (1922 p.
116), (1923 p. 10), Ball (1919 p. 78). 336).
1765. Coplin and Bevan's Glycerol Extract 1767. Fawcus' Dye Bile Salt Agar
Agar (Bezanfon)
1. Water 1000.0 cc.
1. Water 900.0 cc.
2. Agar 30.0 g.
3. Peptone (albumin) 10.0 g.
3. Meat extract, Liebig's 20.0 g.
4. Glycerol 62.0 g.
4. Peptone (10.0% soln.) 100.0 cc.
5. Agar (1.5%) 15.0 g.
5. Brilliant green (1 1000) : 10.0 cc.
Preparation 6. Picric acid (1:100) 10.0 cc.
(1) Dissolve 2, 3, 4 and 5 in 1. 7. NaCl 20.0 g.
(2) Clear and filter in the same way as 8. Sodium taurocholate 5.0 g.
other media. (Method not given) Preparation
(1) Dissolve 2, 3, 4, 7 and 8 in 1.
Use: Cultivation of Micrococcus pyogenes (2) Make alkaline (Indicator not speci-
aureus. Authors reported that 3 or 4 fied).
days after inoculation the medium To 1.5 liters of agar add 10.0 cc. of
(3)
became opaque, and after 5 or 6 days the 1-1000 brilliant green and 10.0 cc. of a
medium appeared as if tapioca was mixed 1-100 picric acid solution.
with it. Sterilization: Method not given.
Reference: Coplin and Bevan (1892 p. 70). Use: Cultivation of colon typhoid group.
1766. Piettre and de Souza's Citric Acid Reference: Bezangon (1920 p. 345).
Extract Agar
1768. Percival's Urea Extract Agar
Constituents:
2. Meat extract 0.7 g. 1. Water 1000.0 cc.
3. Peptone (Chapoteaut) 10.0 g. 2. Beef extract (Lemco's) 5.0 g.
4. NaCl 5.0 g. 3. Peptone (Witte's) 10.0 g.
5. Citric acid 5.0 g. 4. Agar 15.0 g.
6. Agar 18 to 20.0 g. 5. Urea 20.0 g.

Preparation 1770. Wilson and Darling's Brilliant Green

(1) Dissolve 2, 3, 4 and 5 in 1. Bile Salt Agar
(2) If acid, neutralize with (NH4)2C03.
Constituents
Sterilization : Sterilize in the usual manner.
1. Water 1000.0 cc.
(Method not given).
2. Lemco 10.0 g.
Use: Cultivation of organisms using urea.
3. Agar 30.0 g.
Urobacillus Pasteurii. Author reported
4. Peptone 20.0 g.
that urea fermenting colonies were sur-
5. Sodium taurocholate 5.0 g.
rounded with small white crystals of
6. NaCl 5.0 g.
calcium carbonate.
7. Lactose 5.0 g.
8. Brilliant green (1.0%
solution) 4.0 cc.
1769. Gaehtegen's Caffeine Endo Agar Preparation
Constituents: (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
1. Water 1000 cc. (2) Adjust the reaction to a +10 (Eyre)
2. Meat extract (Liebig's) 20.0 g. (3) Distribute in 100.0 cc. quantities.
3. Peptone (Witte siccum) 20.0 g. (4) Add 0.4 cc. of a 1.0% solution of
4. NaCl 10.0 g.
brilliant green to each 100.0 cc. of
5. Agar 80.0 g.
melted medium, cooled to 50C. when
6. Lactose 20.0 g.
ready to pour in plates.
7. Fuchsin (10.0% alcoholic Sterilization: Not specified.
solution 10.0 cc. Use: Cultivation of colon typhoid group.

8. Sodium sulphite (in 50.0 cc.
Authors reported that the reaction must
water) 5.0 g.
be +10 (Eyre). If more acid B. coli will
9. Caffeine 6.6 g.
grow. If more alkaline B. typhosus is
inhibited. Some B. coli may grow.

Preparation
These produce acid and allow for the
(1) Add2, 3, 4and5tol.
development of others.
(2) Place in the autoclave at 110C. for
Reference: Wilson and Darling (1918 p.
two hours.
105).
(3) When agar is completely dissolved,
filter thru cotton.
Add N/1 NaOH until it is 1.5% 1771. Olszewski and Kohler's Endo Bile
(4)
alkaline to phenolphthalein. Salt Agar
(5) Add 20.0 g. lactose, 10.0 cc. of a 10.0%
Constituents:
alcoholic fuchsin solution and a
1. Water 2000.0 cc.
sodium sulphite solution (5.0 g. of 2. Agar 60.0 g.
sodium sulphite in 50.0 cc. water). 3. Meat extract 20.0 g.
(6) Dissolve 0.33% chemically pure 4. Peptone 20.0 g.
crystalline caffeine in (5).
5. NaCl 10.0 g.
(7) Pour into sterile Petri dishes.
6. Sodium taurocholate 2.0 g.
Sterilization: Not specified. 7. Lactose 20.0 g.
Use: Diagnosis of typhoid fever. After 24 8. Fuchsin (alcoholic) 10.0 cc.
hours transparent typhoid colonies ap- 9. Sodium sulphite (10.0%).... 50.0 cc.
peared about 1 to 1.5 mm. in diameter. Preparation
After 30 hours the colonies were 2 to 3 (1) Soak 60.0 g. agar over night in
mm. in diameter, round, plate form, 1000.0 cc. of water.
completely colorless, by transmitted (2) Boil for two to two and a half hours.
light, a delicate pink in reflected light. (3) Add 3, 4 and 5 to 1000.0 cc. of water
B. coli colonies, if any, were red. and boil an hour.
Caffeine inhibited B. coli. (4) Filter.
Reference: Gaehtegens (1905 p. 636). (5) Mix (4) and (2).
(6) Neutralize with 10.0% soda solution. Preparation

(7) Boil one hour. (1) Dry agar at 150C. for 30 minutes.
(8) Filter and sterilize. (Method not (2) Dissolve 6.0 g. of (1) in 500.0 cc.
given). distilledwater by boiling and make
(9) Add 10.0 cc. of 10.0% soda solution up the loss in weight by adding dis-
and 1.0 g. of sodium taurocholate tilled water.
to one liter of (8). Dissolve
(3) 3, 4, 5, 6 and 7 in 500.0 cc.
(10) Heat at 100C. in the steamer for of distilled water.
20 minutes. Mix and
(4) (3) (2) and boil 30 minutes.
(11) Add 10.0 g. lactose, 5.0 cc. of a (5) Make up the loss in weight by the
saturated alcoholic solution of fuch- addition of distilled water.
sin, and 25.0 cc. of a 10.0% sodium
(6) Filter.
sulphite solution to each liter of (7) Adjust the reaction to neutrality
(10). (indicator not specified).
(12) Distribute in small flasks. (8) Add sufficient azolitmin to give the
Sterilization: Method of sterilization not desired color.
specified. (9) Tube in 9.0 cc. lots.
Use: Water analysis. Enrichment medium Sterilization: Autoclave at 15 pounds pres-
for Bacterium coli. sure for 20 minutes.
Reference Olszewski and Kohler (1923 p. 3)
:
Use: Isolation of typhoid bacillus and
cholera spirillum. The author reported
1772. Bacto Saccharose-Mannitol Agar
that typhoid colonies were pink. They
(Dehydrated)
had an opaque nucleus surrounded by a
Constituents translucent area. Bacillus alcaligenes
1. Distilled water formed a blue concentric colony. Other
2. Beef extract (Bacto) 3.0 g. motile organisms produced a blue colony.
3. Peptone (Bacto 5.0 g. Variants:The authors prepared a similar
4. Agar (Bacto) 25.0 g. medium as follows:
5.Sucrose (Bacto) 10.0 g. (1) Dry agar at 150C. for 30 minutes.
6. Mannitol (Bacto) 1.0 g. (2) Dissolve 5.5 g. of (1) in 500.0 cc. of
7. Andrade Indicator (Difco) . 0.0275 g. distilled water.
Preparation Add 5.0 g. beef extract to 500.0 cc.
(3)
(1) Dissolve 44.0 Bacto Saccharose-
g. of of distilled water.
Mannitol Agar (Dehydrated) in (4) Filter (3) into a sterile flask and
1000.0 cc. of distilled water by boiling inoculate with the colon bacillus and
or autoclaving, preferably the latter. incubate at 37C. for 24 hours.
(2) Tube. (5) Filter the sugar-free bouillon and
(3) Final pH = 7.5. dissolve 10.0 g. Witte's peptone,
Sterilization Sterilize in the usual manner,
:
10.0 g. lactose and 8.5 g. NaCl in it.
avoiding excessive heat. (6) Make up the loss due to evaporation
Use: Differential tube medium. by addition of distilled water.
Reference: Digestive Ferments Co. (1925 (7) Mix and (2) and boil for 30
(6)
p. 12). minutes, making up the loss in
weight by the addition of distilled
1773. Hesse's Lactose Glycerol Aâr
water.
(Stokes and Hachtel)
(8) Filter.
Constituents (9) Adjust the reaction to neutral.
1. Distilled water 1000.0 cc. (10) Add 50.0 cc. of glycerol.
2. Agar 6.0 g. (11) Color with Kahlbaum's azolitmin
3. Beef extract (Liebig's) 5.0 g. solution.
4. Peptone (Witte's) 10.0 g. (12) Distribute in 10.0 cc. lots.
5. NaCl 8.5 g. (13) Autoclave at 16 lbs. pressure for 20
6. Lactose (1.0%) 10.0 g. minutes.
7. Glycerol (5.0%) 50.0 g. (14) Store in the ice box until ready for
8. Azolitmin use.
Dilutions of a bile culture of a typhoid Sterilization: Sterilize in the usual manner.

suspect was incubated for 24 hours and a Use: Differentiation of Esch. coli, S.
series of made. These were
dilutions paratyphi, S. schotmuelleri, Ebert typhi
then plated on the medium and incubated and Ebert dysenteriae.
for 24 hours. Typhoid bacillus or para- Reference: Digestive Ferments Co. (1925
typhoid bacillus colonies were medium p. 12).
sized, with concentric rings and showed a
distinct pink color. The pink color en- 1776. Nichols and Woods' Russel's Double
abled one to distinguish them from Sugar Agar
Bacillus typhosus or Bacillus alcaligenes.
Constituents:
Colon colonies were small, moist and Extract agar (3.0%) .".
1000.0
1. cc.
concentrated. The spirillum of cholera
Lactose
2. (1.0%) 10.0 g.
and Nassik spirillum formed a blue
Glucose
3. (0.1%) 1.0 g.
colony. Colonies were bluish, concentric
4. Phenol red (0.02% soln.)
with an opaque nucleus-like center and 50.0 cc.
(5.0%)
varying diameters from 5 to 15 milli- Preparation
meters. Prepare
(1) extract agar containing 3.0%
Reference: Stokes and Hachtel (1909 p. shred agar.
41), (1913 p. 346). Clear
(2) (1).
1774. Schniirer's Saponin Glycerol Agar (3) Add 1.0% lactose 0.1% glucose and
5.0% of a 0.02% watery solution of
Constituents:
phenol red.
1. Beef extract agar
pH 7.2-7.4 hot.
(4) Correct the reaction to
(3.0%) 1000.0 cc.
Use: To study fermentation or respiration
3. Saponin (Merck's) 10.0 to 80.0 g.
by the typhoid bacillus.
. .
Preparation
Reference Nichols and Wood (1922 p. 322).
:
(1) Prepare a beef extract agar 3.0%

(contains 1.0% peptone and 0.5%
1777. Bailey and Lacey's Phenol Red Lead
NaCl).
Acetate Agar
(2) Add 3.0% glycerol and 1.0 to 8.0%
saponin to (1). Constituents:
Sterilization: Not specified. 1. Tap water 1000.0 cc.
Use: To study acid fastness of tubercle 2. Beef extract (Bacto) 5.0 g.
bacilli and timothy bacilli. Author re- 3. Peptone (P. D.) 10.0 g.
ported that acid fastness was not changed 4. NaCl (B. & A.) 5.0 g.
by the presence of saponin in medium. 5. Agar 15.0 g.
Reference: Schniirer (1922-23 p. 15). 6. Lactose 10.0 g.
7. Glucose 10 g.
1775. Bacto Russell Double Sugar Agar Phenol red (0.02%) 50.0 cc.
8.
(Dehydrated)
9. Lead acetate 0.5 g.
1. Distilled water (1) Wash the agar in running water.
2. Peptone (Bacto) 10.0 g (2) Heat in 1000.0 cc. tap water until
3. Agar (Bacto) 15.0 g dissolved.
4. Lactose (Bacto) 10.0 g (3) Dissolve 2, 3 and 4 in (2).
5. Glucose (Bacto) 1.0 g (4) Adjust to pH = 7.4.
6. NaCl 5.0 g (5) Boil for 5 to 8 minutes.

7. Andrade Indicator 0.025 g (6) Readjust the reaction to pH = 7.4.
Preparation (7) Allow to stand and decant the clear
(1) Dissolve 42.0 g. of Bacto Russell supernatant agar.
Double Sugar Agar (Dehydrated) in (8) Coolto50C. (to prevent fiocculation)
1000.0 cc. distilled water by boiling or and add 6, 7, 8 and 9.
autoclaving. Avoid excess heat. (9) Tube.

(2) Tube. Sterilization: Sterilize in the autoclave by
heating at 15 pounds pressure for 15 6. Lactose 10. g.

minutes. 7. Glucose 1.0 g.
Use: Differentiation of colon-typhoid-dy- 8. Brilliant green (0.1%
sentery group. solution) 2.0 or 3.0 cc.
Reference: Bailey and Lacy (1927 p. 185). 9. Andrade Indicator. . . 10.0 cc.
1778. Holt, Harris and Teague's Eosine Preparation

Methylene Blue Agar (1) Dissolve 2, 3, 4 and 5 in 1 in the
autoclave.
Constituents
(2) Adjust so that the reaction is slightly
1. Water 1000.0 cc. alkaline to litmus.
2. Agar 15.0 g. Distribute in 100.0 cc. lots.
(3)
3. Peptone (Witte's) 10.0 g. Before use readjust so that
(4) the
4. NaCl 5.0 g. medium be set to Andrade Indicator.
5. Beef extract (Liebig's) 5.0 g. The reaction to phenolphthalein
6. Sucrose 5.0 g, (hot titration) is 0.6 to 0.7% acid.
7. Lactose 5.0 g. Add Andrade
(5) indicator (amount not
8. Eosin (2.0% yellowish) 20.0 cc. given).
9. Methylene blue (0.5%) 20.0 cc. (6) Add 1.0% lactose and 0.1% glucose to
Preparation
sterile (5), using sterile 25.0%
solutions.
(2) Clear with egg white.
(7) Add appropriate amounts of 0.1%
(3) Flask.
solution of brilliant green (0.2 cc. or
(4) Adjust sterile (3) to -fO.8.
0.3 cc. of a 0.1% solution per 100.0
(5) To melted (4) add 6 and 7 and heat cc. agar).
for 10 minutes in the Arnold.
(8) Pour in sterile Petri dishes.
(6) To every 60.0 cc. of the medium add Allow plates to stand open
(9) until
1.0 cc. of 2.0% yellowish eosin in surface is dry. Inoculate as with
distilled water and then 1.0 cc. of Endo plates.
0.5% methylene blue in distilled
Sterilization: Sterilize (3) in the autoclave.
water
Use: Isolation of typhoid bacilli. Author
(7) Mix thoroughly, and pour in sterile
reported that fecal types were restrained.
Petri dishes.
Typhoid colonies were large and pre-
(8) Dry the surface in usual way and
sented a snow flaky appearance with
inoculate.
black background by light passing
Sterilization: Sterilize in Arnold on three
obliquely thru agar. Medium gave 36.0%
successive days.
Use: Isolation of Bacillus typhosus from
positive increase over Endo agar.
stools. Author reported that typhoid Variants

colonies were colorless and transparent. (a) Krumwiede, Kohn, Kuttner and
Colon colonies were black and did not Schumm prepared the medium as
transmit light. Sugar-free infusion may follows:
be substituted for Liebig's meat extract. (1) Dissolve 15.0 g. agar in 500.0 cc.
References: Holt-Harris and Teague (1916 of water by heating over gas
stove.
p. 597), Stitt (1923 p. 49), Park, Williams
and Krumwiede (1924 p. 128). (2) Dissolve 3.0 g. of Liebig's extract
peptone and 5.0 g.
of beef, 10.0 g.
1778a. Krumwiede, Pratt and McWilliams NaCl in 500.0 cc. of 1 by heating
Brilliant Green Agar over gas stove.
Constituents (3) After complete solution mix (1)
1. Water 1000.0 cc. and (2).
2. Agar 15.0 g. (4) Adjust to 0.6 or 0.7% acid to
3. Salt (NaCl) 5.0 g. phenolphthalein (neutral to And-
4. Peptone (Witte) 10.0 g. rade indicator).
5. Beef extract (Lie- (5) Prepare Andrade indicator by
big's) 3.0 g. adding 16.0 cc. of a N/1 NaOH
555
To determine the amount of dye

solution to 100.0 cc, 0.5% aqueous (13)
to add prepare four dilutions of
solution of acid fuchsin.
dye: 1 to 500,000, 1 to 330,000, 1 to
(6) Cool (4) and add beaten egg white.
250,000 and 1 which
to 200,000,
100.0 cc. lots in corresponds to 0.2, 0.3, 0.4 and 0.5
(8) Distribute in
cc. of a 0.1% solution of dye to
bottles.
100.0 cc. Inoculate these plates
(9) Autoclave for 30 minutes at 15
with a freshly isolated strain of
pounds pressure.
melt (9) and B. tj/phosus or a positive stool.
(10) When ready for use
Choose the dilutions of dye that
add to each 100.0 cc, 1.0 cc. of
(1) at which the typhoid
colonies
(5), 1.0 g. lactose (or 5.0 cc. of
sterilized in the are of good size and undiminished
20.0% solution,
glucose (or 5.0 cc. in number compared with the
Arnold), 0.1 g.
solution, sterilized in
control plate, but many other fecal
of a 2.0%
Arnold sterilizer), and 0.5, types are excluded, (2) a lower
the
of a 0.1%, stock dilution where the typhoid colo-
0.3 or 0.2 cc.
water of nies are reduced in size and num-
solution in distilled
but almost all the other
ber,
brilliant green.
the medium as flora have disappeared.
<b) Kligler prepared
indicated above. The reaction was References: Krumwiede, Pratt and Mc-
adjusted between pH 7.0 and 7.2. Williams (1916 p. 4), Krumwiede, Kohn,
When adding the brilliant green (see Kuttner and Schumm (1918 p. 275),
Kligler (1918 p. 320), Giltner (1921 p.
step (7) above) add 0.25 cc. of a 1.0%
solution of neutral red to each 100.0 366), Park, Williams and Krumwiede
cc. of agar. (1924 p. 128).
<c) Park, Williams and Krumwiede pre-

pared the medium as follows: 1779. Aronson's Fuchsin Sulphite Agar
in 1000.0 cc.
(1) Dissolve 30.0 g. of agar
Constituents
of water in the autoclave.
1. Water 1000.0 cc.
(2) Dissolve 6.0 g. meat extract, 10.0
2. Agar 35.0 g.
g. NaCl and peptone in
20.0 g.
1000.0 cc. of water by heating in
4. Peptone 100 g.
the Arnold.
5. NaCl 5.0 g.
(3) Mi.x (1) and (2).
6. Na2C03 (10 0%) 60.0 cc.
(4) Add normal soda so that the final 50.0 cc.
7. Sucrose (20.0%)
reaction will be neutral to Andrade
8. Glucose (20.0%) 50.0 cc.
indicator (pH 6.9).
9. Fuchsin (saturated) 4.0 cc.
10. Sodium sulfite (10.0%) .... 20.0 cc.
(6) Cool and clear with egg.
Preparation
(7) Filter until clear.
(1) Add 2 to 1 and allow to soak over
(8) Bottle in 100.0 cc. quantities.
night.
Autoclave.
(9)
agar Add 3, 4 and 5 to (1) and boil in
<10) When ready for use melt the
(2)
streaming steam for 4 to 5 hours.
and to each 100.0 cc. add 1.0 cc. of
5.0 cc. of a Reaction slightly acid.
Andrade indicator,
water (3) Slant and remove the insoluble
sterile solution in distilled
material.
of20.0% lactose and 2 0% glucose,
Distribute in 100.0 cc. lots by means
(1.0% lactose and 0.1% glucose) (4)
of a graduated flask into sterile
and the proper amount of a 0.1%
brilliant green in 200-250.0 cc. Erlenmeyer flasks.
solution of
(5) Prepare a 10.0%
solution of Na2C03
distilled water. (See (13)).
(siccum).
(11) Mix well.
Pour thick plates. Use porous (6) Prepare a 20.0% solution of sucrose.
(12)
Prepare a 20.0% solution of glucose.
tops as the plates must be dry. (7)
(8) Prepare an alcoholic saturated solu- 1780, Hesse's Malachite Green Agar
by pulverizing com-
tion of fuchsin (Klimmer)
mercial fuchsin in a mortar and
Constituents
adding absolute alcohol and placing
1. Water 1000.0 cc.
in the incubator for a day shaking
2. Peptone 10.0 g.
occasionally,
3. NaCl 5.0 g.
(9) Prepare a 10.0% solution of sodium
sulphite. Boil several times to
5. Agar 35.0 g.
sterilize.
6. NazCOa (10.0%)
(10) To 100.0 cc. of liquid and sterile (4)
soln.) 30.0 to 40.0 cc.
add 6.0 cc. of sterile (5).
7. Sucrose (20.0%)
(11) Heat in streaming steam for 15 min-
soln.) 50.0 cc,
utes. The agar becomes dark brown
8. Dextrin (20.0%
and turbid.
soln.) 50.0 cc.
(12) Add 5.0 cc. sterile (6), 5.0 cc. sterile
9. Malachite green
(7),0.4 cc. (8) and 2.0 cc. (9) to each
(chlorzinc salt c.p.
100.0 cc. lot immediately after it is
sat. ale. soln.) 4 cc.
removed from the steamer.
10. Na-^SOs (10.0%
(13) Slant the flasks so that the precipi-
soln.) 25.0 cc.
tate settles quickly to the bottom.
Preparation
(14) Pour into plates, but leaving the
(1) Soak 35.0 g. of agar in a liter of water
settled precipitate in the flasks.
over night.
(15) Dry in the incubator or at 50C. for
(2) Add 2, 3 and 4 in (1).
30 minutes.
(3) Boil for 4 or 5 hours in the steamer.
(16) The plates are transparent and
(4) Distribute in 100.0 cc. lots in 200.0
yellowish brown in color.
cc. flasks.
Sterilization: Sterilize (5), (6) and (7) by
(5) Add 3.0 to 4.0 cc. of a 10.0% water
heating in steam for 30 minutes.
free soda solution, 5.0 cc. of a 20.0%
Use: Diagnosis of Author re-
cholera.
sucrose solution, 5.0 cc. of a 20.0%
ported that cholera organisms gave large
dextrin solution, 0.4 cc. of a con-
red colonies. Other organisms were
centrated alcoholic solution of mala-
inhibited. The more alkali added, the
chite green (chlorzinc, double salt,
more coli colonies were inhibited.
c.p.) and 2.5 cc. of a
crystalline,
Variants : Klimmer prepared the medium as
10.0% NajSOg solution to each 100.0
follows
cc. of agar.
(1) Soak 35.0 g. agar in a liter of water Sterilization: Not specified.
over night.
Use: Cholera diagnosis. Author reported
(2) Add 10.0 g. peptone, 5.0 g. NaCl and that cholera colonies were green after 24
10.0 g. meat extract to (1).
to 48 hours.
(3) Boil for 4 or 5 hours in the steamer. Reference: Klimmer (1923 p. 219).
(4) Distribute in 100.0 cc. lots in 200.0
cc. flasks. 1781, Bacto Krumwiede Triple Sugar Agar
(5) Add 6.0 cc. of a 10.0% solution of (Dehydrated)
water free soda, 5.0 cc. of a 20.0%
Constituents:
sucrose solution, 5.0 cc. of a 20.0%
1. Distilled water
dextrin solution, 0.25 cc. of a satu-
rated alcoholic fuchsin solution and
2.5 cc. of a 10.0% NasSOa solution to 4. Lactose (Bacto) 10.0 g,
each 100.0 cc. of agar.
5. Sucrose (Bacto) 10.0 g.
6. Glucose (Bacto) 1.0 g.
References: Aronson (1915 p. 1028), Klim- 7. NaCl 5.0 g.
mer (1923 p. 218). 8. Andrade indicator 0.025 g.
557
Used especially to isolate the inter-

Preparation
Bacto Krumwiede mediates of the colon-typhoid group.
References: Krumwiede and Kohn (1917 p.
Triple Sugar Agar (Dehydrated) in
226), Park, Williams and
Krumwiede
1000.0 cc. distilled water by boiling
(1924 p. 128).
or autoclaving. Avoid excess heat.
(2) Tube. 1783. Amoss' Four Sugar Agar
Constituents
Use: To detect intermediates of the colon- 1000.0 cc.
1. Distilled water
typhoid group.
2. Beef extract 3.0 g.
3. Peptone 100 g.
p. 12).
4. NaCl 5.0 g.
1782. Krumwiede and Kohn's Triple Sugar 5. Agar 150 g.
Agar 6. Lactose 2.5 g.
7. Rafhnose 2.5 g.
Constituents
8. Sucrose 2.5 g.
1. Water 1000.0 cc.
9. Salicin 2.5 g.
2. Agar 15.0 g.
3.0 g. 10. Lead acetate (0.25%
3. Meat extract
lO'O S- soln.) 250.0 cc.
4. Peptone
NaCl (salt) 5.0 g. Preparation
5.
(1) Dissolve 2, 3, 4, 5, 6, 7, 8, 9 and 10 in 1.
6. Lactose 100 g-
Sucrose 10-0 g- (2) Adjust to pH = 7.2.

7.
1-0 g- (3) Distribute in 4.0 cc. lots.
8. Glucose
9. Andrade indicator 10.0 cc. (4) Add to each tube 1.0 cc. of a 0.25%
solution of lead acetate.
Preparation
and 4 in 1. Sterilization: Not specified.
(1) Dissolve 2, 3
murium.
Adjust slightly alkaline to litmus Use: Cultivation of Bacillus typhi
(2)
(or 0.6% acid to phenolphthalein hot
Author reported that Bacillus typhi
7miritim produced browning of medium.
titration).
Distribute in 100.0 cc. lots. Reference: Amoss (1922 p. 28).
(3)
(4) At the time of use, add 1.0% lactose, 1784. Amoss' Sucrose Salicin Agar
(Topley
1.0% saccharose and 0.1% glucose to and Ayrton)
melted sterile (3). (These amounts
Constituents
may be conveniently added from a 1000.0 cc.
stock solution containing 20.0% lac- 1. Water
Beef extract (Lemco) 5.0 g.
tose, 20.0% saccharose and 2.0%
2.
Peptone (Witte) 10 g.
glucose that has been sterilized inter- 3.
NaCl 5.0 g.
mittently. Five cc.'s of this solution 4.
Lactose 10 g.
gives the desired concentration. 5.
Sucrose 10-0 g-
(5) Add 1.0% Andrade indicator and 6.
Salicin 100 g.
adjust reaction. The medium is to 7.
Andrade indicator 10.0 cc.
be red when hot and colorless when 8.
Agar 25.0 g.
cold. (If it is not red when warm the 9.
Lead acetate (1.0%) 50.0 cc.
medium is too alkaline, and if it fails 10.
11. Na2HP04 (1.0%) 50.0 cc.
to decolorize when cool it is too acid.)
Tube from a sterile covered funnel, Preparation
(6)
(1) Dissolve 2, 3, and
4 in 1 by heating
preferably in narrow tubes.
in the steamer.
(7) Steam for 20 minutes.
(2) Add 25.0 g. agar
and steam until
(8) Slant leaving a generous butt for
dissolved.
inoculation.
sterility. Remove from the steamer and add
(9) Incubate over night to test (3)
the white of an egg.
Coagulate in the steamer.
Use: Isolation of para-typhoid bacilli. (4)
(5) Filter. (b) Lohnis added 0.5% glucose, dissolved

(6) Adjust to pH = 7.1. in a littlewater to sterile melted
(7) Add Andrade's indicator.
10.0 cc. of nutrient agar.
(8) Autoclave at 107C. for 20 minutes. (c) Lignieres added 0.25% agar and
(9) Dissolve 5, 6 and 7 in the minimum 0.25% glucose to peptone bouillon
quantity of distilled water (about and sterilizedat 120C. for 15
30 cc). minutes. ,
(10) Mix sterile (9) with (8) and stir with (d) Giltner added 1.0% glucose and 1.0%
a sterile glass rod. CaCOs to hot agar, and sterilized
(11) Distribute into small flasks or tubes. by the discontinuous method.
(12) Place in the steamer for 10 minutes. (e) Harvey added sufficient sterilized
pH at this point should be near pH precipitated chalk to render glucose-
= 7.4. agar white and opaque.
(13) Prepare a 1.0% neutral lead acetate (f) Puder cultivated Rhabditis pellio, a
(lead diacetate) by rapidly raising nematode, on a medium prepared as
30.0 cc. of sterile distilled water to a follows:
boil, and adding 0.3 g. of neutral
(1) Prepare bouillon. Reaction to be
lead acetate (lead diacetate). alkaline.
(14) Add (13) to (11) using a sterile (2) Mix 10.0 cc. of with 90.0
(1) cc.
pipette in the proportion of 5.0 cc. water.
of (13) to each 100.0 cc. of (11).
(3) Dissolve 1.5 g. of agar in (2).
(15) Shake the flask quickly and Add 2.0 g. of glucose and
(4) drops of 8
vigorously. concentrated (strength not given)
(16) Add with a sterile pipette, 5,0 cc. of a alkaline to (3).
1.0% solution, Na2HP04 for each (5) Sterilize on three successive days
100.0 cc. of medium. for 30 minutes each day in a
(17) Shake again. steamer.
(18) Place in the water bath at 55C. Pour into sterile Petri dishes.
(6)
(19) Distribute into tubes. References: Frost (1903 p. 64), Heinemann
Sterilization Sterilize (9) by placing in the
:
(1905 p. 20), Lohnis (1913 p. 17), Lignieres
steamer for 20 minutes. See step (8) for (1919 Giltner
p. 1091), (1921 p. 365),
sterilization of agar. Final sterilization Harvey (1921-22 p. 89), Puder (1923 p.
oftubed medium not specified. 99),Stitt(1923p.38).
Use: Enumeration of B. aertryke in feces.
The authors reported that 1.0% glucose 1786. Mankowaki's Indigo Carmine Glucose
gave better results than lactose, sucrose Agar
and salicin, if B. aertryke outnumber the Constituents
other organisms in the feces. 1. 0.33 to 0.5% glucose agar.
Reference: Topley and Ayrton (1923-24 2. Fuchsin (acid,
p. sat. solution in 1.0%
230). KOH).
3. Indigo carmine (sat. aqueous solution).
1785. Frost's Glucose Agar
Preparation
Constituents: Prepare a 0.33 to 0.5% glucose agar.
(1)
1. Nutrient agar 1000.0 cc. Reaction to be neutral.
2. Glucose (1.0%) 10.0 g. (2) Prepare a saturated solution of acid
Preparation fuchsin in a 1.0% solution. KOH
(1) Add 1.0% glucose to nutrient agar. (Acid fuchsin may be added to a 1.0%
(2) Tube. KOH solution until a dark black
Sterilization: Sterilize in the steamer. brown color is reached.)
Use: General culture medium. (3) Prepare a watery saturated solution
Variants of indigo carmine.
(a) Heinemann added 1.5% glucose to (4) Add 2.0 cc. of (2) and 1 cc. of (3)
nutrient agar and sterilized the to 22 cc. of distilled This
water.
medium in the autoclave at 120 'C. for solution is dark blue and reaction
5 minutes. slightly alkaline.
559
Add 40.0 cc. of (1) to 40.0 cc. of water.

(5) Add (4), drop by drop, to (1) until the (2)
Pour into plates.
medium is colored blue and then (3)
(4) When the medium has solidified cover
violet blue.
the surface with a layer of (1) mixed
with an equal volume of sterile water.
(7) Add to each test tube a drop of a
watery saturated solution of indigo
carmine. Use: To study reduction of sulphite in
Not specified. water analysis. Author reported that a
Sterilization:
Differentiationbetween typhoid pure water showed no black colonies using
Use:
40.0 cc. of the water as an inoculum, a
bacilli and Bacterium coli. Author re-
potable water showed not more than 4, a
ported that typhoid bacilli colored the
sand-filtered water showed not more than
blue medivim a raspberry or carmine red
The author used a similar medium
color. Bacterium coli colored the me- one.
for the typhoid bacilli and
enrichment of
dium bluish green and finally decolorized
detection of sulphite reducers in water.
it.
Variants: The author prepared a similar
Reference: Mankowaki (1900 p. 22).
medium as follows:
1787. Rivas' Glucose Agar (1) Add 1.0 cc. of an 8.0% ferric chloride
but with solution, 0.6 cc. of a 10.0% sodium

Same as medium 924, solidified
hydrate solution and 10.0 cc. of a
agar.
20.0% anhydrous NajSOs solution to
1788. Hall and EUefson's Gentian Violet freshly boiled melted nutrient glucose
Glucose Agar agar containing 3.0% agar.
Add from 1 to 1000, to 1 to 20,000
Constituents: (2)
1000.0 cc. brilliant green to (1).
1. Glucose agar
100,000 Add 40.0 cc. of (2) to 40.0 cc. of water
Gentian violet 1 (3)
2. :
Preparation
under investigation.
Prepare glucose agar in sufficient (4) Pour into plates.
(1)
When the medium has solidified cover
quantity. (5)
be pres- the surface with a layer of (1) mixed
(2) Add gentian violet so that it
ent in 1: 100,000 dilution. with an equal volume of sterile water.

Sterilization: Not specified. Reference: Wilson and Blair (1925 p. 112).
Use : Cultivation of gram positive sporulat-
1790. Scheffler's Neutral Red Glucose Agar
ing anaerobes.
Variants: Authors used 1:1000 and 1:10,000 Constituents
of gentian violet, but best results were 1. Nutrient agar. . . 1000.0 cc.
obtained with 1:100,000 concentration of 2. Glucose 3.0 or 10.0 g.
gentian violet. 3. Neutral red (con-
Reference: Hall and Ellef son (1918 p. 336). centrated solu-
10.0, c.O 2.5 cc.
1789. Wilson and Blair's Sulphite Glucose tion)
Agar Preparation:
(1) Prepare a nut' lent agar.
Constituents:
100.0 cc. (2) Add 0.3 or 1.0% dextrose to (1).
Glucose agar (3.0%)
1.
1.0 cc.
(3) Add varying amounts of a concen-
2. Ferric chloride (8.0% soln.)
trated solution of neutral red to 100.0
.
3. NaoSOs (20.0%) 10.0 cc.

cc. of (2). Amounts used were 1.0,
Preparation
0.5 or 0.25 cc.
(1) Add 1.0 cc. of an 8.0%
ferric chloride
Steri'ization: Not specified.
solution, 0.6 cc. of a 10.0% sodium
hydrate solution and 10.0 cc. of a Use: Detection of Bact. coli. Author
20.0% anhydrous sodium sulphite reported that all coli strains investigated
solution to freshly boiled melted gave a fluorescence in a greater or less
nutrient glucose agar containing 3.0% degree after 48 hours. Fluorescence may
agar. usually be obtained after 24 hours using
0.3% glucose and 1.0 cc. dye per 100.0 cc. 3. Litmus (soln.) 30.0 cc.
agar. 4. Nutrient agar (3.0%)
Variants Preparation
(a) Makgill added 1.0% of a saturated (1) Dissolve 3.0 g. of glucose and 30.0
solution of neutral red to glucose cc. of litmus in 200.0 cc. distilled
agar. water.
(b) Heinemann added sufficient 0.5% (2) Add five parts sterile (1) to six parts
neutral red to give a red color to 1.0% of sterile nutrient 3.0% agar in tubes.
glucose agar. The mixture should be a violet tint.
(c) Bezangon prepared a similar medium Sterilization: Sterilize (1) by filtering.
as follows: Method of sterilization of 3.0% agar not
(1) Dissolve 5.0 g. of agar in 1000.0 cc. given.
of bouillon. Use: Differentiation of diphtheria and
(2) To melted (1) nearly solid, add 2.0 pseudo diphtheria. True diphtheria
g. glucose and 0.7 cc. of a aqueous bacilli changed the color of the medium
saturated solution of neutral red. to red and grew only anaerobically.
(3) Tube. Pseudodiphtheria strains did not change
(4) Sterilization not specified. the color and grew only on the surface.
(d) Rothberg and Scheffler(Klimmer) Reference: Martin and Loiseau (1919 p.
prepared a medium as follows: 73). Taken from (1919 p. 185).
(1) Add 0.3% glucose to melted sterile
nutrient agar. 1792, Frost's Lactose Agar
(2) Add 1.0 cc. of a saturated (cold)
solution of neutral red sterilized in Constituents
the steamer to each 100.0 cc. of (1). 1. Nutrient agar 1000.0 cc.
(e) Cunningham prepared a medium 2. Lactose (1.0%) 10.0 g.
as
follows: Preparation
(1) Add 1.5% agar to bouillon. (1) Add 1.0% lactose to nutrient agar.
(2) Steam for 30 minutes to dissolve (2) Tube.

the agar. Sterilization: Sterilize in the steamer.
(3) Boil over an open flame for 15 Use: General culture medium.
minutes, stirring constantly. Reference: Frost (1903 p. 64).
(4) Adjust the reaction to a slight
alkalinity using turmeric paper as 1793. Wurtz's Litmus Lactose Agar
an indicator (distinctly brown).
Constituents
(5) Filter while hot thru a plug of clean
cotton-wool in the bottom of an 2. Lactose (2.0%) 20.0 g.
enamelled funnel.
3. Litmus
(6) Add 0.5% glucose dissolved in a Preparation
few cc. of water.
(1) Add 2.0% lactose to sterile infusion
(7) Stir to thoroly mi.x.
agar.
(8) Tube.
(2) Tube.
(9) Sterilize intermittently in steam.
(3) Melt sterile (2) and add suflicient
References: SchefHer (1900 p. 202), Magkill tincture of litmus to give a violet
(1901 p. 431), Heinemann (1905 p. 127), color.
Tanner (1919 p. 50), Bezangon (1920 p. (4) Pour sterile (3) into plates.
114), Klimmer (1923 p. 211), Cunningham Sterilization: Method of sterilization of
(1924 p. 16), Savage (1901 p. 437), Irons or not given.
(1) (2) Sterilize (3) at
(1902 p. 315).
100 C.
Use: Differentiation of Bad. coli and
1791. Martin and Loiseau's Glucose Litmus
Eberth's bacillus. Author reported that
Agar
Bad. coli fermented lactose with the
Constituents production of acid. Typhoid bacillus
1. Distilled water 200.0 cc. colonies were colorless, colon colonies
2. Glucose 3.0 g. were red.
Variants (17) Sterilize for 15 minutes in the

(a) Smith specified that the reaction of autoclave at 120C., or for 30
the agar be slightly alkaline (0.5%). minutes on each of 3 successive
(b) Smith (1905) also gave the following days.
method of preparation: (e) Ball (1919) prepared a similar medium
(1) Add 10.0 g. of c.p. lactose to 1000.0 as follows:
cc. of sugar-free meat infusion (1) Add 1.0% lactose to nutrient agar
agar. just before sterilization.
(2) Add 20.0 cc. of a saturated watery (2) Reaction of (1) to be neutral.
solution of lime-free blue litmus (3) Boil 1.0% Kahlbaum's azolitmin
to (1). for 5 minutes.
(3) Sterilization not specified. (4) Add (3) to the tubes just before
(c) Heinemann prepared a similar me- sterilization, or if to be used in
dium as follows: plates, add at the time of plating.
(1) Add 1.0% lactose to sugar-free (f) Abbott (1921) gave
the following
infusion agar. method of preparation:
(2) Tube in 8.0 cc. quantities. (1) Prepare a nutrient agar so that the
(3) Add 1.0% sterile litmus
1.0 cc. of alkalinity is such that it requires
solution to each tube before using. 0.1 cc. of a 1:20 normal H2SO4
(d) Committee A. P. H. A. (1913). solution to neutralize 1.0 cc. of
(1) Boil 10.0 or 15.0 g. of thread agar medium. Indicator not specified.
in 500.0 cc. of water for half an (2) Add 3.0% lactose.
2.0 to
hour and make up weight to 500.0 (3) Decant into test tubes.
g. or digest for 15 minutes in the (4) Sterilize in the usual way (method
autoclave. Cool to 60C. not given)
(2) Infuse 500.0 g. of lean meat for 24 (5) Add sufficient sterile litmus tinc-
hours with 500.0 cc. distilled water ture to each tube to give a decided
in a refrigerator. but not intense blue color. Add
(3) Make uplost weight. the litmus under aseptic con-
(4) Strain thru cotton flannel. ditions.
(5) Weigh. (g) Giltner gave the following methods
(6) Add 2.0% Witte's peptone and of preparation. The media were
warm on the water bath until used in water analysis.
solution is complete. Do not heat (1) (1) Prepare infusion agar using
above 60 C. equal parts meat infusion and
(7) Mix 500.0 cc. (6) and 500.0 cc. of water, with 1.0% peptone and
(1), keeping the temperature below 1.5% agar.
60 "C. (2) Adjust the reaction to -f 1.0.
(8) Titrate and adjust the reaction to (3) Add 1.0% lactose and 2.0%
neutral to phenolphthalein, add- azolitmin solution just before
ing normal HCl or NaOH. tubing.
(9) Heat on a water bath for 40 (4) Tube.
minutes. (5) Sterilize for 30 minutes on
(10) Make up lost weight. 3 successive days.
(11) Readjust to neutrality (2) (1) Preparation of meat infusion

if neces-
sary and boil 5 minutes. not given.
(2) Strain (1) thru a piece of clean
(12) Restore lost weight.
cheese cloth.
(13) Filter thru absorbent cotton and
(3) Place 2.0% washed agar in
cotton flannel.
(14) Titrate and record final reaction.
(4) Weigh (3).
(15) Add 1.0% lactose and sufficient (5) Digest over a free flame.
azolitmin solution. Add distilled water to make up
(6)
(16) Tube in 10.0 cc. quantities. the loss in weight.
Add 2.0% lactose and 2.0% (2) Place 1.5 g. of lactose into a sterile
(7)
peptone to hot (6) and mix flask.
until solution is complete. (3) Pour approximately 100.0 cc. of the
(8) Add t6 (7).

(2) liquid agar over the lactose and mix
(9) Adjust the reaction to 0.
well.
(10) Add 2.0% azolitmin solution. (4) Boil 100.0 g. of distilled water 15
(11) Boil over a free flame. minutes.
(12) Distribute in 100.00 cc. lots in (5) Dissolve 0.1 g. crystal violet in (4).
250.0 cc. Florence flasks. (6) Add a weak soda solution that has
(13) Sterilization not specified. been boiled several times to (3) until
References: Wurtz (1897 p. 43), Smith the desired degree of alkalinity is
(1905 p. 94), Heinemann (1905 p. 127), obtained.
Committee American Public Health (1913 (7) Dissolve 0.04 g. azolitmin in a little
p. 129), Ball (1919 p. 78), Abbott (1921 p.

water and boil.
142), Giltner (1921 pp. 379, 380). (8) Add 1.0 cc. of (5) and the azolitmin
solution to (6) (the melted lactose
1794. Gassner's Metachrome Yellow Water agar) that has been cooled to 45C.
Blue Lactose Agar (Klimmer) (9) Mix well and after about ten minutes,
pour into sterile Petri dishes. Allow
Constituents the agar to solidify with the covers
1. Nutrient agar 2000.0 cc.
removed.
2. Metachrome yellow (2.0% Sterilization: Method not given.
125.0 cc.
soln.) Use: Diagnosis of cholera. Authors re-
3. Water blue (6B extra ported that cholera colonies were deep
"Afga" 1.0% soln.) 175.0 cc.
blue.
4. Lactose 100.0 g. Hirschbruch and Schwer
Reference:
Preparation (1904 p. 150).
(1) Make two liters of agar prepared from
yeast or meat and peptone, slightly 1796. Ramond's Rubine Acid Lactose Agar
alkaline. Constituents
(2) Add 125.0 cc. of a 2.0% solution of 1. Nutrient agar 1000.0 cc.
metachrome yellow (boiled two 2. Lactose 40.0 g.
minutes). 3. Eubine acid
(3) Dissolve 100.0 g. lactose in 175.0 cc. Preparation
of a 1.0% solution of water blue (6B Add 4 parts per 100 of lactose to
(1)
extra "Afga") and boil 10 minutes. nutrient agar.
(4) Add (3) to (2).
(2) Add several grains of rubine
acid
Sterilization: Not specified. until the medium is colored red.
Use: Detection of typhoid bacilli. Author add a satu-
(3) Heat to 70 or 80C. and
reported that B. were deep
coli colonies
rated solution of Na2C03 until the
blue. Typhoid and dysentery colonies color disappears.
lightened up the medium, coloring it a
(4) Filterthru paper.
greyish yellow. (5)Distribute in tubes.
Reference: Klimmer (1923 p. 216). Sterilization: Sterilize at 115C. for five
minutes.
1795. Hirschbruch and Schwer's Azolitmin Use Detection of typhoid bacilli. Author
:
Crystal Violet Lactose Agar reported that Bad. coli colonies colored
Constituents: the medium red. Typhoid colonies did
Nutrient agar (1.0 to 1.5%). 1000.0 cc.
1. not change the color.
15.0 g. References: Ramond (1896 p. 884), Wurtz
Lactose
2.
3. Crystal violet (0.1%, soln.) 10.0 cc. (1897 p. 43).
4. Azolitmin 0.4 g.
1797. Delta's Fuchsin Lactose Agar
Preparation
(1) Prepare nutrient agar containing 1.0
Constituents
Distilled water. .0 cc.
1.
to 1.5% agar.
2. Agar (3.0%) 100.0 cc. Use: Detection of typhoid fever and dys-
3. Lactose 1-5 g. entery. Author reported that typhoid,
4. Fuchsin acid (0.5%) 10.0 cc. paratyphoid and dysentery bacilli gave
5. NaaCOs light colorless colonies. Coli forms gave
Preparation small red colonies. They were inhibited.
(1) Prepare a 3.0% nutrient agar with a Cholera colonies were colorless but the
reaction slightly alkaline to litmus. medium was not selective for this
(2) Dissolve 1.5 g. lactose in 4.0 cc. of organism.
distilled boiling water. Boil for 30 Reference: Kindborg (1915-16 p. 445).
seconds.
(3) Prepare a 0.5% solution of acid 1799. Bitters' China Blue Malachite-green
fuchsin. Agar (Klinimer)
(4) Boil (3) and decolorize by adding four
Constituents
drops of a normal NaaCOs solution.
Boil again until it assumes a port wine
1. Agar (2.0 or 3.0%) 1000.0 cc.
(5)
2. Lactose (2.0%) 20.0 g.
color.
Add and 3. China blue (Hochst. sat.
(6) (2) (5) to 100.0 cc. of (1).
solution)
Use: Examination of feces. 4. Malachite green (Hochst.
Variants: The author reported that any 0.1% soln.) 25.0 cc.
Preparation
sugar may replace lactose. The medium
may be made more differentiating by the (1) Prepare a 2.0 or 3.0% nutrient agar.
addition of "nutrose or caffeine or mala- (2) Neutralize to litmus by the addition
chite green (in tested solution as for Lentz ofNaOH.
and Tietz's medium) or crystal violet
(3) Add 2.0% lactose.
(4) Boil several minutes.
(10 drops of a 1 to 1000 solution). In the
lasttwo combinations the background is (5) Add 9 drops of a saturated solution
green (or blue) and the coli colonies are of China blue (Hochst.) to each 100.0
cc. of agar.
violet (or red)."
Reference: Delta (1915 p. 1053).
(6) Add 2.5 cc. of a 0.1% solution of
Hochst. crystalline extra malachite
1798. Kindborg's Fuchsin Malachite Green green to each 100.0 cc. (The mala-
Agar chite green may be omitted.)
Constituents: (7) Heat for 10 minutes in a steamer.

Agar (8) Pour in plates.
1. 1000.0 cc.
2. Lactose 14.0 g.
Fuchsin acid (3.0%) Use: Differentiation of colon typhoid

3. 50.0 cc.
4. Malachite green (1:10,000 group. Author reported that coli colo-
nies were blue; typhoid colonies yellow
soln.) 10.0 cc.
Preparation or colorless.
Prepare nutrient Reference: Klimmer (1923 p. 217).

(1) agar, neutral to
litmus.
(2) Prepare a 3.0% stock solution of acid 1800. Liebermann and Acels' Congo Red
fuchsin. Agar (Klimmer)
(3) Prepare a solution of malachite green Constituents:
1:10,000 (from a 0.1: 100). 1. Nutrient agar 1000.0 cc.
(4) Dissolve 14.0 g. lactose in about 40.0 2. Lactose 15.0 g.
cc. water. 3. Congo red 3.0 g.
(5) When the agar has clearedsomewhat Preparation
by settling, add 50.0 cc. of the red and (1) Add 15.0 g. of lactose and 3.0 g. of
10.0 cc. of the green liquid and finally Congo red to a liter of slightly alka-
the lactose solution. line nutrient agar.
(6) Pour sterile (5) into sterile plates. (2) Boil.
Sterilization : Sterilize (5) one-half hour in (3) Mix thoroly.
the autoclave. Sterilization: Not specified.
Use: Differentiation of colon typhoid to nutrient agar and sterilized the

group. Author reported that typhoid medium in the autoclave.
colonies were red and transparent; colon References: Lange (1916 p. 153), Tanner
colonies bluish black. (1919 p. 60), Allen (1918 p. 15).
1803. Hoffmann's Nitrate Starch Agar
1801. Massini's Triple Dye Lactose Agar Constituents
1. Nutrient agar 1000.0 cc
Constituents
1000.0 cc.
2. Starch 5.0 g.
1. Nutrient agar (3.0%)
2. Lactose 30.0 g.
3. KNO3 (0.1%) 1.0 g.
Preparation
3. Egg chrome yellow 2 G 1.0 g.
(1) Prepare 1000.0 cc. nutrient agar.
4. Helvetia blue 0.65 g.
(2) Add 0.5%o starch and 0.1% KNO3 to
Preparation
(1).
(1) Prepare nutrient 3.0% agar.
(2) Add 30.0 g. lactose, 1.0 g. egg chrome
Use: Differential count of bacteria in soil
yellow 2 G. and 0.65 g. helvetia blue
nitrate reduction. Treat the seeded
to 1000.0 cc. of (1).
the Arnold on plates with a dilute solution of KI in
dilute Author reported that colo-
HCl.
each of three consecutive days for 15
nies reducing nitrate to nitrite had a blue
minutes.
halo around them following this treat-
Use: Detection of typhoid and dysentery.
ment.
The medium was turned blue by B. coli
Reference: Hoffmann (1912 p. 386).
and other acid forming organisms while
the typhoid-dysentery group gave a
1804. Hoffmann's Nitrate Starch Agar
yellow color.
Reference: Massini (1918 p. 887) taken Same as medium 1803 but using KNOj
from (1918 p. 204). instead of KNO3. The author reported
that colonies reducing nitrite produced a
1802. Lange's Starch Agar clear halo around the colony when a dilute
Constituents
KI solution in dilute HCl was added. If
1000.0 cc. a yellowish halo was produced around a
1. Nutrient agar
50.0 g.
colony when a dilute solution of Nessler's
2. Starch (rice) (5.0%)
reagent be added, ammonia was formed.
Preparation
(1) Add 40.0 cc. of a 10.0%, soda solution
1805. Khouvine's Cellulose Agar
to of agar neutral to litmus.
1 liter
(2) Prepare a 5.0% rice starch paste by Constituents

boiling rice starch in water. 1. Nutrient agar.
Mix 6 parts sterile alkaline agar with 2. Cellulose.

(3)
one part sterile rice starch paste. Preparation
(4) Pour into Petri dishes. (1) Add very finely divided Berzelius
Sterilization: Method of sterilization of filter paper or precipitated cellulose

agar not given. Sterilize (2) in the to nutrient agar.
autoclave. Sterilization: Not specified.
Diagnosis of cholera. Author re- Use: Isolation and enrichment of B.

Use:
ported that cholera vibrio showed char- cellulose dissolvens.
acteristic growth after from 14 to 20 Variants: The author prepared the agar
hours. Also used to determine the as indicated:
production of diastase by bacteria. (1) Place a layer of sterile agar in a sterile
Variants Petri dish.

(a) Tanner added 2.0% paste of potato (2) Add a sheet of sterile filter paper.
starch to melted and cooled nutrient (3) Cover with a layer of melted agar con-
agar. taining the organisms.
(b) Allen added 2.0% water soluble starch Reference: Khouvine (1923 pp. 714, 715).
1806. Scales' Salt Cellulose Agar 1807. Cantani's Basal Glycerol Agar
1. Water. 1. Nutrient agar.
2. Filter paper 2. Glycerol.
3. Nutrient agar. Preparation
4. Salts. (1) Mi.x equal amounts of glycerol and
Preparation one of the added nutrients. Place in
(1) Dilute 100.0 cc. of concentrated an Erlenmeyer flask.
H2SO4 with 60.0 cc. distilled water in (2) Melt tubes of sterile nutrient agar and
a two liter Erlenmeyer flask. add 0.5 to 0.75 cc. of sterile (1) to
(2) Cool to about 60 or 65C. each tube.
(3) Moisten 5.0 g. of filter paper with (3) Incubate 24 hours to test sterility.
water. Sterilization: Storing the fluid in glycerol
(4) Add (3) to (2) and shake until the tends to sterilize After a time test the
it.
paper is dissolved. sterility of mixture. Method of
the
(5) When solution is complete fill the sterilization of agar not given.
flask as quickly as possible with cold Use: Cultivation of parasitic and patho-
tap water. The task of dissolving genic forms.
the filter paper and filling the flask Added nutrients: The author added one
requires about one minute. of the following: Urine, pus, sperm, milk,
(6) The rapid addition of cold water egg white and other albuminous materials.
precipitates the cellulose in small Reference: Cantani (1910 p. 471).
flocks.
Throw the 1808. Wurtz's Glycerol Agar
(7) precipitate on a filter
paper and wash with distilled water Constituents
until free from H2SO4. This re- 1. Nutrient agar 1000.0 cc.
quires about 3 hours time and only 5 2. Glycerol (6.0%) 60.0 g.
liters of distilled water. A 12 inch Preparation
funnel with a folded filter is the best (1) Add 6.0% sterile glycerol to sterile
apparatus to filter with. nutrient agar under aseptic con-
(8) During the washing do not allow the ditions.
volume of water in the funnel to Sterilization: Method not specified.
get below 100.0 cc. Use: Cultivation of tubercle bacilli. Simi-
(9) When the wash water is free from lar media have been used to cultivate a
H2SO4 as shown by the addition of large variety of other pathogenic forms.
BaCl2 solution, allow the volume in Variants
the funnel to drain to about 200.0 cc. (a) Gllicksmann (1897) added 7.7% gly-
(10) Brush any cellulose particles cling- cerol to 1.5% nutrient agar and
ing on the dry filter paper into the cultivated diphtheria bacilli.
suspension. (b) Thoinot and Masselin added 4.0 to
(11) Punch a hole in the bottom of the 6.0% glycerol to nutrient agar,
filter, collecting the cellulose sus- (c) Dalton and Eyre added 5.0% glycerol
pension. to nutrient agar and adjusted the
(12) Wash the filter with a stream of reaction to -}-10 on Eyre's scale.
water from a wash bottle. They cultivated Micrococcus meli-
(13) Make the suspension to 500.0 cc. tensis.
(14) Prepare 500.0 cc. of nutrient agar (d) Smith cultivated plant parasites in a
with salts (composition not spe- medium prepared by the addition of
cified). 50.0 cc. of Schering's c.p. twice dis-
(15) Mix (13) and (14). tilled glycerol to nutrient agar.
Sterilization: Not specified. (e) Roux and Rochaix added 1.0 to 5.0%
Use: Cultivation of organisms capable of glycerol to agar. They suggested
utilizing cellulose. the addition of several drops of a
Reference: Scales (1916 p. 662). saturated gum arable solution. This
favored the adherence of the agar to 1811. Heinemann's Litmus Mannitol Agar
the walls of the containing vessels. Constituents
Reference: Wurtz (1897, p. 46), Glucks- 1. Sugar free agar 1000.0 cc.
mann (1897 p. 436), Thoinot and Masselin 2. Mannitol (1.0%) 10.0 g.
(1902 p. 35), Smith (1902 p. 92), Dalton 3. Litmus
and Eyre (1904 p. 159), Heinemann (1905 Preparation
p. 128), Smith (1905 p. 196),
Roux and (1) Add 1.0% mannite to sugar-free agar.
Rochaix (1911 p. 117), Roddy (1917 p. 43), (2) Tube in 8.0 cc. quantities.
Ball (1919 p. 74), Besson (1920 p. 43), (3) Add 1.0 cc. of 1.0% sterile litmus
Dopter and Sacquepee (1921 p. 128), solution to each tube before use.
Bitfield (1922 p. 117), Stitt (1923 p. 38), Sterilization: Not specified.
Cunningham (1924 p. 165) Use: General culture medium.
1809 . Scheffler's Indicator Glycerol Agar

1812. Penfold's Phenylacetate Agar
1. Glycerol agar
1000.0 cc. 150.0 cc.
1. Nutrient agar
2. Neutral red (Concentrated 2. Bhenylacetate (10.0% soln.)
aqueous soln.) 15.0 cc.
Preparation
Preparation (1) Dissolve phenylacetate
in saturated
(1) To 100.0 cc. of glycerol agar add 1.5
Na2C03 solution until the solution is
cc. of a concentrated watery solution just faintly alkaline to neutral litmus
of neutral red. paper.
(2) Add distilled water to render the
Use: Detection of iBac^m'MW coli. Author solution of 10.0%, strength.
reported that after 24 hours in^ neutral (3) Filter thru a Doulton filter.
red, a slight greenish coloration was (4) Pour ascending quantities of the
formed, in 48 hours a distinct green solution into Petri dishes and 15.0 cc.
fluorescence. At the end of 24 hours with of melted agar added to each plate.
saffarin, the under surface was lighter Not given.
Sterilization:
and after 48 hours decolorization occurred. Use To show inhibition of bacterial growth
:
Variants: The author substituted a con- using members of the colon-aerogenes

centrated watery solution of saffarin for group. Author reported that B. coli
neutral red. produced papillated colonies and showed
Reference: Scheffler (1900 p. 201). marked variability in the size of its
colonies.
1810. Mandelbaum's Rosolic Acid Glycerol Reference: Benfold (1913-14 p. 38).
Agar
1813. Wurtz's Phenol Agar (Copeland)
1. Glycerol agar 6.0%.
1. Wurtz agar.
2. Rosolic acid 1.0% alcoholic soln. Phenol 2.0% solution.
2.
(1) Brepare a 6.0% glycerol
agar. or composition
(1) Method of preparation
(2) Brepare a 1.0% alcoholic
solution of
ofWurtz agar not given.
rosolic acid. Add 0.2 cc. of a 2.0% solution of car-
(2)
(3) Melt sterile (1), cool to 50C. and to
bolic acid to the agar. Amount of
each tube add 0.3 cc. of (2). agar not specified.
Use: Differentiation of typhoid and meta- Use: Isolation of Bacillus coli communis
typhoid bacilli. Author reported that from water.
metatyphoid bacilli gave red colonies. Variants: Behmer's (Klimmer) added 2.0
cc. of a 5.0% phenol solution to
each
The typhoid colonies were yellow.
Reference: Mandelbaum (1912 p. 48). 100.0 cc. agar.
Reference: Copeland (1901 p. 493), Klim- Use: CultivatioQ of typhoid and inter-
mer (1923 p. 227). mediate group.
Reference: Hurler (1912 p. 357).
1814. Penfold's Monochlorhydrin Agar
p , , 1817. Finger, Ghon and Schlagenhaufer's
, , " Urea Agar
^
1. Agar.
2. Monochlorhydrin 20.0% solution. Constituents:
Preparation: 1. Nutrient agar (2.0% agar,
(1) Prepare a 20.0% solution of mono- 1.0% peptone) 1000.0 cc.
chlorhydrin. 2. Urea (2.0%) 20.0 g.
(2) Place ascending quantities of sterile Preparation:
(1) in Petri dishes. (1) Prepare nutrient agar containing
(3) Add 15.0 cc. of sterile agar to each 2.0% agar with 1.0% peptone,
plate. Mi.xwell. (2) Add 2.0% urea to (1).
Sterilization: Filter (1) to sterilize. Sterilization: Not specified.
Method of sterilization of agar not given. Use: Cultivation of gonococci. Author
Use To show inhibition of bacterial growth
: reported that growth was not as luxuriant
using members of colon aerogenes group. as in serum agar. Also used by other in-
Author reported that B. coli produced vestigators to study urea decomposition.
various sized colonies and papillae may Variants:
be formed. (a) The author used 3.0 or 5.0% urea
Reference: Penfold (1913-14 p. 39). instead of 2.0%.
(b) Lohnis studied urea decomposition
1815. Jacobson's Ethylcinnamic Ether Agar
^^ ^^^.^^ ^ ^^ ^^ ^ J5 0^^ ^q^^^^^
Constituents: solution of urea to each tube of
1. Agar. nutrient agar. The medium was then
2. Ethylcinnamic ether. heated in the steam sterilizer.
Preparation: (c) Cunningham added 2.0, 5.0 or 10.0%
(1) Liquify 10.0 cc. tubes of nutrient agar urea to nutrient agar, and sterilized
by heating on the salt water bath. by heating intermittently in the
(2) Add one small drop of ethyl cinnamic steamer. The medium was used to
ether to each tube by means of a fine study urea decomposition,
pipette. This is equivalent to 0.025 g. Reference: Finger, Ghon and Schlagen-
Sterilization: Not specified. haufer (1894 p. 14), Lohnis (1913 p. 95),
Use: Differentiation of dysentery bacilli. Cunningham (1924 p. 143).
Author reported that the Hiss type of -^^ ,,. -^ , , r^
, r -1 J .
J 4.U 4. 1818. Russell's Double Sugar
^ Agar
to develop in the pres-
1
dysentery failed ^
ence of ethyl cinnamic ether while the Constituents:
Flexner showed good development. 1. Nutrient agar (2.0 or
Reference Jacobson (1919 p. 726).
: 3.0%) 1000.0 cc.
2. Litmus solution
1816. Hurler's Caffeine Agar
^^ 0^^ ^q^^^^^ ^^1^.
Constituents: tion) 30.0 to 50.0 cc.
1. Nutrient agar 1000.0 cc. 3. Lactose 10.0 g.
2. Caffeine (0.3%) 3.0 g. 4. Glucose 1.0 g.
(1) Prepare nutrient agar. (1) Prepare2.0or 3.0% nutrient agar with
(2) Adjust the reaction of (1) to slightly a reaction of about 0.8% acid to
alkaline. phenolphthalein.
(3) Add 0.3% caffeine to (2). The (2) Add enough of a 5.0% aqueous solu-
caffeine is dissolved in 5.0 cc. of tion of litmus to (1) to give a distinct
water by heating over the
distilled purple violet, the amount required
water bath before adding to the agar. depending on the original color of the
Sterilization: Method not given. agar.
(3) Adjust the reaction by adding sodium and Bacillus murium pro-
enteritidis
hydrate until the mixture is neutral to duced browning while A type did not.
litmus. (b) Giltner prepared a similar medium
(4) Add 1.0% lactose and0.1% glucose as follows:
(dissolved in a small amount of hot (1) Prepare a 1.5% nutrient agar.
water) to (3). (2) Add 1.0% glucose and 1.0%o lactose
(5) Slant the tubes following sterilization to (1).
and store in small quantities in a (3) Make up a 0.5% solution of basic
dark place. lead acetate.
Sterilization: Sterilize in the Arnold. (4) Sterilize (3). (Method not given.)
Pack the tubes loosely in the sterilizer (5) Tube.
basket to allow good circulation of the (6) Add the necessary amount of (4)
steam. Under these conditions 10 to each tube of agar to give 0.05%

minutes on the first day and 15 minutes basic lead acetate.
the second are sufficient. (7) (method not given).
Sterilize
Use: Isolation of typhoid, paratyphoid (c) Pitfield Andrade's in-
substituted
and dysentery from feces and urine. dicator for litmus as an indicator.
Variants : References: Russell (1911 p. 226), Roddy
(a) Kligler gave the following variant: (1917 p. 43), Kligler (1918 p. 321), Tanner
(1) Prepare the nutrient agar from (1919 p. 52), Ball (1919 p. 84), Giltner
meat infusion (or beef extract). (1921 pp. 385, 386), Harvey (1921-22 p.
(Preferably clear sugar free beef 109), Pitfield (1922 p. 120), Stitt (1923
infusion.) Exact composition not pp. 50, 51).
given.
1819. Thoinot and Masselin's Glucose
(2) Adjust to pH = 7.4 or neutral to
Glycerol Agar
Andrade's indicator.
(3) Add 1.0% by volume of Andrade's Constituents
indicator. 1. Agar 1000.0 cc.
(4) Tube in 5.0 cc. lots and sterilize. 2. Glucose (5.0 to
(5) Prepare a solution containing 20.0% 10.0%) 50.0 to 100.0 g.

lactose and 20.0% glucose and 3. Glycerol (4.0 to
sterilize. 6.0%) 40.0 to 60.0 g.
(6) Add 0.25 cc. of sterile (5) to each Preparation

tube of melted sterile (4), cooled to (1) Add 5.0 to 10.0% glucose and 4.0 to
60C. 6.0% glycerol to agar.

(7) Prepare and sterilize a 0,25% basic Sterilization: Not specified.
lead acetate solution and add 1.0 Use: General culture medium.
cc. to each tube of (4) cooled to Variants
60C. (a) Besson dissolved 20.0 g. agar in a
(8) Slant so as to have a butt of at liter of bouillon, and added 2.0 to
least to f inches and a slant of 4.0% glucose and 5.0% glycerol.
about IJ inches. The medium was sterilized at 115C.
Kligler gave the following special for 20 minutes.

reactions B. coli reddened whole tube
: (b) Dopter and Sacquepê solidified
and gas was produced. Bacillus bouillon with agar, and added 5.0%
typhosus reddened butt, colorless glycerol and 2.0% glucose.
slant and produced browning particu- References: Thoinot and Masselin (1902 p.
larly near the surface of the stab. 35), Besson (1920 p. 43), Dopter and
The dysentery bacillus reddened the Sacquepee (1921 p. 128).
butt but did not produce browning.
1820. Thoinot and Masselin's Sucrose
Paratyphosus bacilli produced gas
Glycerol Agar
while Bacillus hjphosus and Bacillus
dysenteriae did not. Bacillus para- Same as medium 1819 but substituting
typhosus B. and allied bacilli, Bacilli 5.0 to 10.0 g. sucrose for glucose.
1821. Kligler and Defandorfer's Double or inositol. Paratyphoid B fermented

Sugar Agar inositol with the formation of gas.
Prepared in the same manner as Russell's Reference: Hulton-Frankel and Mac-

Double Sugar Medium (see 1818) using the Donald (1917 p. 31).
same constituents but substituting 0.5% 1824. Kitasato's Glucose Formate Agar
mannitol for 1.0% lactose. The authors
(Tanner)
reported they used the medium for the
differentiation of B. typhosus from dys-
Constituents
entery bacilli. Nutrient agar
1. 1000.0 cc.
Reference: Kligler and Defandorfer (1918 2.Glucose 20.0 g.
3. Sodium formate 4.0 g.
p. 439).
Preparation
(1) Prepare nutrient agar.
1822. Kendall and Ryan's Sucrose Mannitol
Agar
Constituents (4) Tube.
1. Nutrient agar 2.5% 1000.0 cc. Sterilization: Method not given.
2. Mannitol 1.0 g. Use: General culture medium.
3. Sucrose, 10.0 g. Reference: Tanner (1919 p. 49).
4. Andrade indicator 10.0 cc.
Preparation :
1825. Sohngen and Fol's Glucose Butyrate
(1) Prepare nutrient 2.5% agar.
Agar
(2) Adjust to a reaction so that when Constituents
Andrade indicator is added that the 1. Water agar 50.0 cc.
medium be a faint pink while hot. 2. Nutrient agar 50.0 cc.
(3) Add 2 and 3 to (2). 3. Calcium butyrate 1.0 g.
(4) Tube. 4. Glucose 0.25 g.
(5) Slant so that slanted surface begins Preparation
1.0 cm. from the bottom. Medium is (1) Prepare tap water agar and nutrient
colorless when cold. agar.
Sterilization: Not specified. (2) Mix 50.0 cc. of tap water and 50.0 cc.
Use: Detection of intestinal and other of nutrient agar.
bacteria. (3) Dissolve 3 and 4 in (2).
Reference : Kendall and Ryan (1919 p. 403). Sterilization: Not specified.
Use: Cultivation of actinomyces, Acti-
1823. Hulton-Frankel and MacDonald's nomyces elastica and Actinomyces fuscus
Inositol-Dextrin Agar Reference: Sohngen and Fol (1914 p. 95).
Constituents 1826. MacDonald's Glucose Lactic Acid

1. Nutrient agar (3.0%) 1000.0 cc. Agar
2. Inositol (1.0%) 10.0 g. Constituents
3. Dextrin (1.0%) 10.0 g. 1. Nutrient agar 1000.0 cc.
4. Litmus 2. Glucose 10.0 g.
Preparation 3. Lactic acid 1.0 to 10.0 g.
(1) Add 1.0% inositol and 1.0% dextrin Preparation
to 3.0% nutrient agar. (1) Prepare nutrient agar using Armour's
(2) Add litmus as an indicator. peptone.
Sterilization: Not specified. (2) Titrate carefully so that after one
Use: Differentiation of typhoid group. hour sterilization in the autoclave at
The authors reported that typhoid 15 pounds pressure, the reaction is
fermented dextrin with acid formation in neutral to phenolphthalein.
the butt of the tube, decolorizing entirely (3) Tube in 10.0 cc. lots.
in 24 hours with a violet slant. Para- (4) Cool sterile (3) to 50C. and add
typhoid A did not ferment either dextrin lactic acid in varying amounts to
each tube undiluted so that 0.1 cc. Variants: The author used one of the
equals 1.0% of acid. following instead of 10.0 cc. of fuchsin:
Safranin 10.0 cc.
(5) Add glucose.
Sterilization: Sterilize in autoclave at 15
Bismark brown 50.0 cc.
G. orange 60.0 cc.
pounds pressure one hour.
Neutral red 60.0 cc.
Use To show effect of lactic acid on growth
:
of organisms. Author reported that lactic

Reference: Miiller (1922 p. 1251).
acid had a varying degree of inhibitory

action on bacteria and fungi. Generally 1828. Kligler's Lead Acetate Glucose Agar
1.0% lactic acid inhibited growth. Constituents
Reference: MacDonald (1917 p. 322). 1. Nutrient agar (0.5%). 1000.0 cc.
2. Glucose (0.2%) 2.0 g.

1827. Miiller's Lactose Tartrate Agar 3. Lead acetate (0.05 to
0.1%) 0.5 to 1.0 g.
Constituents
Preparation
1. Bouillon
1000.0 cc.
(1) Add 0.05% to 0.1% lead acetate and
2. Lactose 25.0 g.
0.2% glucose to 0.5% nutrient agar.
3. Agar 30.0 g.
4. Potassium ferric tartrate
Use: Differentiation of typhoid and para-
5. Potassium ferrocyanide
typhoid bacilli. The author reported
6. Fuchsin
that B. typhi produced browning without
Preparation gas. B. paratyphi B. produced browning
(1) Dissolve 30.0
of agar and 25.0 g.
g.
with gas, B. paratyphi A. produced no
lactose in 1 liter of bouillon.
browning and gas and B. dysenteriae
(2) Neutralize and then add 10.0 to 12.0 produced no browning and no gas.
cc. of normal NaOH to (1).
(3) Prepare a 10.0% solution of potassium
ferrocyanide. 1829. MacConkey's Lactose Bile Salt Agar
(4) Prepare a 10.0% solution of potassium (Heinemann)
ferric tartrate.
cc. of sterile (4) to 1000.0 cc.
Constituents
(5) Add 50.0
Nutrient agar 100.0 cc.
of sterile (2). Mix well. 1.
Mix 2. Sodium taurocholate (0.5%). 0.5 g.

(6) Add 50.0 cc. of sterile (3) to (5).
3. Peptone (2.0%) 2.0 g.
well.
Lactose (2.0%) 2.0 g.
(7) Color the agar by the addition of 4.
Preparation
10.0 cc. fuchsin solution.
Distribute as desired in sterile con- (1) Prepare nutrient agar.
(8)
tainers. (2) Add 2 and 3 to (1).
the usual (3) Boil.

Sterilization: Sterilize (2) in
The lactose (4) Clarify.
manner (method not given) .
(5) Filter.
may be sterilized at 100C. in water and
(6) Add 2.0% lactose.
then added to the sterile agar if desired.
Tube.
Sterilize (3) by heating at 100 for 30
(7)
minutes. Sterilize (4) by heating at
100 C. for 30 minutes.
typhoid Reference: Heinemann (1905 p. 128).
Use: Differentiation colon
of
bacilli. The author gave the following 1830. Fleming's Oleic Acid Glycerol Agar
reactions:
Constituents
With fuchsin, typhoid blue; coli red. 100.0 cc.
Nutrient agar
With safranin, typhoid blue; coli orange.
1.
Glycerol (2.0%) 2.0 g.

With Bismark brown, typhoid brown,
2.
3. Oleic acid (0.1%) 0.1 g.
coli bright green.
brown, coli Preparation
With G. orange, typhoid
(1) Neutralize nutrient agar
with HCl.
bright green.
(2) Add 2.0% glycerol and 0.1% oleic 1832. Rosenow's Glucose Brain Agar
acid to (1).
(Haden)
(3) Tube. Constituents
Sterilization: Sterilize in the autoclave in 1. Water 1000.0 cc.
the usual way. (Details not given.) 2. Bacto nutrient broth 8.0 g.
Use: Cultivation of the bacillus of acne 3. NaCl 8.0 g.
vulgaris (Bacillus acne). 4. Glucose (c.p.) 2.0 g.
Reference: Fleming (1909 p. 1036). 5. Andrade indicator 10.0 cc.

6. Brain, calf
1831. Rosenow and Towne's Ascitic Fluid 7. Marble
Kidney Agar 8. Agar 7.0 g.
Preparation
Constituents
1. Water 1000.0 cc.
Cool and add 4 and 5.
(2)
2. Peptone (Witte's) Tube in 6 by 0.75 inch test tubes so
(3)
3. Meat extract (Liebig's)
that the depth is at least 3.4 to 4.0
4. Glucose 10.0 g.
inch.
5. Ascitic fluid (10.0%) 100.0 cc.
(4) Add 3 pieces of crushed calf brain
6. Rabbit kidney, sterile
about 1 cmm. square and two or three
7. Agar 25.0 g.
pieces of crushed marble to each tube.
Preparation Sterilization: Autoclave for 20 minutes at
(1) Prepare an extract broth from water, 15 pounds pressure.
Liebig's Beef Extract and Witte's Use: Isolation of bacteria from infected
peptone (amounts not given). teeth.
(2) Titrate and adjust the reaction of Reference: Haden (1923 p. 831).
(1) to 0.6-0.8 acid to phenolphthalein.
(3) Add and dissolve 1.0% glucose and 1833. Goldberger's Glucose Alkaline Egg
2.5% agar in (2). Agar (Abbott)
(4) Add 10.0% sterile ascitic fluid to Constituents
sterile (3). 1. Distilled water 1000.0 cc.
(5) Distribute in tall test tubes (column 2. Egg 1.0
of medium measuring 0.8 cc. in
3. Meat extract (Liebig's) 3.0 g.
diameter and 13.0 cc. in height. 4. Peptone (Witte's) 10.0 g.
(6) Add a piece of fresh sterile rabbit 5. NaCl (c.p.) 5.0 g.
kidney. 6. Glucose 1.0 g.
(7) Add a layer of sterile mineral oil.
7. Agar 30.0 g.
Sterilization: Method of sterilization of Preparation
(3) not given. (1) Mix a whole egg with an equal
Use: Cultivation pleomorphic strepto-
of volume of distilled water.
cocci causing poliomyelitis. The author (2) Mix one volume of (1) with an
reported that initial growth could best equal volume of 6.5% solution of
be obtained without the addition of the anhydrous NajCOa.
kidney. (3) Steam for 30 to 60 minutes.
Variants: The authors prepared the (4) Mix 3, 4, 5, 6 and 7 in 1000.0 cc. of
medium as indicated in step (1) thru (4), water.

omitting the agar. One part of this (5) Steam for 3 hours to insure complete
was mixed with 1.5%
ascitic fluid broth solution.
agar, tubed and a piece of kidney added (6) Decant or filter thru cotton.
to each tube. A layer of sterile mineral (7) Distribute in 100.0 cc. quantities in
oil was added to each tube. flasks.

Reference: Rosenow and Towne (1917 p. (8) Mix one volume of (3) with five
177). volumes of sterile (7). The agar

should be completely liquified by Sterilization: Sterilize (4) in the Arnold for

steam. 30 minutes. Sterilization of extract agar
(9) When thoroly mixed, pour into not specified.
Petri dishes. Use: Cultivation of cholera organisms.
(10) Place in the incubator with covers Stitt reported that cholera colonies
partially removed. were clear, round and showed a brownish
Sterilization: Sterilize (7) by steaming for center. They did not show the striking
90 minutes. bluish opalescence as on ordinary plates.
Use: Diagnosis of cholera. Abbott re- Reference: Stitt (1923 p. 50).
ported that cholera vibrio and cholera-
1835. Dimitroff's Egg Agar
like organisms grew luxuriantly while
other organisms were restrained. Constituents
Variants: Krumwiede (Park, Williams and 1. Extract agar 750.0 cc.
Krumwiede) prepared a similar medium 2. Extract broth 250.0 cc.
as follows: 3. Egg white
(1) Mix equal parts egg and water. Preparation

(2) Mix equal parts (1) and a 12.0% (1) Place a small cube of hard boiled egg
Na2C03 (crystalline) solution. white in 10.0 cc. of beef extract broth.
(3) Steam in the Arnold sterilizer for 20 (2) Following sterilization of (1), mix
minutes. 25.0 cc. of (1) in 75.0 cc. of beef extract
(4) Prepare a 3.0% beef extract peptone agar.
(Fairchild's) agar. Do not adjust Sterilization: Method of sterilization not
the reaction. The size of the colonies given.
is increased if 0.2% glucose be added. Use: Cultivation of Spirillum virginianum,
(5) Mix 20 parts (3) to 80 parts (4). The author reported that the spirilla
Sterilization not specified. produced small dew-drop, convex, glisten-
(6) Pour in plates. ing, slightly opaque colonies.
(7) Dry for 20 minutes. Reference: Dimitroff (1926 p. 22).

Reference: Abbott (1921 p. 571), Park,
1836. Dunschmann's Bile Salt Gelatin Agar
Williams and Krumwiede (1924 p. 130).
Constituents
1834. Goldberg's Meat Infusion Extract
1. Meat extract 1000.0 cc.
Agar (Stitt)
Agar
2. (3.0 to 4.0%) . . . 30.0 to 40.0 g.
Constituents 3. Gelatin (0.5%) 5.0 g.
1. Extract agar (3.0%) 1500.0 cc. 4. Sodium taurocholate
2. Water 500.0 cc. (1.5 to 2.5%) 15.0 to 20.0 g.
3. Beef 500.0 g. 5. Lactose (4.0%) 40.0 g.
Preparation 6. Peptone (5.0%) 50.0 g.
(1) Treat 500.0 g. lean beef with 500.0 g. 7. Litmus

water. Temperature not specified. Preparation
(2) After 3 hours strain. (1) Prepare a meat extract with Liebig's
(3) Adjust the reaction to neutral by the beef extract (or by extracting 500.0 g.
addition of 5.3% anhydrous Na2C03 of veal with 1 liter of water).

solution. (2) Dissolve 2, 3, 4, 6 and 6 in (1).
(4) Add 2.5 cc. of 5.3% anhydrous NaoCOa (3) Distribute in flasks or Petri dishes
solution for each 100.0 cc. of infusion. and add 10.0% sensitive litmus
(5) Filter sterile (4). (strength of solution not specified).
(6) Prepare a 3.0% meat extract agar. Sterilization: Not specified.
(7) Mix one volume of (5) with 3 volumes Use: Enrichment medium for typhoid
of hot melted sterile (6). bacilli. Author reported that mineral
(8) Pour plates. salts may be used as a basis for this
(9) Cover the plates with a piece of filter medium instead of extract or infusion
paper and place in the incubator for broth.
30 minutes until they are dry. Reference: Dunschmann (1909 p. 64).
1837. Paneth's Glucose Ascitic Fluid Agar 1838. Plotz, Olitsky and Baehr's Ascitic
Fluid Agar
Constituents
2. Liebig's meat extract 12.0 g. 1. Glucose agar (2.0%) 1000.0 cc.
NaCl 15.0 g. 2. Ascitic fluid 200.0 cc.

3.
4. Peptone (Witte's) 30.0 g. 3. Plain agar
5. N/IH2SO4 17.5 cc. Preparation
6. Agar 85.0 g. (1) Prepare nutrient agar using Liebig's
7. Glucose 50.0 g. meat extract and containing 2.0%

glucose and 2.0% agar.
8. Ascitic fluid
(2) Distribute in 20.0 cc. lots in tubes
Preparation
15.0 cm. X 2 cm.
(3) Cool the sterile melted agar tubes to
(2) Add 6 to (1).
40C. and add to each tube 4.0 cc.
(3) Allow to stand for several hours.
ascitic fluid and 2.0 cc. of infected
(4) Boil in the steamer for two hours.
blood.
(5) Filter thru gauze.
(4) Mix by pouring back and forth into
(6) Mix the whites of eight eggs with
avoiding air bubbles.
sterile test tube,
(5) When the agar has solidified add
(7) Cool the liquid agar to 50, add (6)
sterile plain agar to a depth of 2.0
and mix well.
cm. to each tube.
(8) Pour into flasks and heat at 100 in
(6) Incubate at 37.5C.
the steamer for two hours.
(9) Filter thru wet cotton. Gauze is
Use: Cultivation of B. typhi exanthematici
placed on top of the cotton.
(supposed cause of typhus fever), from
(10) Adjust the reaction from 1.8 to 2.2.
blood.
(11) Add 2.0% glucose (about 50.0 g.).
Variants Baehr and Plotz gave the follow-
:
(12) Heat for 20 minutes in a steamer.

ing method of preparation:
(13) Distribute into large test tubes in
(1) Mix 12.0 g. Liebig's meat extract,
about 15.0 to 20.0 cc. lots.
15.0 g. NaCl, 30.0 g. Witte's peptone,
(14) Distribute sterile ascitic fluid into
17.5 g. N/1 H2SO4, and 85.0 g. agar
large sterile test tubes.
shreds in 3000.0 cc. water.
(15) Liquify the agar and cool to 40 to
(2) Place in an Arnold steam sterilizer
42C.
for two hours.
(16) Draw, under aseptic conditions,
(3) Filter rapidly thru several layers of
about 2.0 cc. of infected blood di-
gauze to remove water.
rectly into the liquefied agar and
(4) Mix the whites of eight eggs with
add one tube of ascitic fluid (amount
not specified) to the agar. Mix.
(5) Cool (3).
(In the work of Plotz, Olitsky and
(6) Mix and (4) and shake thoroly.
(5)
Baehr, 2.0 cc. of ascitic fluid is
(7) Place in Arnold sterilizer (100C.)
added.)
fortwo hours.
(17) After cooling cover with plain agar
(8) Decant clear agar and filter thru
or paraffin.
moistened absorbent cotton.
minutes
Sterilization: Sterilize (13) for 20 Adjust to acidity of 0.9 to 1.1 to
(9)
in the autoclave at 10.0 kilograms pres- phenolphthalein.
sure or in the steamer on two or better (10) Add 2.0% glucose to (9).
four successive days for 20 minutes each (11) Distribute into 2 x 20 cm. test tubes
day. in about 20.0 cc. lots (half full).
Use: Cultivation of Bacterium typhi (12) Sterilize (11) at 10.0 kg. pressure for
exanthematis. 20 minutes or in an Arnold on 3 or
Reference: Paneth (1916 p. 647). 4 successive days for 20 minutes.
(13) Remove blood aseptically with a cal changes did not appear except near
syringe from the median basilic or the limiting pH values.
median cephalic veins of patient Reference: Reed and Orr (1923 p. 104).
(15.0 cc).
(14) Divide the 15.0 cc. blood among eight 1840. Becker's Defibrinated Blood Agar
sterile melted tubes of (11), 2.0 cc.
per tube. Constituents
(15) Add ascitic fluid to each tube of 1. Extract agar 1000.0 cc.
(13), i to J volume (i.e., 6-10.0 cc.) 2. Blood, defibrinated 175.0 cc.

ascitic fluid must be clear, free from Preparation
bile or blood pigment and have a (1) Prepare extract agar according to
specific gravity of more than 1.015. Standard Methods, see medium 1695,
Must not be filtered, sterilized or and distribute in 40.0 cc. lots in 100.0
contain preservatives. cc. flasks.
(16) Mix the content of each tube thoroly (2) Melt agar and cool to 45-50 C.
by pouring once or twice into (3) Add 1.0 cc. of defibrinated human
another sterile tube. Avoid air blood per 6.0 cc. agar.
bubbles. (4) Mix thoroly.
(17) After thoro solidification cover each Sterilization: Method not specified.
one of the (15) with 2 or 3 cm. of Use: To isolate streptococci, pneumococci

sterile melted agar (11). and gonococci.
(18) To prevent drying, paraffin the Reference: Becker (1916 p. 759), Tanner
cotton stopper of each tube. (1919 p. 69).
References: Plotz, Olitsky and Baehr

(1915 p. 6), Baehr and Plotz (1917 p. 203). 1841. Esch's Alkaline Hemoglobin Ragit
Agar
1839. Reed and Orr's Blood Agar
Constituents
1. Water 1000.0 cc. 2. NaOH (normal 7.5 cc.
2. Peptone 5.0 g. 3. Hemoglobin (Merck) 2.5 g.
3. Agar 15.0 g. 4. Ragit agar 85.0 cc.
4. Beef extract 3.0 g. Preparation
5. Rabbits blood (whole) 10.0 cc. (1) Grind 2.5 g. Merck's hemoglobin to a
6. Phosphates to 0.05M powder in a mortar.
Preparation (2) Place in a balloon flask and add 7.5
(1) Prepare beef extract peptone agar cc. of N/1 caustic soda solution and
according to Committee A. P. H. A. 7.5 cc. of distilled water.

(1916-1917). (See medium 1695.) (3) Heat by means of a gas burner and
(2) Adjust with NaOH to pH = 7.4. stir strongly to obtain complete
(3) Add phosphate mixture of pH = solution of the hemoglobin which
7.4 to make finished medium 0.05M requires about 10 minutes.
phosphate. (4) Heat (3) for one hour in a Koch
(4) Add whole rabbits blood to sterile steamer, or for 15 minutes in an auto-
(3) cooled to 80C. to make finished clave at one atmosphere pressure.
medium 1.0% blood. (5) Prepare "Ragit agar."
(5) Maintain at 80C. for 10 minutes. (6) Melt sterile (5) and add (4) to 85.0
(6) Tube or plate. cc. of the agar.

Sterilization: Sterilize in the autoclave. (7) Mix well and distribute into sterile
Use: To maintain cultures of Hemophilus petri dishes.

influenzae. Author reported that pH Sterilization : Step (4) above gives steriliza-
range of 6.8 to 7.4 gave very nearly tion of alkaline hemoglobin. Method of
maximum growth. Marked morphologi- sterilization of Ragit agar not given.

Use: Enrichment medium for cholera parent and light green. A great many
vibrio. of the intestinal forms were entirely
Reference: Galli-Valerio (1912 p. 550). inhibited.
Reference: Padlewsky (1908 p. 543).
1842. Padlewsky's Malachite Green Bile
Agar 1843. Fildes' Body Fluid Agar
1. Meat extract agar 1. Extract agar.
(3.0%) 1000.0 cc. 2. Ascitic fluid or other body fiuid.

3. Bile 35.0 cc. (1) Collect ascitic fluid in a large glass
4. Lactose (c.p.) 10.0 g. bottle and allow to stand in the ice
5. Malachite green chest for a day or two.

(1.0% aq. solution) (2) Decant or filter thru glass wool.
(Hochst) 5.0 cc. (3) Adjust the reaction to slightly

6. Sodium sulphite alkaline or neutral to litmus by the
(10.0% aq. pur. solu- addition of alkali or acid.

tion) 7.5 to 10.0 cc. (4) Distribute in measured glass stop-
Preparation pered bottles in 200.0 cc. lots. (The
Prepare a 3.0% meat extract agar. fit of the glass stopper must be
(1)
(Meat infusion may be used instead perfect.)
of meat extract). (5) Add chloroform to each bottle until
(2) Treat the ox bile with steam in a 0.5% chloroform has been added.
Koch's apparatus. (Time not (6) .Add a drop of sterile oil to the stop-
specified). per and fasten a dust cover tightly
(3) Filter the bile thru cotton. over the stopper.
(4) Add 2.0% peptone, 3.0%, (3) and (7) Place the bottles in the water bath
1.0% c.p. lactose to (1). for one hour at 45C. Shake
Adjust the reaction to slightly occasionally.
(5)
alkaline to litmus. (8) When bottles have cooled, remove a
(6) It is not necessary to filter the agar sample under aseptic conditions and
after the addition of the filtered bile. mix with agar. Incubate the mix-
(7) Distribute into flasks in 100 to 200 cc. ture at 37C. for 5 days to test
lots. sterility.
(8) When ready for use melt the sterile (9) Prepare ordinary lemco (or meat
agar, cool to 60 to 65 C. infusion) agar containing 2.5 to 3.0%
(9) Add to each 100.0 cc. of (8) 0.5 cc. agar.

of a 1.0% watery solution Hochst of (10) Measure (9) into 200.0 cc. flasks in
chemically pure crystalline mala- 150.0 cc. lots.
chite green, 0.5 cc. bile and 0.75 to (11) When ready for use melt in the
1.0 cc. of a 10.0% watery solution of steamer and treat in one of the
sodium sulphite (pur.) by means of a following two ways
pipette. (a) Pour into a sterile distributer
(10) Mix thoroly. maintained at 50C. by means

sterile Petri dishes and
Pour into of a water bath and add 50.0
(11)
each
cc. of ascitic fluid (8) for
allow the agar to solidify with the
cover of the dish removed. flask of agar. Mix and tube
(12) Dry in the incubator.
thru a "hooded" pipette.
Sterilization: Sterilize (7) by the fractional (b) Pour 50.0 cc. of ascitic fluid
method. (8) directly into a flask of

melted agar. Mix well, and
Use: Detection of typhoid group bacilli.
The author reported that typhoid colonies distribute into tubes or Petri
were colorless. The medium was trans- dishes.

Mix the ascitic fluid and agar at as not sterilize with the method
low a temperature as possible. This employed.)
aids the production of a transparent (5) Add chloroform to each bottle
medium. until 0.5% chloroform has been
Sterilization: Sterilize (10) in the auto- added.
clave. (6) Add a drop of sterile oil to the
Use: Cultivation of meningococci and stopper and fasten a dust cover
other pathogenic cocci. tightly over the stopper.
Variants (7) Place in the air incubator at 37 C.
(a) Fildes substituted serum for ascitic for 24 hours, shaking constantly.
fluid. The serum was prepared as (8) After this time, remove a sample
follows: under aseptic conditions and test
(1) Collect horse or ox blood in tall its sterility by mixing it with agar
sterile jars from the slaughter and incubating. (Time not speci-
house. fied.) If sterile do not heat longer
(2) Allow to stand until all the cor- for heating tends to darken the
puscles have deposited. It may medium.
require 14 days for this to take (9) Prepare ordinary lemco (or meat
place. After 5 days examine the infusion) agar containing 2.5 to
jars and pipette or siphon off the 3.0% agar.
clear serum. Do not disturb the (10) Measure (9) into 200.0 cc. flasks in
sediment. 150.0 cc. lots.
(3) Distribute the clear serum in 200.0 (11) Autoclave (time and pressure not
cc. lots in perfectly fitting glass given).
stoppered flasks. (12) Add 25.0% of sterile laked blood
(4) Add chloroform each bottle
to (8) to (11). (Method of distribu-
until 0.5% chloroform has been tion not specified.)
added. Reference: Fildes (1917 p. 492).
(5) Add a drop of sterile oil to the
stopper and fasten a dust cover
1844. Haner and Frost's Milk Body Fluid
tightly over the stopper.
Agar
(6) Place the bottles ip the water
bath for one hour at 45C. Shake Constituents
occasionally. 1. Nutrient agar 1.0%
(7) When the have cooled,
bottles (dehydratedDif co) 1000.0 . cc.
remove a sample under aseptic 2. Milk, sterile 1000.0 cc.
conditions and mix with agar. 3. Serum (horse or
Incubate the mixture at 37C. for rabbit 6,0 to 12.0%). 120.0 to 240.0 cc.
five days to test sterility. Preparation
(b) Fildes also prepared the medium as (1) Prepare 1.0% agar from Difco de-
follows: hydrated product.
(1) Collect ox or horse blood from a (2) Add equal part of sterile milk to
slaughter house. sterile (1).
(2) Defibrinate the blood by strirring (3) Add 6.0% to 12.0% of sterile horse or
with a sterilized large wooden rabbit serum to (2).
stick wrapped in gauze. Sterilization: Method of sterilization not
(3) Lake the blood by the addition of given.
an equal volume of distilled water. Use: Cultivation of pneumococci and
(4) Distribute in 200.0 cc. lots into streptococci.
perfectly fitting glass stoppered Variants: The authors used whole blood
bottles. (The
author mentions instead of serun.
that the medium should contain Reference: Haner and Frost (1921
no suspended material, or it will p. 270).
1845. Bacto Conradi-Drigalski Agar (3) Add 1.0% peptone, 5.0% beef extract
(Dehydrated) and 1.2% agar to the filtrate.
(4) Heat to boiling point until all the
Constituents:
agar is dissolved.
1. Distilled water.
(5) Correct the reaction to 1.2 using
phenolphthalein as an indicator.
3. Sodium caseinate, Bacto. . 10.0 g.
(6) Cool to 45C. and add the whites of an
4. Lactose, Bacto 10.0 g.
egg.
5. Agar, Bacto 15.0 g.
(7) Heat to lOO^C. for 45 minutes and
6. NaCl 5.0 g.
filterthru cotton.
7. Dibromcresolsulphoneph-
(8) Correct the reaction if necessary and
thalein 0.03 g.
tube.
8. Crystal violet 0.02 g.
Sterilization : Sterilize three successive days
Preparation
in the Arnold sterilizer.
(1) Dissolve 50.0 g. of Bacto
Conradi-
Use: Cultivation of bacteria found in
Drigalski Agar (dehydrated) in 1000.0
cheese. Author reported that bulgaricus
cc. of distilled water by boiling or
types grew well on this medium.
autoclaving.
Reference: Eldredge and Rogers (1914
(2) Restore any lost weight.
p. 8).
20 minutes pH = 6.8. 1848. Ayres and Mudge's Milk Powder
Sterilization: Sterilize at 15 pounds pres- Agar
Use: The author reported that acid produc- Constituents:
ing colonies were yellow. 1. Water 500.0 cc.
Reference: Digestive Ferments Co. (1925 2. Skimmed milk powder. 5.0 g.
p. 12).
3. Na.HPOi + 2HoO
(Sorensen's phosphate). 1.0 g.
1846. Haner and Frost's Milk Agar 4. Peptone (Difco) 2.0 or 5.0 g.
Constituents 5. Liebig's extract 1.0 or 3.0 g.
1. 1.0% nutrient agar (De- 6. Agar 3.0% 500.0 cc.
hydrated Difco) 1000.0 cc. Preparation

2. Milk, sterile 1000.0 cc. (1) Pour 5.0 g. skimmed milk powder
Preparation into 20.0 cc. cold distilled water.
Prepare 1.0% agar from dehydrated Stir until dissolved.
(1)
Difco product. (2) Dissolve by heat, 1.0 g. Sorensen's
(2) Add an equal part of sterile milk to phosphate in 5.0 cc. distilled water.
sterile (1). (3) Add (2) to (1), place in 30C. water
Sterilization: Method not given. bath and heat to 60 in 10 minutes.

Use: Cultivation of staphylococci. A flocculent grayish precipitate
Reference: Haner and Frost (1921 p. 270). appears.
(4) Steam until precipitate gets white.
1847: Eldredge and Roger's Whey Agar Dilute about \ with distilled water
(5)
Constituents and steam 5 more minutes.

1. Whey 1000.0 cc. (6) Decant solution and filter, keeping
2. Lactic acid (10.0%) 20.0 cc. most of the precipitate in beaker
3. Peptone (1.0%) 10.0 g. until most of the liquid has passed
4. Beef extract (5.0%) 50.0 g. thru the filter. Then wash precipi-
5. Agar (1.2%) 12.0 g. tate with a little distilled water.
Preparation The filtrate is cloudy.
(1) Heat one liter of skim milk to boiling (7) Make up (6) to 250.0 cc. with dis-
and add 20.0 cc. of a 10.0% lactic acid tilled water.
solution. (8) Dissolve 5.0 g. peptone and 3.0 g.

Liebig's extract in 100.0 cc. distilled
(2) Filter.
water by steaming in Arnold, or (5) Heat in an Arnold sterilizer 10

boiling over flame. minutes to obtain thoro mixing.
(9) Filter until clear and make up to (6) Distribute in tubes or bottles.
250.0 cc. with distilled water. Sterilization: Sterilize in the Arnold
(10) Mix (7) and (9). sterilizer.
(11) Place 30.0 g. agar in 2000.0 cc. dis- Use: Differentiation of colon-typhoid
tilled water and let stand 24 hours. group. Author reported that acid pro-
(12) Pour ofE water and add 2000.0 cc. duction indicated by black colonies, or
more water. colonies having black centers. Omit the
(13) Allow to stand 24 hours, pour on saccharose when used for dysentery
cotton flannel cloth in funnel and isolation. B. dysenteriae (Shiga) may
wash once more with one liter dis- fail to grow on this medium. Omit the
tilled water. Press out as much crystal violet if to be used for dysentery.
water as possible. Reference: Park, Williams and Krumwiede
(14) Add enough water to have weight of (1924 p. 128).
1030.0 cc. of agar and water.
(15) Dissolve in Arnold and filter thru 1850. Tausz and Peter's Ragit Nutrose Agar
cotton flannel until clear.
(16) Add 500.0 cc. of (15) to (10). Constituents
Sterilization: Method not given. 1. Water 1000.0 cc.
Use: To determine number of bacteria in 2. Ragit nutrose agar 52.0 g.
milk. Authors reported that the medium 3. Congo red tablets (Merck) . 18.0 g.
gave high count. Colonies were large Preparation
and easy to count. (1) Boil 52.0 g. ragit nutrose agar in one
Variants: The authors specified that indi- liter of water for one hour in the
cators and glucose or lactose might be steamer. Shake occasionally.
added if desired. (2) Filter.
Reference: Ayres and Mudge (1920 p. 568). (3) Distribute in 100.0 cc. lots.
(4) To each 100.0 cc. of (3) add 1.8 g. of
1849. Conradi-Drigalski's Crystal Violet
Congo red tablets (Merck). The
Litmus Agar (Park, Williams
tablets contain lactose and congo red.
and Krumwiede)
(5) Boil for 15 or 20 minutes.
Constituents: (6) Pour into Petri dishes.
2. Agar 20.0 g. Use: Cultivation of Bacterium aliphaii-
3. NaCl 5.0 g. cum, Bacterium aliphaticum liquefaciens,
4. Peptone 20.0 g. paraffin bacteria. Authors reported that
5. Nutrose 10.0 g. ragit nutrose agar is a commercial
6. Beef extract (Liebig's) 4.0 g. powder. Congo red tablets contain lac-
7. NaOH (N/1) 50.0 cc. tose and Congo red.
8. Litmus (Kubel and Thie- Reference: Tausz and Peter (1919
mann's) 130.0 cc. p. 508).
9. Crystal violet (1 1000) ....: 10.0 cc.
10. Lactose 15.0 g. 1851. Hunter's Trypsinized Casein Extract
Preparation Agar
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1 in an
autoclave. Constituents
(2) Cool and clarify with eggs. 1. Water 1000.0 cc.
(3) Adjust the reaction moderately but 2. Casein 15.0 g.
distinctly alkaline to litmus. 3. Peptone 15.0 g.
(4) To each add 130.0 cc. of
liter of (3) 4. Beef extract 3.0 g.
Kubel and Thiemann's litmus solu- 5. Agar 15.0 g.
tion, 10.0 cc. of a 1 to 1000 solution 6. Lactose 15.0 g.
of crystal violet, and 15.0 g. of lactose. 7. Sodium oleate (2.0% soln.) . 50.0 cc.
Preparation (9) Filter the milk phosphate solution
(1) Dissolve 2, 3, and 4 in 1. thru a small piece of absorbent

(2) Add 1.5 g. trypsin and make slightly cotton placed in the neck of a funnel.
alkaline. (10) Add 5.0 g. lactose to filtered (9) and
(3) Add chloroform. make up to one liter by the addition
Incubate at 37C. for 48 hours and of distilled water.
(4)
then boil several minutes to remove (11) Mix (10) and the agar from (8).
the chloroform. (12) Distribute as desired.
(5) Add 15.0 g. agar. Sterilization: Autoclave at 15 pounds pres-

(6) Adjust the reaction to pH 7.0 and add sure for 15 minutes, or sterilize frac-
15.0 g. lactose and 50.0 cc. of a 2.0% tionally by heating at 5 pounds pressure
sodium oleate solution. (In another for 10 minutes on each of 2 successive days
part of the article the author states holding the medium at 30C. between
that best growth was obtained with heating.
about sodium
1.0% oleate. The Use: Qualitative milk counts.
percentage used above is only 0.1%. Reference: Zoller (1923 p. 385).
(7) Filter. Agar
1853. Smillie's Tissue Infusion
(8) Tube.
Sterilization: Sterilize in the autoclave. Constituents
Use: Cultivation of Lactobacillus hulgari- 1. 2.0% veal infusion agar 100.0 cc.
cus and Lactobacillus acidophilus. 2. Ascitic fluid 200.0 cc.
Reference: Hunter (1924 p. 3). 3. Rabbit tissue (kidney)

Preparation
1852. Zoller's Citrate Milk Agar
(1) Prepare 2.0% veal infusion agar.
Constituents: (2) Adjust (1) to 4-0.5 to phenolphthalein
1. Distilled water 5000.0 cc. (3) When ready for use, melt sterile (2),
2. Bacto Nutrient Agar (1.0%) 120.0 g. boil and cool rapidly to 50C.
3. Sodium citrate 2.0 g. (4) Warm, straw colored, bile

clear,
4. Milk (skimmed) powder. . . 25.0 g. free, ascitic having a specific

fluid
5. Na2HPOr2H20 6.0 g. gravity of 1.015 or higher to 45C.

6. Lactose 5.0 g. (5) Place in a vacuum to remove air.
Preparation (6) Mix one part melted (3) at 50C. and
(1) Stir 120.0 g. of Bacto nutrient agar two parts (5) at 45C.
(1.0%) into 4 liters of distilled water. (7) Add 15.0 cc. of (6) to a sterile tube
Add 2.0 g. sodium citrate and set in containing sterile rabbit tissue
(2)
streaming steam in the autoclave (kidney) (and the poliomyelitis
for about 5 minutes. materials).
(3) Stir 25.0 g. of any good grade of Sterilization: Method not specified.
milk powder in 100.0 cc. distilled Use: Cultivation of globoid bodies of
water. poliomyelitis. Author reported that the

(4) Stir 6.0 g. Na2HP04-2H20 in 30.0 organisms did not attack any added
cc. of distilled water and heat to carbohydrate. Other investigators used
boiling. similar media for the cultivation of
(5) Add (4) to (3) and steam for 5 anaerobes.

minutes at the same time the agar is Variants
being steamed. (a) Olitsky and Gates cultivated ana-
(6) Remove the agar from the autoclave erobic organisms found in influenza
and stir. on a medium prepared as follows:
(7) Add 50.0 cc. distilled water to (5) (1) Prepare a 2.0% beef infusion agar.
following steaming. (2) Adjust (1) to pH = 7.4.
(8) Replace (6) and (7) in the autoclave (3) Mix one part (2) with two parts
and heat for 5 minutes at 5 pounds sterile human ascitic fluid in a
pressure. flask at 40C.
(4) Pipette (3) into sterile tall test (8) Add sterile chalk in excess (about 5
tubes containing pieces of sterile spoonfulls) and distribute into sterile
rabbit kidney and the inoculum. tubes.
(5) Seal with sterile melted vaseline, (9) Add a sterile piece of liver to each
(b) Harvey cultivated anaerobic spiro- tube.
chaetes on the following medium: Sterilization: Sterilize at 100C. for one
(1) Transfer small portions of organs hour and slant.
aseptically removed from a rabbit, Use: Cultivation of Bad. butyricus and
specially killed for the purpose other anaerobes.
(2514 variant (a), to each sterile Reference: van Riemsdyk (1922 p. 248).
test tube 20 x 2 cm.
Mix one part infusion agar, 1855. Williams' Tissue Infusion Agar
(2)
1.0% acid to phenolphthalein at Constituents
45C. (see medium 1661 variant (v) 1. Water, tap 90.0 cc.
for preparation) with one part 2. Agar (1.0%) 1.0 g.
sterile ascitic fluid heated to 45C. 3. Infusion broth (10.0%) 10.0 cc.
(3) Fill, with sterile precautions, about 4. Brain
20.0 cc. of (2) into each test tube Preparation
(1). (1) Mix 90.0 cc. tap water, 10.0% ordinary
(4) Cover the medium to a depth of infusion broth and 1.0% agar.
3 cm. with sterile liquid paraffin. (2) Dissolve.
(5) Test sterility by incubation 48 (3) Reaction neutral to phenolphthalein.
hours. (4) Cut fresh sterile brain in small pieces
References: Smillie (1918 p. 324), Olitsky and place on the surface of sterile (3).
and Gates (1921 p. 715), Harvey (1921-22 Sterilization: Sterilize as usual (method
p. 97). not given).
Use: Cultivation of amoeba.
1854. van Riemsdyk's Liver Infusion Agar
Variants: Park, Williams and Krumwiede
Constituents substituted liver or kidney for brain.
1. Water 500.0 cc. Reference: Park, Williams and Krumwiede
2. Beef liver 0.5 lb. (1924 p. 134).
4. NaCl 2.5 g.
1856. Olitsky and Gates' Ascitic Fluid
5. Agar 10.0 g.
Kidney Agar
6. 2.0% glucose agar 500.0 cc. Constituents
7. Liver tissue 1. Beef infusion broth.
8. Glucose 10.0 g. 2. Beef infusion (2.0%) agar.
9. Chalk 3. Human ascitic fiuid.
Preparation 4. Rabbit kidney.
(1) Pass raw beef liver thru a fine meat Preparation
grinding machine. (1) Prepare beef infusion broth and 2.0%
(2) Add 500.0 cc. of water to pound of beef infusion agar.
(1). (2) Adjust both the broth and agar to
(3) Boil for 30 minutes with occasional pH = 7.4.
stirring. Allow to settle. (3) Place in each 50.0 cc. Florence flask
(4) Pour off the liquid and force the liver i of a moderate size rabbit kidney.
thru a very fine metallic sieve. Sections are cut across the entire
(5) Add the pulverized liver and 3, 4 and kidney and placed with the cut sur-
5 to the liquid. face parallel to the base of the flask.
(6) Heat for one hour at 110C. and make (4) Inoculate the kidney.
alkaline to phenolphthalein using (5) Mix one part (2.0%) infusion agar,
N/10 NaOH. (2), with two parts sterile human
(7) Add 10,0 g. of glucose and mix with ascitic fluid of pH = 7.8 to 8.0, in a
an equal volume of 2.0% glucose agar. flask at 40C.
each 1858. Avery's Oleate Blood Cell Agar

(6) Add 100. cc. of (5) to flask.
the agar by immersing the (Harvey)

(7) Solidify
flask in cold water for 15 minutes. Constituents
(8) Mix one part infusion
broth (2) and 1. Infusion agar 950.0 cc.
two parts sterile ascitic fluid. 2. Sodium oleate (2.0% neutral
(9) Fill each flask (7) to the neck
with solution) 50.0 cc.
the mixture of (8). 3. Erythrocytes, human 10.0 cc.
(10) Seal with a layer of sterile vaseline Preparation

1.0 cm. deep. (1) Prepare infusion agar according to
Sterilization: Methods of sterilization not Harvey's method (see variant (v)
specified. medium 1661).
Use: Mass cultivation of anaerobic (2) Adjust (1) to 0.4% acid to phenol-
organisms found in influenzae. phthalein and heat to 45 C.
Reference: Olitsky and Gates (1921 p. 716). (3) Mix 950.0 cc. (2) with 50.0 cc. of a
2.0% neutral sodium oleate solution,
1857. Pelouze and Viteri's Brain Veal and 10.0 cc. of sterile washed human
Infusion Agar erythrocytes, both heated to 45C.
Constituents Use: Cultivation of B. influenzae.
1. Dist. water 500.0 cc. Variants: Park, Williams and Krumwiede
2. Brain, calf 500.0 g. prepared a similar medium as follows:
3. KH2PO4 (0.5%) 2.5 g. (1) Remove the corpuscles from sterile
4. Peptone (1.0%) 5.0 g. defibrinated human or rabbit blood
5. Veal infusion agar (2.5%) by centrifugation.
Preparation (2) Discard the supernatant fluid by
(1) Force 500.0 g. of calf brain thru a pipetting off.
wide meshed gauze into 500.0 cc. (3) Make up the original volume of (1)
distilled water and place in the ice by the addition of sterile infusion
box for 24 hours. broth.
(2) Filter thru cotton of varying degrees (4) Prepare a 2.0% solution of neutral
of compactness. The filtrate will sodium oleate. (Kahlbaum's pre-
not be clear. ferred.)
(3) Add 0.5%, KH2PO4 and 1.0% peptone. (5) Sterilize (4) in the autoclave.
(4) Prepare standard 2.5% agar from veal (6) To 94.0 cc. of hot 2.0%, nutrient agar
infusion with the addition of 0.5% (vitamin agar, see variant (e) medium
NaCl and 1.0% peptone. 1863 is recommended) add 5.0 cc. of
(5) Mix one part (2) with three parts (4). (5) and add 1.0 cc. of (3) under
(6) Adjust to pH 7.8. aseptic conditions.
(7) Tube. References: Harvey (1921-22 p. 87), Park,
(8) Following sterilization and solidify- Williams and Krumwiede (1924 p. 130).
ing replace the cotton plug with a
1859. Brown and Orcutt's Blood Cell Agar
sterile rubber cork.
Sterilization: Sterilize in the autoclave. Constituents:
Use: Cultivation of gonococci. 1. Veal infusion agar.
Variants: Following the mixing of the 2. Blood corpuscles.

brain and agar the medium may be Preparation
(1) Prepare veal infusion
agar.
placed in the autoclave, brought to 15
pounds pressure quickly and then filtered (2) Defibrinate blood and wash re-
before tubing and sterilizing. This proc- peatedly with sterile physiological
ess gives a clear medium but gives also a salt solution.
scanty growth. The author does not (3) Add some of the washed blood cor-
recommend the medium.

clear puscles to melted (1), (amounts not
Reference: Pelouze and Viteri (1926 p. given) and pour into plates.
685). Sterilization: Not specified.
Use: To determine food requirements for (8) Dilute the red blood cells, freed
Bacillus pyogenes. Authors reported from serum, with 0.5% NaCl solution
that this medium supported the growth to the original blood volume.
of Bacillus pyogenes nearly as well as did (9) Shortly before use melt the desired
blood agar. tubes of sterile agar, cool to 60C.
Variants: The authors prepared a similar and add two volumes of (8) to each
medium as follows: tube.
(1) Prepare veal infusion agar. (10) Mix well and solidify in slanting
(2) Defibrinate blood and wash repeat- position.
edly with sterile physiological salt Sterilization: Sterilize (6) in autoclave by
solution. heating to 105-108 for 15 minutes.
(3) Lake thewashed corpuscles with Use: Special culture medium for Trypano-
distilled water and remove the cor- soma Brucei.
puscle stroma by centrifugation. Reference: Behrens (1914 p. 29).
(Centrifuge the laked blood cor-
1861. Sacquepee and Delater's Egg Albumin
puscles until the supernatant hemo-
Infusion Agar
globin solution no longer gives a
clouding reaction with salt.) Constituents
(4) Wash the corpuscle stroma repeatedly 1. Distilled water
in sterile distilled water until no 2. Egg white
visible trace of hemoglobin remains. 3. Meat 500.0 g.
(5) Add the stroma suspension to melted 4. Peptone 10.0 g.
agar (amount not given) and pour into 5. NaCl 5.0 g.
plates. 6. Agar 30.0 g.
This medium was inferior to blood cell Preparation
medium above. (1) Place the whites of two eggs in a
Reference: Brown and Orcutt (1920 pp. graduated glass cylinder.
222-223). (2) Shake constantly and add little by
littlethree times the volume of
1860. Behren's Blood Cell Agar
distilled water.
Constituents (3) Make alkaline by the addition of
1. Water 1000.0 cc. about 0.5 cc. of a 10.0% soda solution
2. Chopped beef 125.0 g. for each 100.0 cc. of (2).
3. Peptone 20.0 g. (4) Shake well.
4. NaCl 5.0 g. (5) Heat in the autoclave at 115 for 15
5. CaCl, 0.1 g. minutes.
6. N/1 NaaCOs solution 10.0 cc. (6) Macerate 500.0 g. of meat in a liter of
7. Agar 20.0 g. water.
8. Red blood cells from 2000.0 (7) Allow to stand in the cold for 24
cc. Rabbit blood hours and then bring slowly to boil.
(1) Digest chopped beef in 250.0 cc. (9) Dissolve 10.0 g. peptone, 5.0 g.
water over night in the cold, or for NaCl in (8).
one hour at 55C. (10) Dissolve 30.0 g. agar in (9).
(2) Strain (1), boil the infusion and (11) Exactly neutralize to litmus and
filter then add 9.0 cc. of a 10.0% soda
(3) Dialyze the filtrate in a large col- solution.
lodium sac against running distilled (12) Clarify by the addition of an egg
water for 24 to 48 hours. white to the agar at 60C. Heat in
(4) Dilute (3) to 1000.0 cc. the autoclave and filter thru paper.
(5) Dissolve 3, 4, 5, 6 and 7 in (4). (13) Mix (5) and (12). Approximately
(6) Distribute in test tubes in 1.0 cc. one part (5) to five parts (12).
lots. (14) Distribute as desired.
(7) Centrifuge defibrinated rabbit blood Sterilization: Sterilize for 15 minutes at
and draw off serum. 112C.
583
Meningococci, Micro- (5) Remove the vessel from the sterilizer

Use: Cultivation of
and Diplococcus flavus. and separate with a glass rod, the
coccus catharralis
firm clot which has formed from the
References Sacquepee and Delater (1914 p.
:
side of the vessel.

224), Dopter and Sacquepee
(1921 p.
Return to the Arnold sterilizer at
(6)
139).
100C. for U
hours.
1862. Emile-Weil Egg Yolk Infusion
Agar Remove the vessel and allow to stand
(7)
at room temperature for about 10
Constituents
minutes in a slightly inclined
750.0 cc. position.
2. Sea water or
(8) Pipette off the fluid portion
3. Veal 500.0 g.
decant. If it is poured thru a fine
4. Glycerol 40.0 g.
wire sieve, many of the fine pieces of
5. Glucose 80 S-
100 S-
meat clot may be
caught. (Avoid
6. Peptone
filteringthru cheese cloth, cotton or
7. Agar 20.0 g.
250.0 cc. other adsorptive materials.)
8. Egg yolk
(9) Allow (8) to stand in tall cylinders
Preparation
broth from 500.0 g. for 15 to 20 minutes until the fat
(1) Prepare infusion
cc. distilled water and present has risen to the surface and
veal, 250.0
sea water. Details of removed. The medium may be
750.0 cc.
further cleared by filtering thru
method not given.
glass wool, asbestos wool, sedimenta-
(2) Make distinctly alkaline.
tion or centrifugation.
(3) Add and dissolve 4, 5, 6 and 7 in (2).
Tube in 10.0 cc. lots.
(4) Add to cool agar, one part egg yolk to (10)
Sterilization Sterilize intermittently in the
four parts agar. (Not clearly stated :
solid state.) steamer.

if the agar is in a liquid or
Not specified. Use: Substitute for media containing ser-

Sterilization:
ous fluids. Author reported that the
Use: Cultivation of B. leprae.
Reference: Emile-Weil (1905 p. 799). medium had a growth value 10 times as
great as standard agar and was at least
1863. Huntoon's Hormone Agar as good as the average grade serum agar.
Variants
Constituents
Water 1000.0 cc. (a) Huntoon prepared a semi-solid hor-
1.
Beef, fresh heart or steak. 500.0 g. mone agar as indicated above but
2. .
10.0 g. using only 5.0 g. agar instead of 16.0 g.

3. Peptone (Bacto)
Huntoon reported that this medium
4. Agar (Bacto) or soaked
16.0 g. was as good as average grade serum
thread
agar, can be employed as stab cul-
5. Salt 5.0 g.
tures, and very useful both for
6. Whole egg 1
anaerobic and aerobic cultures. It
Preparation
and is especially suitable for preservation
(1) Chop 2 and mix 1, 2, 3, 4, 5 6.
of stock cultures.
Place in an enameled ware vessel or a
large coffee pot.
(b) Huntoon added 5.0% glycerol to the
Heat over a free flame with constant medium just before tubing and
(2)
reported that after ten days growth of
tubercle bacilli almost as heavy as in
meat infusion changes to brown at a
about 68C. Do not Dorset's egg medium.
temperature of
this temperature. (c) Harvey gave the following method
go beyond
of preparation:
Prepare finely minced ox heart
with N/1 NaOH and then add 1.0 (1)
500.0 g.; 10.0 g. peptone, sodium
cc.per liter of medium.
chloride 5.0 g., contents of one
(4) Cover the vessel and place in an
egg; prepared agar (see medium
1401) 16.0 g., water 1000.0 cc.
at 100C. for one hour.
(2) Heat at a temperature not exceed-

Separate clot from the sides of the
(6)
ing 68C. until the red color of the
receptacle and replace in the
mixture turns brown.
sterilizer for another hour.
(3) Make faintly alkaline to litmus. (7) Clear by centrifugation, or by
(4) Add after this alkalinization 1.0 straining thru wire mesh and then
CO.N/1 sodium hydroxide per liter. thru glass wool Or the meat resi-
.
(5) Steam 60 minutes. due can be deposited on the glass

(6) Separate the clot formed from the wool in a funnel and the fluid be
sides of the vessel.
allowed to percolate thru it several
(7) Steam 90 minutes. times. Never use cotton or any
(8) Allow to stand at room tempera- other adsorptive materials to
ture 10 minutes. clarify.
(9) Pipette offthe fluid and place in a
tall cylinder.
(9) Reheat and tube in 7.0 cc. lots.
(10) Leave 20 minutes.
(10) Sterilize in Arnold sterilizer.
(11) Skim off the fat from the surface.
(11) If ascitic fluid is to be used, add
each test tube.
1.0 cc. to
(13) Sterilize at 100C. for three days.
(e) Park, Williams and Krumwiede pre-
Harvey reported that the medium is pared a similar vitamin agar for the
not filtered at any stage thru any
cultivation of meningococci as
cloth, filter paper or cotton wool. If follows:
filtration is needed it should be thru (1) Add 500.0 g. tap water to 500.0
glass wool. A less amount g
of agar, of chopped fat and tendon free
2. g., 0.5% instead of 1.6% will give a beef heart.
medium in which, if sealed and kept Add
(2) 15.0 g. of peptone, 5.0
in the incubator, g.
meningococcus and NaCl, and one well beaten egg to
gonococcus sown by the stab culture
(1).
method will live for two or three
(3) Heat in a water bath, double
months.
boiler or
over the open flame,
(d) Torrey and Buckell called the follow-
stirring constantly until the color
ing medium a semi solid agar, but did
changes to brown (at about 68
not mention the addition of agar.
to 70C.).
Ascitic fluid may
be used to ad- (4) Strain thru a wire sieve or wire
vantage in rejuvenating delicate gauze. Do not use cheese cloth,
strains of gonococci or recovery of
cotton or paper.
old stock strains. Also in primary
(5) Dissolve 15.0 g. of agar in 500.0 cc.
fishing of gonococci colonies.
tap water.
(1) Mix 500.0 g. of finely chopped fresh
(6) Cool(5)to70C.
beef heart and one whole egg with
(7) Mix (4) and (6).
water.
(8) Adjust the reaction to +0.2
(2) Place in a double boiler over a
phenolphthalein or to about pH
free flame and maintain at
60C. = 7.4.
for 5 minutes. Stir constantly.
(9) Heat an autoclave at 15 pounds
in
(3) Add 10.0 g. peptone and 5.0 g. pressure for 30 minutes.
NaCl and raise temperature until
(10) Remove the kettle carefully and
the mixture assumes a brownish
set aside for sedimentation to take
color.
place or run in Sharpies centrifuge.
(4) Adjust reaction to slightly alka-
(11) If the sedimentation method is
line to litmus using
10.0% NajCO, used, turn the solid agar out of the
solution.
kettle in mould form and trim off
(5) Place (4) in flask or preferably in a the layer of sediment and discard
coffee pot and heat in Arnold (12) Melt.
steam sterilizer for an hour. (13) Tube.
(14) Sterilize in an autoclave at 15 1865. Meyer's Basal MoUusk Infusion Agar

pounds for 30 minutes. This Constituents:
medium was recommended by the 1. Tap Water 1000.0 cc.
authors to be used in Avery's 2. MoUusks 500.0 g.
Oleate Blood Cell Agar, see 3. Peptone 10.0 g.
medium 1858. 4. Agar 20.0 g.
References: Huntoon (1918 pp. 170, 172), 5. Egg 2
Harvey (1921-22 p. 120), Torrey and Preparation
Buckeil (1922 p. 127), Park, Williams and (1) Place 500.0 g. of finely chopped mol-
Krumwiede (1924 p. 126). lusks (or beef heart), two whole eggs
and 1000.0 cc. of tap water in a
1864. Harvey's Alkaline Egg Agar double boiler over a free flame and
Constituents: infuse at 60C. with constant stirring
1. Water 500.0 cc. f,or 5 minutes.
Whites of 2 eggs (2) Add 20.0 g. of flaked agar and 10.0 g.

2.
3. Yolk of 1 egg peptone (if desired) and raise the
4. N/1 NaOH 6.0 cc. temperature until the mixture assumes
5. Infusion agar 5000.0 cc. a brownish color.
Preparation (3) Make slightly alkaline, using a 10.0%
(1) Mix the whites of two
eggs, the yolk Na2C0j solution.
normal Heat in the steam sterilizer for one
of one egg and 6.0 cc. of (4)
NaOH. hour.
Add 500 .p cc. of water. (5) Separate the clot from the sides of
(2)
Heat very slowly to 95C. the container and place in the steri-
(3)
Keep at 95C. for 60 minutes. lizer for another hour.
(4)
Filter thru cotton wool. (6) Clarify by straining thru glass wool.
(5)
(6) Distribute into flasks. (7) Adjust to pH = 7.2.
(7) When ready for use mix 10 parts (8) Tube or flask.
melted infusion agar, cooled to Sterilization: Sterilize the fractional
sterile
45''C. (see variant (v) medium 1661 method at 100C. in flowing steam.
for preparation) with one part sterile Use: Cultivation of Cyclostoma elegans
and other organisms living in symbiosis
(6) heated at 45C.
Sterilization: Sterilize (6) in the autoclave. with moUusks.
Use: General culture medium. Added nutrients:
Variants (a) The author used the basal medium
(a) Harvey reported that a clearer without additional material.
medium was obtained if the whites (b) The author added 10% defibrinated
of two eggs were mixed with 4.0 cc. rabbit blood to the melted agar just
of normal NaOH and 330.0 cc. of before use.
water instead of the quantities given (c) The author added 1.0 to 2.0% of
above. filteredHelix pomatia or Cyclostoma

(b) Harvey prepared a similar medium as infusion prepared as follows:
follows: (1) Grind 10 to 20 large sterilized
(1) Mix the whites of two eggs, yolk of Cyclostomas in a sand mortar.
one egg and 6.0 cc. of normal NaOH. (2) Mix (1) with an equal weight of
Add 500.0 cc. of water. Ringer's solution. The weight of
(2)
Heat very slowly to 90C. (1) to be as weight of tissue and not
(3)
(4) Distribute into flasks. ofsand and tissue.
(5) Sterilize (4) in the autoclave. (3) Shake for ten minutes.
When ready for use, mix one part (4) Centrifuge at low speed.
(6)
(5) heated to 45C. and ten parts (5) Remove the supernatant fluid and
sterile melted infusion agar cooled mix with the hemolymph of about
to 45C. (see variant (v) medium 20 to 40 moUusKs.
1661 for preparation). (6) Pass thru a diatomatous filter, or
Reference: Harvey (1921-22 p. 86). sterilize by the addition of chloro-
form and preserve in the ice chest (3) Adjust to slightly alkaline to litmus
for four weeks or sterilize in flowing with N/1 NaOH and then add 1.0
steam at 100C. for 30 minutes. cc. per liter of medium.
Reference: Meyer (1925 p. 47). (4) Cover the vessel and place in an
1866. Farcy and Chavaillon's Egg Albumin
at 100 for one hour
Serum Agar
(5) Remove the vessel from the sterilizer
Constituents and separate with a glass rod the
1. Infusion agar firm clot, which has formed, from the
2. Serum 100.0 cc. side of the vessel.
3. Egg white 20.0 cc. (6) Return to the Arnold sterilizer and
Preparation: heat at 100 for 1 hours.
(1) Add 100.0 cc. of horse serum obtained (7) Remove the vessel and allow to
under aseptic conditions to a medium stand at room temperature for about
sized sterile flask containing glass ten minutes in a slightly inclined
beads. position.
(2) Add 20.0 cc. of egg white to (1). (8) Pipette off the fluid portion or
Remove the egg white by means of a decant. If it is poured thru a fine
sterile pipette, and add to (1) under wire sieve, many of the fine pieces of
aseptic conditions. meat clot may be
caught. (Avoid
(3) Shake the flask vigorously for 5 to 10 filteringthru cheese cloth, cotton or
minutes. other adsorptive materials.)
(4) Prepare a 2.5% infusion agar. (9) Allow (8) to stand in tall cylinders
(5) Exactly neutralize (4) and add 0.2 for 15 to 20 minutes until the fat
cc. of a 10.0% soda solution per liter present has risen to the surface and
of agar. removed. The medium be may
(6) Mix one part (3) with 3 parts (5), further cleared by thru
filtering
melted and cooled to 50C. glass wool, asbestos wool, sedimenta-
(7) Distribute as desired. tion or centrifugation.
Sterilization: Not specified. (10) Tube in 10.0 cc. lots.
Use: Cultivation of meningococci. (11) Add a small amount (1.0 to 100.0
Variants: Authors suggested that sugars cc.) of blood (just
defibrinated
maj' be added if desired. enough to give a pinkish tinge to the
Reference: Faroy and Chavaillon (1915 p. poured plate) to each tube of sterile
455). (10).
SteriUzation : Sterilize (10) intermittently
1867. Huntoon's Hormone Blood Agar
in the steamer.
Constituents: Use: To cultivate highly parasitic organ-
1. Water 1000.0 cc. isms. Author reported the medium es-
2. Beef heart or steak (fresh). 500.0 g. pecially adapted to the cultivation of the
3. Peptone (Bacto) 10.0 g. meningococci. The medium is superior
4. Agar 16.0 g. to glucose ascitic agar.
5. Salt 5.0 g. Variants: Huntoon reported better results
6. Whole egg (one) were obtained using laked blood instead
7. Blood, defibrinated of defibrinated.
Preparation Reference: Huntoon (1918 p. 171).
(1) Chop 2 and then mix with 1, 3, 4,
5 and 6. Place in an enamel ware 1868. Torrey and Buckell's Ascitic Fluid
vessel or a large coffee pot. Egg Agar
(2) Heat over a free flame with constant Constituents
stirring until the red color of the 1. Distilled water 1000.0 cc.
meat infusion changes to brown at a 2. Beef heart 500.0 g.
temperature of about 68C. Do not 3. Whole egg 1
go beyond this temperature. 4. Peptone (Difco) 15.0 g.

587
18-0 g. 1869. Williams-Burdick's Modified Petroff's

5 Agar (Flaked)
500.0 cc. Egg Infusion Agar (Roddy)
6 Ascitic fluid
7. Methyl violet
-
Preparation: ^'"*."!mI'; .
(preferably directly 1 Distilled water
(1) Place beef heart
.
Eggs
from slaughter house) and one whole 2.
3. Glycerol
egg in water.
Veal ^^^"^ S-
(2) Place (1) in a double boiler over a 4. ...
Gentian violet (1.0% soln.) 1.0 cc.
free flame and maintain at 60C. for
5.
5 minutes with constant stirring.

6. NaCl

Agar
(3) Add 4 and 5 and raise temperature 7.
Preparation:
until the mixture assumes a brown- , j j^, .
. , ,
(1) Dilute egg whites
obtained under
aseptic conditions with 10 parts
(4) ld]usi\o slightly alkaline to litmus
distilled water. Shake thoroly
using 10.0% solution of Na.COa-
To clear, pass the Auid thru a thm
(5) Place (4) in flask, or preferably in a (2)
layer of cotton and then heat to
coffee pot and keat at 100C. in
100C.
Arnold steam sterilizer for one hour.
(6) Separate clot from sides of the _
(4) Dilute egg yolk, obtained unde

receptacle and replace in sterilizer
aseptic conditions wxth 10 parts
for another hour.
distilled water. Mix well.
(7) Clear by centrif uging or by straining
^ Clarify the cloudy emulsion by the
thru a fine wire mesh and then thru (5)
addition of norma NaOH. Too
glass wool. (Do not use cotton
much NaOH - lârmfuL The emul-
cloth or any other material with
absorbent
medium may
properties.)
also be obtained
A clear
by
^^ n
Usually I'n ^
LO cc. of
-^^^J^^^^^^
tl( Nlont
required for each 100.0 cc. of the
depositing the meat residue on glass
emulsion
wool in a funnel and the fluid portion
(6) Heat to 100C. and filter
allowed to percolate thru.
ôv- 500.0 g.o finely chopped lean
(8) AdjustreactiontopH = 6.8. (7)
veal with one liter of water contain-
(9) Heat and tube in 10.0 cc. lots.
each tube ing 15.0% glycerol.
(10) Add 5.0 cc. ascitic fluid to
Infuse for 24 hours and filter
of sterile melted medium just before (8)
(9) Add 5.0 g. NaCl to the filtrate ot
and heat to boiling.
SterilizTonfSterilize (9) in autoclave at (8)
12 pounds pressure minutes but

for 10 (10) Filter.
x i nor
+1.0%
,
Adjust the reaction to

preferably at 100C. in flowing steam (11)
alkaline.
using the fractional method.
Use: Isolation of gonococcus. Authors (12) Place 300.0 cc. of (3) m a liter
r+ . flask flo=t-
Place 300.0 cc. of (6)m another

reported that colonies after 24 hours were (13)
about1.0 mm. in diameter. May show a flask.

j ir n ^ ..f
Place 400.0 cc. of 11) and 15.0 g. of
smoky tinge by transmitted light. May (14)
show heaped up or flat centers. The

agar amthird flask
be very thin and slightly (15) Rernove (/4) f-- th-t-^---^

may
Jdges
serrated or may be raised well defined
and smooth.
while hot add ^-^ /
alholic solution of gentian violet
to the infusion agar.
J ^
Variants: 1.0 cc. of a 1:100,000 dilution
Pour the conten s of the infus on
of methyl violet in distilled water may be (16)
the
agar gentian violet flask into
added to each tube containing 10.0 cc. of and
the ascitic flask containmg the egg white,
the medium and 5.0 cc. of
g .^ then the egg yolk is added.
Reference: Torrey and Buckell (1922 p. (17) Pour from one flask to another to
insure thoro mixing.
j2g)
(18) Tube and slant. 3. NasCOs (6.5% Solution) 10.0 cc.

(19) Leave the tubes in a slanted position 4. Neutral red (0.5% soln.)
for 72 hours at room temperature (1-0%) l.Scc.
until the contents have well set. 5- Egg 10.0 cc.
Then seal the tubes with cork and Preparation
paraffin. Mix
(1) the contents of one egg with an
under preparation.
Sterilization: See equal quantity of water.
Use: Cultivation of B. tuberculosis. (2) Add to this mixture an equal amount
Reference: Roddy (1917 p. 46). of6.5% anhydrous NasCOg.
(3) Add 1 volume of the sterile mixture to
1870. Chapin's Egg Yolk Urine Agar
5 volumes neutral 1.0% sucrose agar.
Constituents (4) Add to the mixture while melted 1.0%
1. Meat infusion 1000.0 cc. freshly prepared 0.5% neutral red.
2. Urine 200.0 cc. (5) Slope or pour on plates.
Peptone Sterilization: Sterilize (2) for one

3.
50g hour at
4. N/1 H2SO4 (1.2%) 12.0 cc. 100C. Method of sterilization of 1.0%
5. Agar to solidify sucrose agar not given.
6. Pea flour (0.5%) 5.0 g.
Use: Isolation of cholera vibrio. Harvey
7. Glucose (0.5%) 5.O g.
reported that the medium had orange tint
8. Egg yolk (5.0%) 50.0 cc.
and V. cholerae colonies had deep red
Preparation centers.
(1) Prepare meat infusion. Reference: Harvey (1921-22 p. 86).

(2) Heat to remove coagulated proteid.
1872. Leboeuf's Egg White Liver Infusion
(3) Add 2, 3, and 4 (a normal dilution of
Agar
H2SO4, 1.2%).
Constituents
(4) Divide into 5 flasks and add varying
amounts of N/1 NaOH, 0.0, 1. 2/2.8, 1- Water looO.O cc.
2. Liver, horse or beef 500.0 g.
3.7and 4.7% respectively having the
following
3. Peptone (Chapoteaut) 20.0 g.
reactions: 3.4, 2.6, 1.8,
0.5 and alkaline.
4. Egg white
5. Starch, potato
(5) Add a sufficient quantity of agar,
0.5% 6. Agar
of 6 and 0.5% of 7.
Preparation
(6) Add 5.0% egg yolk to the cooled
(1) Boil 500.0 g. of finely chopped horse
medium just before pouring the
or beef liver in 1000.0 cc. of water
plates.
slowly for 90 minutes.
Use: To study influence of CO2 and reac- (2) Filter thru paper and make up the
filtrate to the original volume.
tion of medium on cultivation of gono-
coccus. The author incubated the (3) Add 20.0 g. of Chapoteaut's peptone
cultures in a 10,0% CO2 atmosphere.

He
reported good growth in the medium con- (4) Neutralize to litmus.
taining 4.7% NaOH the reaction being
alkaline, and (6) Mix one part egg white with ten
also in media whose reac-
parts distilled water.
tions were -|-2.6 and 1.8. A lighted
candle
left in the jar beside the plates at the time (7) Boil (6) and filter.
of sealing the chamber gave a favorable (8) Mix 100.0 cc. of sterile (7) with
1000.0 cc. of and add 5.0
atmosphere. (5), g.
Reference: Chapin (1918 potato starch.
p. 342).
(9) Readjust the reaction to neutral if
1871. Harvey's Sucrose Egg Agar necessary.

(10) Add 20.0 g. of agar per 1000.0 cc. of
Constituents and autoclave at 115C.
(9) for 35
1. Sucrose (1.0%,) Agar 150.0 cc. minutes.
2. Water 10.0 cc. (11) Tube.
Sterilization: Sterilize (7) by heating in the (1) Extract one pound of lean chopped
autoclave at 120C. for 20 minutes. beef or veal in 500.0 cc. of water
for 18 hours.
Use: Cultivation of gonococci.
Reference: Leboeuf (1924 p. 768). (2) Add and dissolve in this infusion,
made up to 1 liter with water,
Agar 10.0 g. agar, 20.0 g. gelatin, 20.0 g.
1873. Besson's Gelatin Infusion
Witte peptone and 5.0 g. salt.
Constituents (3) Adjust to the neutral point using
1. Infusion broth 1000.0 cc. phenolphthalein as an indicator.
2. Gelatin 80.0 g. (4) Tube.
3. Agar 5.0 g. (b) Dopter and Sacqu6pê prepared the
Preparation : medium as follows:
(1) Dissolve 80.0 g. of gelatin in 1000.0 (1) Add 1000.0 cc. of water to 500.0
cc. of infusion broth. g. of finely chopped fat and tendon
(2) Neutralize. free beef.
(3) Dissolve 5.0 g. of agar in (2). (2) Allow to stand in the ice box for
(4) Allow to cool to 55 or 50C. 12 hours, or heat at 50 to 55 for

(5) Beat the white of an egg in 100.0 cc. 30 minutes.
of water and add to (4) (3) Heat slowly to boiling.
(6) Mix well. (4) Boil slowly for 10 minutes, stirring
(7) Autoclave at 115C. for 20 minutes. constantly.
(8) Filter thru a moistened Chardin filter (5) Strain thru a cloth.
paper using a hot water funnel. (6) Dissolve 80.0 g. gelatin, 5.0 g.
(9) Tube. NaCl and 20.0 g. peptone in (5).
Sterilization: Sterilize at 115 for 20 Neutralize.

(7)
minutes. (8) Dissolve 5.0 g. agar in (7).
Use: General culture medium. (9) Clarify by the addition of egg

Variants: Author reported that 50.0 g. of white.
gelatin and 8.0 g. of agar may be used (10) Autoclave.
instead of the amounts indicated. (11) Filter.
Reference: Besson (1920 p. 43). (12) Distribute
1874. Frothingham's Gelatin Infusion Agar Guarniari (Abbott) gave the follow-
(c)
Constituents: ing method of preparation

(1) Dissolve 3.0 to 4.0 g.
agar sep-
Agar 7.5 g. arately in about 100.0 cc. of water
2.
3. Gelatin 50.0 g. while the other materials are being
Preparation: prepared.
of (2) Prepare meat infusion.
(1) Dissolve 7.5 g. agar in 1000.0 cc.
infusion broth. (A 0.5% solution of (3) Dissolve 5.0 g. NaCl, 25.0 to 30.0
Liebig's meat extract solution may g. peptone and 40.0 to 60.0 g.
be used.) gelatin in (2)
(2) Dissolve 50.0 g. of gelatin in (1). (4) Mix (3) and (1).
Method not given. (5) Sterilization not specified.

Sterilization:
main- Harvey solidified infusion broth, see
Use : General culture medium and to (d)
tain stock cultures of streptococci, variant (bb) medium 779 by the

pneumococci and other pathogens. North addition of 10.0 or 12.0%, gelatin
reported that the addition of 1.0% glucose and 0.5% agar.
made the growth more favorable at times, (e) Park, Williams and Krumwiede gave
but reduced vitality. the following method of preparation:
(1) Prepare a meat infusion
using 1.0
Variants
pound of meat to 500.0 cc. of water
(a) North gave the following method of
preparation: (Method not given).
(2) Dissolve 20.0 g. gelatin and 20.0 Preparation

g. peptone in warmed (1). (1) Prepare usual infusion broth.
(3) Dissolve 10.0 g. of agar in 500.0 (2) Dissolve 2 and 3 in (1) by heat and
cc. of water and cool to below tube.
50C. (3) Cool sterile (2) to 45C.
(4) Mix (2) and (3). (4) Add 4. (i volume of 4 to (3)).
(5) Adjust the reaction. (5) Inoculate while still a liquid.
(6) Heat to coagulate the albumin (6) Mix medium and solidify by immers-
and clear the medium. ing in cold water.
(7) Readjust the reaction and reheat Sterilization: Sterilize (2). (Method not
if necessary. given).
(8) Filter. Use: Culture medium for BaczVûs abortus.
(9) Sterilization not specified. The authors reported a growth zone 5 mm.
References: Frothingham (1895 p. 53), beneath the surface of the medium and
North (1909 p. 361), Tanner (1919 p. 51), extending down for 1.0 to 1.5 cm.
Dopter and Sacqu^pee (1921 p. 128), Reference: MacNeal and Kerr (1910 p.
Abbott (1921 p. 144), Harvey (1921-22 469).
p. 71), Stitt (1923 p. 39), Park, Williams
and Krumwiede (1924 1877. Supplee's Nutrose Gelatin Agar
p. 118).
(Ayres and Johnson)
1875. Krause's Gelatin Urea Agar
Constituents
Constituents: 1. Meat infusion 1000.0 cc.
1.Infusion agar (3.0%) 1000.0 cc. 2. Gelatin 20.0 g.
2. Infusion gelatin (20.0%).... 2000.0 cc. 3. Agar lO.O g.
3. Urea (2.5%) 75.0 g. 4. Peptone 20.0 g.
Preparation 5. Nutrose 5.0 g.
(1) Prepare a 3.0% meat infusion agar. Preparation
(2) Prepare a 20.0% meat infusion gelatin. (1) Prepare meat infusion.
(3) The NaCl content of (1) and (2) (2) Dissolve 2, 3, 4 and 5 in (1).
should be about 0.7 to 0.8%. The (3) Adjust to neutral with phenol-
reaction is not adjusted. phthalein.
(4) Mix one part (1) with two parts (2). Sterilization: Not specified.
(5) Adjust the reaction to 0.27 to 0.3% Use: Used especially in carrying stock
lactic acid. cultures of streptococci and similar
(6) Dissolve 2.5% urea in the least organisms.
amount of water and thoroly mix with Reference: Ayers and Johnson (1924 p.
(5). 111).
(7) Distribute into sterile tubes.
(8) Before use, melt sterile (7) and pour 1878. Kinsella, Brown and Garcia's Nutrose
into sterile plates. Gelatin Agar
Sterilization: Steam (7) for 15 minutes in Constituents:
flowing steam. 1. Distilled water 1000.0 cc.
Use: Detection of typhoid bacilli.
Author 2. Beef 500.0 g.
reported that typhoid colonies had a 3. Peptone 10.0 g.
nucleus surrounded by thread-like ap- 4. NaCl 5.0 g.
pendages, they were grey in color when 5. Agar 5.0 g.
young and brown when old. 6. Gelatin 50.0 g.
Reference: Krause (1902 p. 94). 7. Nutrose 10.0 g.
Preparation
1876. MacNeal and Kerr's Gelatin Agar
(1) Mix finely chopped beef in water and
Constituents: set in the ice chest for 24 hours.
1. Infusion broth 1000.0 cc. (2) Strain thru linen towel.
2. Agar 7.5 g. (3) Add and
dissolve 3 and 4 in (2).
3. Gelatin 50.0 g. (4) Boil to clear and filter thru filter
4. Blood serum (Sterile) 500.0 cc. paper.
591
3.0 g. of sodium citrate in the melted

(5) 5, 6 and 7 to (4).
Add
Boil over saturated solution of NaCl
agar.
(6)
Mix (12) and (10) and make up to
in a double boiler for 45 minutes. (13)
1000.0 cc. by adding distilled water.
(7) Strain thru absorbent
cotton.
= 7.3. Tube.
(8) Titrate to pH
(14)
Tube. (15) Final pH = 7.5.

(9)
minutes at 15
Sterilization: Sterilize in autoclave for 20
pounds (A precipitate
in the autoclave.
minutes.
Use: To maintain cultures of gonococci. may be formed. It will settle out or be
Authors reported that growth started as a absorbed in a short time. In either case
film on the surface and radiated from
the medium is not effected).
point of inoculation. The gonococci Use: To carry stock cultures of strepto-

cocci, DipJococcus pneumoniae, Hemo-
survived on this medium for from 3 to 4
philus pertussis, Pasteurella bovis, Ery-
weeks.
Brown and Garcia sipelothrix porci.
Reference: Kinsella,
Reference: Ayares and Johnson (1924 p.
(1923 p. 4).
112).
1879. Ayers and Johnson's Casein Gelatin
1880. Frazier's Gelatin Agar
Agar
Constituents
Constituents
1. Distilled water. 1000.0 cc.
2. NaCl 5.0 g.
3. Peptone (Parke-Davis) 10.0 g. 3. KH2PO4 0.5 g.
4. Gelatin (Difco) 10.0 g. 4. K2HPO4 1.5 g.
5. Gelatin Bacto.. 4.0 g.

5. Casein (pure, prepared ac-
6. Peptone Bacto. 0.1 g.
cording to Hammarsten) 5.0 g.
7. Glucose. . ;
0.05 g.
6. Glucose 0.5 g. .
8.Beef infusion 5.0 cc.

7. Na2HP04-2H20 (Sorrenson's
9.Agar (3.0%) 15.0 g.
phosphate) 4.0 g.
Sodium citrate 3.0 g. Preparation:
8.
cc. distilled
(1) Dissolve 2, 3 and
4 in 100.0
9. Agar 7.5 g.
water.
Preparation
broth. Dissolve 4.0 g. Bacto gelatin in
(1) Prepare meat infusion
(2)
(Parke-Davis) 400.0 cc. distilled water and add 0.05
(2) Add 10.0 g. peptone
Na2HP04-2H20 (Sor- g. glucose, 0.1 g. Bacto peptone, and
and 2.0 g.
phosphate.) Heat until 5.0 cc. of beef infusion.
renson's
(3) Mix (1) and (2) and heat in a steamer.
solution takes place.
pH = 7.8. (4) Prepare 500.0 cc. of a 3.0% washed
(3) Adjust the reaction to
casein (prepared agar.
(4) Dissolve 5.0 g. pure
according to Hammarsten) and 2.0 g, (5) Mix (4) and hot (3).
Na2HP04-2H20 (Sorrenson's phos- (6) Adjust to pH = 7.0.
phate) in 150.0 cc. distilled water by (7) Tube or flask.

heating.
Mix (4) and (3). Use: To determine gelatin decomposition.
(5)
Add 10.0 g. of gelatin (Difco) to (5). Pour plates of the medium, allow to
(6)
Heat (6) in the autoclave at 15 harden, inoculate duplicate plates, on the
(7)
pounds for 10 minutes. surface of the medium, so as to form a
Add 0.5 g. glucose. giant colony. Following incubation for

(8)
Reaction should be pH = 7.6. 2 or 3 days, flood one plate with a 1.0%,
(9)
Filter thru paper.
solution of tannic acid while the duplicate
(10)
is flooded with an acid solution
of
Prepare 250.0 cc. of 3.0% agar (Not plate
(11)
specified if the agar is to be dissolved bichloride of mercury, (HgCl2 15.0 g.,
HCl (con.) 20.0 cc. and water 100.0 cc).
in meat infusion or water).
thru cotton and dissolve If the gelatin has been attacked, a clear
(12) Filter (11)
zone appear about the colony on the

will moist, translucent and somewhat
plate with acid bichloride of
flooded brownish, with uniform slightly
mercury. The plate flooded with tannic granular structure. By transmitted
acid presents a white to clear zone about light they were translucent, homo-
the colony depending on the vigor with geneous and showed no zone of
which the organism attacked the gelatin. hemolysis. Best results were ob-
Reference: Frazier (1926 p. 302). tained by using blood as fresh as
possible. Laking was not necessary.
1881. Duval and Lewis' Glucose Blood Agar
If plates are warmed before use, more
Constituents : rapid and abundant growth of menin-
1. Infusion agar gococci was obtained.
2. Glucose (0.5 to 1.0%)... 5.0 to 10.0 g. (1) Exact method of preparation or
3. Blood, defibrinated exact composition of 2.0% meat
(2.0 to 5.0%) 20.0 to 50.0 cc. infusion agar not given.
Preparation : (2) Dissolve 1.0 peptone in (1).
(1) Prepare beef infusion agar. (3) Adjust to exactly neutral to
(2) Adjust reaction of (1) but with one phenolphthalein.
change if possible between 0.4 and (4) Distribute in 300.0 cc. lots in 500.0
0.8% acid to phenolphthalein (cold cc. flasks.
titration). (5) Autoclave at 15 pounds for 30
(3) Add glucose from 0.5 to 1.0%, also minutes.
from 2.0 to 5.0% "fresh" defibrinated (6) Sterilize 5.0 g. glucose in 50.0%
blood. solution.
Sterilization: Not specified. (7) Cool the melted agar to 45C.
Use: Cultivation of pneumococci. Similar and add the slightly warmed glucose
media were employed to cultivate a large solution and 50.0 cc. sterile defibri-
variety of organisms. nated sheep's (or whole) blood.
Variants : (8) Mix by a whirling motion avoiding
(a) Novy (Hagemeister) cultivated air bubbles.
pathogenic trypanosomes on a (9) Pour in sterile plates.
medium prepared as follows: (c) Hertig and Wolbach cultivated Rick-
(1) Prepare an extract of 125.0 g. beef ettsia melpohagi on a medium pre-
(or horse meat) in one liter of water. pared as follows
(2) Dissolve 20.0 g. agar, 20.0 g. peptone (1) Prepare beef bouillon in the usual
and 5.0 g. NaCl in (1). manner (exact procedure not
(3) Neutralize to litmus by the addition given).
of N/1 NaOH. (2) Dissolve 20.0 g. glucose and 25.0 g.
(4) Add 5.0 cc. of N/1 NaOH per liter. agar in (1).
(5) Dissolve 2.0% glucose in (4). (3) Method of sterilization not given.
(6) Distribute in 3.0 or 4.0 cc. lots in (4) To melted at 100C. add sterile
(3)
test tubes. defibrinated rabbit blood in equal
(7) Add a double or triple amount of volume to double volume.
sterile defibrinatedblood to each (5) Slant the tubes and seal with rubber
tube of sterile agar, melted and caps to prevent evaporation.
cooled to 60C. Mix well. References: Duval and Lewis (1905 p.
(8) Solidify in a slanted position. 474), Hagemeister (1914 p. 228), Bushnell
(9) After inoculation seal the tubes (1918 p. 2), Hertig and Wolbach (1924 p.
with paraffin. 337).
(b) Bushnell used the following medium
for the isolation of meningococci.
1S82. Torrey's Glucose Blood Agar
Author reported that by reflected Constituents:
light, meningococci colonies appeared 1. Distilled water 1000.0 cc,
raised, somewhat convex, glistening. 2. Liver, beef 500.0 g.
3. Peptone 10.0 g. 4. Glucose 5.0 g.

4. Agar 20.0 g. 5. NaH2P04 2.0 to 3.0 g.
5. Glucose 10.0 g. 6. Agar, granular 25.0 g.
6. K2HPO4 1.0 g. 7. Blood, human 5.0 to 25.0 cc.
7. Blood, sterile defibrinated Constituents
rabbit 100.0 cc. (1) Remove connective tissue from
all
Preparation beef testicles, put thrumeat grinder
(1) Chop 500.0 g. beef liver into small and infuse on ice over night.
pieces and boil for 2 hours in 1000.0 (2) Next morning heat in a double boiler
cc. of distilled water in a double to 50C., allow to stand an hour and
boiler. then bring to the boiling point.
(2) Filter thru flannel and cotton. (3) Let stand for another hour to permit
(3) Add to filtrate peptone and agar. the solid particles to settle.
(4) Heat in Arnold for one hour. (4) Decant off the liquid.
(5) Adjust reaction as desired (for B. (5) Dissolve 3, 4, 5 and 6 in (4) by heating
bifidus, +1 to phenolphthalein). over a free flame stirring constantly.
(6) Clear with eggs if necessary. (6) Titrate using phenol red as an indi-
(7) Add glucose and K2HPO4 to clear cator to pH = 7.4 to 7.8 or a reaction
filtrate. of 0.6 to phenolphthalein.
(8) To each 10.0 cc. of sterile medium (7) Tube and autoclave at 15 pounds for
add about 1.0 cc. of sterile defibri- 20 minutes.
nated rabbit blood just before pouring (8) Check titration.
the plate. (9) While (8) is still liquid, add human
Sterilization: Method not given. blood in proportion of 0.5 to 2.5%.
Use: Isolation of B. bifidus. To isolate Sterilization: See step (7) above.
B. bifidus pour medium in petri dish. Use: Isolation and cultivation of gono-
Prepare nutrient agar seeded with B. cocci. Author reported that using phenol
cereus and pour in cover of the Petri dish. red as an indicator primary acidity and
Allow the liver agar to solidify and secondary alkalinity may be determined.
nutrient only partially. When
agar In a case of mixed infection, methyl violet
nutrient agar semi solid, invert the
is in proportion of 1:200,000 to 500,000
inoculated liver agar plate and place it inhibited the growth of staphylococci.
in the semi solid agar. The solidifying Variants: The authors substituted 1.0 to
agar forms a seal. This gives partial 5.0% defibrinated rabbit blood for human
anaerobiosis. Author reported colonies blood.
were raised, more or less globular, opaque, Reference: Erickson and Albert (1922 p.
1 to 3 mm. in diameter, buff to reddish 277).
brown in color. B. acidophilus gave
serrated edge colony To isolate
flat.
1884. Harvey's Lactose Blood Agar
B. acidophilus use this medium omitting Constituents:
blood and adjusting to +4 acid. Grow 1. Infusion agar 1000.0 cc.
aerobically. 2. Lactose 30.0 g.
Variants: Harvey solidified medium 833 3. Blood, sterile defibrinated
variant (a) by the addition of agar. human
References: Torrey (1922 p. 435), Harvey 4. Rosolic acid (1.0%)
(1921-22 p. 110). Preparation
(1) See variant (v) Medium 1661 for
1883. Erickson and Albert's Testicular
Blood Agar
(2) Mix 0.3 g. lactose with each 10.0 cc.
Constituents: of (1).
1. Distilled water 1000.0 cc. (3) Raise slowly to boiling water
2. Beef testicle 500.0 g. temperature.
3. Peptone 20.0 g. (4) Cool to 45 C.
(5) Add 1.0 cc. defibrinated sterile human (5) Add to each sterile tube of (4) 0.5 cc.
blood at 45C., and 3.0 cc. 1.0% alco- of defibrinated rabbit blood.
holic rosolic acid at 45 C. Sterilization: Sterilize (4) in the autoclave.
(6) Prepare plates. Use: Cultivation of B. tuberculosis.

(7) Keep two days before use. Reference: Wherry and Ervin (1918 p. 194).

and Huntoon's Basal Blood
1888. Elser
Use: Cultivation of colon-typhoid group. Infusion Agar
Just before inoculation of tube of medium
1885. Harvey's Saponin Blood Agar with several
1677, cover the slanted surface
drops of defibrinated human blood which
Constituents
has been allowed to age for some time in
1. Infusion agar.
order to reduce to the minimum its initial
2. Saponin.
bactericidal properties. Authors reported
3. Blood, defibrinated.
this medium especially adapted to the culti-
Preparation
vation of freshly isolated cultures.
(1) See variant (v) medium 1661 for
preparation of infusion agar. 1889. North's Gelatin Blood Agar (Kligler)
(2) Make a saturated solution of de-

Same as medium 1874 with the addition of
fibrinated blood in 1.0% saponin.
1.0 cc. of defibrinated rabbit blood per
(3) Mix (1) and (2), amounts not given.
North's gelatin agar.
10.0 cc. tube of
Use: General culture mediiun.
Reference: Harvey (1921-22 p. 76). 1890. Dieudonne's Alkaline Blood Agar
(Harvey)
1886. Warden's Blood Veal Agar
Constituents
Constituents 1. Infusion agar 140.0 cc.
1. Veal infusion 1000.0 cc. 2. Blood, defibrinated ox 10.0 cc.
2. Agar 20.0 g. 3. NaOH (normal solution) 10.0 cc.
3. Blood, defibrinated rabbit.. 500.0 cc. Preparation
Preparation (1) Mix equal parts defibrinated ox blood
(1) Prepare salt free veal infusion broth. and normal NaOH solution (or 11.4%
(2) Dissolve agar in (1). Na2C03 may be used instead of
(3) Neutralize to phenolphthalein. NaOH).
(4) Add defibrinated rabbit blood. (2) See variant (v) medium 1661 for
Sterilization: Not specified. preparation of infusion agar.
Use: Cultivation of gonococci. (3) Neutralize (2) to litmus.
Reference: Warden (1915 p. 426). (4) Mix 3 parts sterile (1) at 45 C. with 7
parts sterile (3) cooled to 45C.
1887. Wherry and Ervin's Glycerol Blood (5) Distribute immediately into test
Agar tubes or Should be kept
plates.
Constituents freely open after tubing or plating,

1. Beef infusion 1000.0 cc. under sterile sheets of paper, for 48
2. Peptone (Witte's) 10.0 g. hours at 37C., or under a bell jar
3. Na2HP04 5.0 g. containing carbon dioxide gas for 30
4. Glycerol 60.0 g. to 60 minutes.
5. Blood, defibrinated rabbit.. 100.0 cc. Sterilization: Sterilize (1) at 100C. on
Preparation each of 3 successive days.
(1) Prepare lean beef infusion. Use: Isolation of V. cholerae.
Variants: Harvey gave the following
(2) Dissolve2, 3, and4in(l).
(3) Adjust to a reaction from -f-1.5 to variants.
+2.0 to phenolphthalein. (a) (1) Mix equal parts normal NaOH
(4) Tube in 5.0 cc. lots. and defibrinated ox blood.
(2) Steam for 60 minutes. (13) Add 2.0% agar and dissolve.
(3) Desiccate in vacuo over sulphuric (14) Filter thru cotton wool and distrib-
acid or calcium chloride. ute in 600.0 cc. flasks.
(4) Grind the residue to powder. (15) The pH of sterile agar should be
(5) Make for use a 10.0% solution of about 7.2.
blood powder. (16) Add 30.0 cc. of (4) to each 600.0 cc.
(6) When ready for use add 3 parts of melted (15) cooled to 40 to 50C.
(5) at 45C. to 7 parts infusion Sterilization: Sterilize (10) by heating in
agar at 45 C. the steamer on each of 3 consecutive days.
(b) (1) Mix equal parts 12.0% crystalline Use: Cultivation of influenza bacilli,
Na2C03 solution and defibrinated Haemoglobinophilic bacteria.
blood. Reference: Kristensen (1922 p. 229).
(2) Mix 3 parts (1) with 7 parts of
1892. MacNeal's Blood Infusion Agar
4.0% infusion agar.
Reference: Harvey (1921-22 pp. 73, 75). Constituents:
1. Water 1000.0 cc.
1891. Filde's Blood Digest Agar 2. Chopped beef 125.0 g.
(Kristensen)
3. Agar 20.0 g.
1. Water 25,000.0 cc. 5. NaCl 5.0 g.
2. Beef 25,000.0 g. 6. N/1 NaoCOa soln 10.0 cc.
3. Peptone 1.0% 7. Blood, naturally sterile de-
4. NaCl 0.5% fibrinated rabbit 2000.0 cc.
5. Blood, defibrinated sheep Preparation
or horse (1) Extract 125.0 g. beef in 1000.0 cc.
Preparation distilled water.
(1) Add 6.0 cc. of pure concentrated (2) Dissolve 3, 4, 5 and 6 in (1).
HCl to 150.0 cc. of a 0.9% NaCl (3) Tube.
solution, and then 50.0 cc. defibri- (4) Add to sterile (3), cooled to about
nated horse or sheep blood and 2.0 g. 60 two volumes of naturally sterile
Langebek's concentrated pepsin. defibrinated rabbit's blood. Mix
(2) Pour into a sterile flask and incubate thoroly.
at 56C. for 5 or 6 hours. (5) Slant and solidify.
(3) Add NaOH solution until a sample Sterilization: Sterilize (3) in the autoclave.
diluted with distilled water to a Use: Isolation of Nagana parasite, Tr.
light brown color turns red on the Brucei and other trypanosomes. In-
addition of phenol red, without oculate Nagana blood in the small amount
giving the alkaline color change of liquid which collects on the surface
with a-naphtholphthalein. of the blood agar. Thompson cultivated
(4) Add 0.25% chloroform and stopper trypanosomes found in gold fish (T.
the flask with a sterile rubber and danilewskyi)
keep in a cold room. Variants
(5) Mince 25,000.0 g. beef. (a) The author used 4 times as much meat
(6) Add 25 liters of water and allow to (500.0 g. per liter) for succeeding
stand in the cold until the next day. cultures.
(7) Boil for a short time. (b) Tobey used the same medium and
(8) Press the fluid from the meat. added two volumes of defibrinated
(9)Add about 16 liters of water to the rabbit's blood. He inoculated the
meat, heat to boiling once more and water of condensation with a drop of
again press the juice from the meat. blood taken from the heart of a bird
(10) Mix the fluid from (7) and (8). by means of a drawn-out tube pipette.
(11) Add 1.0% peptone and 0.5% NaCl. The cotton plug is then cut short,
(12) Adjust to pH about 7.8 to 7.9. moistened with mercuric chloride
and the tube covered with a rubber (6) Cool sterile (3) to 45C. and add an
cap, after which it is placed at 25C. equal volume of (5).
for about one week, (7) Mix and slant.
(c) Behrens gave the following method Sterilization: Method of sterilization of
of preparation (4)not given.

(1) Digest 125.0 g. choiaped beef in Use Cultivation of Trypanosomata Lewisi
:
250.0 cc. water over night in the and other trypanosomata. Other in-
cold, or for one hour at 55 C. vestigators cultivated a number of differ-
(2) Strain (1), boil the extract and ent organisms on similar media.
filter. Variants
(3) Dialyze the filtrate in a large col- (a) Duval (Gurd) prepared a medium as
lodium sac against running distilled follows:
water for 24 to 48 hours. (1) Prepare a beef infusion.
(4) Dilute (3) to 1000.0 cc. (2) Dissolve 10.0 g. peptone, 20.0 g.
(5) Dissolve 20.0 g. peptone, 5.0 g. agar and 5.0 g. NaCl in (1).
NaCl, 0.1 g. CaCh, 10.0 cc. N/1 (3) Adjust reaction to 0.6% acid to
NajCOa solution and 20.0 g. agar phenolphthalein (hot titration).
in (4). (4) Tube.
Distribute in test tubes in 10.0 cc. (5) Sterilize in the autoclave.
(6)
lots. (6) Cool to 52C. and add a small
(7) Sterilize in autoclave by heating to quantity of defibrinated sterile
105-108 for 15 minutes. human blood. From 4 to 7 drops
(8) Shortly before use, the desired of blood are added to each 6 to
number of agar tubes are melted in 10.0 cc. of agar.
the water bath, cooled to 60C. (7) Shake thoroly.

and two volumes of defibrinated (8) Slant or pour in sterile Petri dishes.
rabbit's blood are added. Gurd used the medium to isolate
(9) Mix well and solidify in slanting

gonococci. He reported that gono-
position. cocci colonies were raised worty-
References: MacNeal (1904 Tobey
p. 535), looking, bluish gray or almost color-
(1906 p. 125), Novy and MacNeal (1905 less semi-transparent and round.
p. 265), Thompson (1908 p. 77), Behrens Gave a bright crimson almost trans-
(1914 p. 27). parent medium. If agar was above
60C. when mixed with blood, the
hemoglobin was destroyed and me-
1893. Smedley's Blood Infusion Agar
dium assumed a dirty brown color.
Constituents (b) Duval cultivated B. leprae on a
1. Bullock heart infusion 1000.0 cc. medium prepared as indicated. He
2. Peptone 10.0 g. reported that cultures without the
3. NaCl 10.0 g. blood and using B. typhosus and
4. Agar 20.0 g. Entameba coli gave growth of B.
5. Blood, defibrinated rabbit.. 1000.0 cc. leprae.
Preparation (1) Prepare nutrient infusion agar.

(1) Prepare an infusion of bullock's heart. (2) Sterilize (Method not given).
(2) Dissolve 1.0% peptone and 1.0% (3) Add 1.0% human defibrinated
NaCl and 2.0% agar in (1). blood to sterile (2).
(3) Clear in the usual way by the addition (4) Slope.

of the white of an egg. (5) Inoculate the surface with pure
(4) Tube. growth cultures of B. influenza
(5) Collect blood from the heart of a and meningococcus.
rabbit by means of sterilized Pasteur (6) Incubate at room temperature for
bulbs and then defibrinated in steri- 48 hours.

lized bottles containing a little broken (7) Inoculate the slant with an emul-
glass. sion prepared from leprous tissue.
(c) Hagemeister cultivated pathogenic (3) Cool the flask to 50C. and thoroly
trypanosomes in the following mix 5.0 cc. of defibrinated human
medium: blood with the fluid agar.
(1) Infuse one pound of finely chopped (4) Pour this mixture into petri dishes
beef or horse meat with one liter for blood agar plates on the surface
of water for 24 hours. of previously prepared agar.
(2) Boil for one hour. (5) The agar for (4) is prepared by
(3) Filter thru a towel. dissolving 1.5% agar in normal
(4) Make up to 1 liter by the addition saline (0.85% NaCl), filtered,
of water. tubed and sterilized.
(5) Add 10.0 g. peptone (Sice), 20.0 g. (6) Pour the blood agar mixture (3)
agar and 5.0 g. NaCl to (4). on the plain agar (5) to a depth of
(6) Heat to boiling, stirring con- about 2 millimeters.
stantly. He reported that it was advisable to
(7) Neutralize by the addition of N/1 add the serum with the red blood cells
NaOH using litmus as an in- as the serum is an important aid in
dicator. hemolysin production.
(8) Add 5.0 cc. of N/1 NaOH per liter. (f) Kohman studied the oxygen tension
(9) Heat in the autoclave at 100C. of meningococci on the following

for one hour. medium
(10) Filter thru a double filter or cotton (1) Prepare a 2.5% meat infusion agar.
and distribute in sterile tubes. (2) Dissolve 10.0 g. of Bacto peptone
(11) Sterilize in the steamer for one in 1000.0 cc. of (1).
hour on each of three successive (3) Add 40.0 cc. of human defibrinated
days. blood to (2).
(12) Add 2 or 3 times the volume of (g) Bernstein and Lowe used the follow-
blood to each
sterile defibrinated ing medium for the isolation of
tube of agar, melted and cooled influenza bacilli:
to 60C. Mix well. (1) Prepare infusion agar with a
(13) Solidify in a slanted position. reaction of pH = 7.1.
(14) Seal the tubes with paraffin after (2) Add gentian-violet so that the
inoculation. content be 1:5,000, taking a con-
(d) Warden isolated and cultivated gono- centrated alcoholic solution of
cocci on the following medium: dye as unity.
(1) Dissolve 22.5 g. agar in 1000.0 cc. (3) Method of sterilization not given.
of salt free broth. (4) Add sterile defibrinated human
(2) Neutralize to phenolphthalein. blood to sterile melted (3).
(3) Add 25.0 cc. of sterile defibrinated (5) Pour in sterile plates and store in
rabbit blood at 60C. to 1000.0 cc. refrigerator for use.
of sterile (2) cooled to 60C. (6) Streak surface with washed
(The blood may be omitted.) sputum or material from naso-
Warden reported that growth was pharynx.
better if agar was completely dry The author reported that pneumo-
(in desiccators over H2SO4) before cocci, streptococci and staphylococci
inoculation. were nearly all inhibited. If ascitic
(e) Holman studied hemolysis by strep- be added, the gram
fluid organisms+
tococci on a medium prepared as were not inhibited by the dye.
follows (h) Wahl, White and Lyall cultivated
(1) Plain infusion agar (+0.6) is influenza bacilli on a medium pre-
sterilized in 100.0 cc. quantities pared as follows:
in flasks. (1) Prepare neutral beef infusion agar.
(2) Heat a flask of (1) in the autoclave (2) To every 100.0 cc. of sterile (1)
and place in the paraffin oven at add 5.0 cc. of sterile defibrinated
5SC. for some time. sheep blood at 45 to 50C.
(3) Mix thoroly and pour in plates. infusion agar at 70C. (See
(i) Bell cultivated influenza bacilli in a variant (v) medium 1661.)
medium containing 4.0% defibrinated (2) Raise the temperature of the
rabbit blood to a 2.0% infusion agar. mixture to boiling point over a
(j) Yoshida cultivated Entamoeba tetra- free flame.
gena and Entamoeba coli from their (3) Shake to mix.
cysts in the water of condensation of (4) Raise to boiling point twice again.
an agar containing one part defibri- (5) Allow to deposit.
nated horse blood to two parts agar. (6) Decant the clear supernatant fluid
The blood and agar were mixed at into test tubes while the agar is
60C. still melted, or filter thru glass

(k) Anderson and Schultz added 2.0% wool.
defibrinated blood to infusion agar (7) Slope.
and heated to 80C. until a brown (q) Harvey prepared a medium as
color produced. They cultivated
is follows:
Bacillus pjeifferi on this medium. (1) Mix one part defibrinated ox blood
(1) Harvey mixed one part of sterile at 70C. with 20 parts of infusion
defibrinated blood at 45C. with two agar at 70C. (See variant (v)
parts infusion agar (see variant (v) medium 1661.)

medium 1661). (2) Steam 45 minutes.
(m) Novy-MacNeal (Harvey) cultivated (3) Allow to solidify.
leishmania on a medium prepared by (4) Steam to melt the medium.

mixing one part melted infusion agar (5) Strain thru glass wool, filled to a
at 45 C. containing 2.0% peptone (see depth of l/8th inch into a Buchner
variant (aa) medium 1375) with two funnel, under slightly reduced
parts sterile defibrinated rabbit blood pressure.
at 45C. (6) Distribute into test tubes.
(n) Harvey cultivated gonococci on a (7) Sterilize at 100C. on each of 3
medium prepared by adding 5 drops successive days.
of sterile defibrinated blood at 45C. (r) Jones cultivated an organism re-
to a tube of infusion agar (see variant sembling Bacillus actinoides from
(v) medium 1661) with a reaction of pneumonic rat lung on a medium
0.6% acid to phenolphthalein at prepared as follows:
45C. Preferably infusion agar in (1) Prepare veal infusion agar.
which sodium chloride is replaced by (2) Slant.
di-sodium phosphate. The substitu- (3) Add 0.5 cc. of defibrinated horse
tion of phosphate is important. The blood, or calf serum, to the water

blood is sterilized by heating 8 days of condensation.
at 57C. Serum may be used instead (4) Remove a small piece of lung
of blood. tissue from an infected rat under
(o) Harvey cultivated B. influenza, etc., aseptic conditions and push the
on a medium prepared as follows: down into the tubes.
tissue
(1) Boil 1.0 cc. blood with 9.0 cc. (5) Seal the tubes with sealing wax.
water. (s) Pitfield added 1 part sterile defibri-
Allow to deposit. nated blood to 5 parts sterile melted
(2)
(3) Add 0.5 cc. clear colorless super- agar.
natant fluid to 5.0 cc. melted (t) Stitt prepared a N. N. N. medium
infusion. (See variant (v) me- for the cultivation of trypanosomes,
dium 1661.) leishmania and protozoa in the

(p) Harvey used the following medium to following manner:
cultivate B. influenza etc.: (1) Cover 125.0 g. chopped beef with
1000.0 cc. of water and place over
(1) Mix 1.0 cc. of defibrinated rabbit
or human blood with 20.0 cc. of night in the refrigerator.
(2) Strain. and fresh defibrinated blood (rab-

(3) Add 20.0 g. of peptone, 5.0 g. NaCl, bit or rat).
10.0 cc. normal NasCOs solution (2) Allow the medium to stiffen
and 20.0 to 25.0 g. agar. slanted so more of the water of
(4) Prepare as for nutrient agar and condensation will settle at the
sterilize (method not given). bottom.
(5) To one part melted and cooled to not specified.
(3) Sterilization
60C. (4) add two volumes of Inoculate with infected blood.
(4)
defibrinated rabbit blood under (y) Soule cultivated Trypanosoma Lewisi
aseptic conditions. and Leishmania tropica on a medium
(6) Slant. prepared as follows:
(7) Cover the cotton plugs with (1) Infuse one part finely chopped
melted paraffin or use rubber choice lean beef in two parts
stoppers to seal the tubes. distilled water in the ice box for
(u) Brown (Stitt) differentiated strepto- 24 hours.
cocci on the following medium: (2) Strain.
(1) Prepare an agar base using 500.0 g. (3) Heat the juice to coagulate the
veal, 5.0 g. NaCl, 15.0 g. agar and proteins. Filter.
10.0 g. peptone in the same manner (4) Dissolve 1.0% Witte's peptone and
as for the preparation of ordinary 0.5% Kahlbaum's NaCl in the
nutrient agar. filtered infusion.
(2) Reaction of (1) to be between (5) Adjust to pH = 7.4.
+0.8 and +1.2. (6) Boil and filter.
(3) Tube in 12.0 cc. amounts. (7) Dissolve 2.0%, agar in (6).
(4) To prepare the blood agar melt the (8) Tube or distribute in flasks.
tubes of agar and place in a water (9) Cool melted tubes of (8)
sterile
bath at 45 C. for 15 minutes. to 50C. and add an equal volume
(5) Add 0.6 cc. defibrinated blood to of sterile defibrinated rabbit
each tube and mix thoroly. blood.
(6) Inoculate. (10)Mix well and slant,
(7) Pour into plates. ai) Autoclave (8) at 110C. for 20
(v) Beilin (Klimmer) cultivated influenza minutes. Blood collected and de-
bacilli on a medium prepared as fibrinated under aseptic con-
follows: ditions.
(1) Heat3.0%infusionagar with 1.0% References: Smedley (1905 p. 28), Gurd
peptone to boiling. (1908 p. 303), Duval (1910 p. 653), Hage-
(2) Add 15.0% of sterile fresh defibri- meister (1914 p. 228), Warden (1915 p.
nated horse blood to boiling (1). 428), Holman (1916 p. Kohlman
381),
(3) Remove from the flame. (1919 p. 574), Bernstein and Lowe (1919
(4) Cool to 50 to 60C. and mi.x p. 78), Wahl, White and Lyall (1919 p.
thoroughly. 420), Yoshida (1920 p. 361), Bell (1920 p.
(5) Pour in plates. 464), Anderson and Schultz (1921 p.
(w) Schottmuller (Klimmer) cultivated 656), Harvey (1921-22 pp. 73, 74, 76),
gonococci, meningococci and strepto- Jones (1922 p. 363), Pitfield (1922 p. 119),
cocci on a medium prepared by mix- Stitt (1923 pp. 43, 44, 52), Klimmer (1923
ing five parts liquified agar at 45C. p. 226), Park, Williams and Krumwiede
with two parts sterile defibrinated (1924 p. 133), Soule (1925 p. 248).
human blood.
1894. Harvey's Peptic Blood Digest Agar
(.\) Novy and MacNeal (Park, Williams
and Krumwiede) cultivated flagellate, Constituents:
trypanosomes and leishmania on a 1. Infusion agar
medium prepared as follows: 2. NaCl (0.85% solution) 150.0 cc.

(1)Mix equal parts of nutrient agar 3. HCl 6.0 cc.
4. Blood, defibrinated sheep . . . 50.0 cc. Preparation

5. Pepsin 1.0 g. (1) See variant (bb) medium 779 for
6. NaOH (20.0%) 12.0 cc. preparation of infusion broth.
Preparation (2) See variant (v) medium 1661 for
(1) Prepare by addition in the order preparation of infusion agar.
given: 0.85% sterile salt solution (3) Mix 100 parts ascitic fluid, 200.0 cc. of
150.0 cc; hydrochloric acid 6.0 cc; defibrinated sheep blood, 10.0 g.
defibrinated sheep blood 50.0 cc, maltose, dissolved in 30.0 cc. (1) with
granulated pepsin B.P. 1.0 g. 600.0 cc. of (2).
(2) Shake to dissolve. Sterilization: Not specified.
(3) Heat in the water bath at 55C. for Use: General culture medium.
2 to 24 hours with occasional shak- Reference: Harvey (1921-22 p. 84).
ing. (Note: The exact time is im-
1897. Harvey's Oxalated Blood Agar
material.)
(4) Add 12.0 cc. of 20.0% sodium Constituents
hydroxide. 1. Infusion agar
(5) Adjust the reaction by addition of 2. Blood, ox or sheep 400.0 cc.
20.0% sodium hydroxide until a 3. NaCl (0.85% solution)
sample gives with cresol red indi- 4. Ammonium oxalate (1.0%
cator solution (0.02%) the color of solution) 30.0 cc.
permanganate (corresponding to Preparation
pH = 7.6). (1) Collect 400.0 cc. ox or sheep blood
(6) Add pure Hydrochloric acid drop by at the slaughter house in a sterile
drop until cresol red indicator solu- flask containing 30.0 cc. of a 1.0%
tion gives practically no change of solution of ammonium oxalate and
color but phenol red gives red. 0.5 cc. formalin.
Note: Corresponding to pH = 7.0 (2) Mix well.
to 7.2. (3) Allow to stand 30 minutes.
(7) Add chloroform to 0.25%. (4) Dilute i with 0.85% sterile salt
(8) Shake to mix. solution.

(9) Keep
in a tightly stoppered bottle (5) Leave for 48 hours.
required for use.
till (6) Mix one part of oxlated blood with
(10) When ready for use, mix at 45C. fifteen parts of infusion agar at 45C.
100 parts melted infusion agar (see (see variant (v) medium 1661 for
variant (v) medium 1661) with 3.5 preparation).
parts 9. Sterilization: Not specified.
Sterilization: Not specified. Use: Cultivation of gonococci, meningo-
Use: Cultivation of B. tnûenzae. cocci and pneumococci.
Reference: Harvey (1921-22 p. 100). Reference: Harvey (1921-22 p. 77).
1895. Bailey's Hormone Blood Agar 1898. Wolbach, Chapman and Steven's
Citrated Blood Agar
Add 5 parts per 100 of fresh defibrinated
human blood to Bailey Hormone Agar Constituents:
(1673). This medium is recommended to 1. Distilled water 1000.0 cc
preserve stock cultures of pneumococci and 2. Veal 500.0 g.
streptococci. 3. Glucose 1.5 g.
4. NaCl 6.0 g.
1896. Harvey's Ascitic Fluid Blood Agar
5. Agar agar 14.0 g.
Constituents: 6. Blood, citrated rabbit

1. Infusion broth 30.0 cc. Preparation
2. Infusion agar 600.0 cc. (1) Prepare a veal infusion from 500.0 cc
3. Blood, defibrinated, sheep... 200.0 cc. of distilled water and 500.0 g. of lean
4. Ascitic fluid 100.0 cc veal. Exact method not given.

5. Maltose 10.0 g. (2) Evaporate (1) to 100.0 cc.
(3) Dissolve 3, 4, and 5 in 900.0 cc. dis- Use: Isolation of Spirochaeta icterohaemr-
tilled water rhagiae. Inoculate with suspected
(4) Add (2) to (3). blood (material) and cover with a thin
(5) Tube. layer of sterile paraffin oil. Author
(6) Add 2.0 to 3.0 cc. of a mixture of reported that growth took place at any
equal parts of sterile rabbits blood temperature from 10C. to 37C. Growth
and citrate saline solution to 4.0 cc. was nearly invisible and about 1.0 to 1.5
of the sterile agar jelly. cm. below the surface.
(7) Heat the mixture to 45C. for 30 Reference: Noguchi (1917 p. 761).
minutes.
and allow to stand until a 1900. Sparkar's Citrated Blood Agar
(8) Slant,
considerable water of condensation (Listen)
has collected, before inoculation. Constituents:
Sterilization: Sterilize (5) method not 1. Water 2000.0 cc.
given. 2. Meat 1000.0 cc.
Use: Cultivation of trj^panosomes. 3. Peptone 40.0 g.
Authors reported that medium afforded 4. NaCl 20.0 g.
rapid growth and the vitality of the 5. Agar 60.0 g.
cultures were not diminished. Gates 6. Blood (human)
cultivated meningococci on a similar Preparation
medium. (1) Boil 1000.0 g. of meat in 2 liters of
Variants : Gates prepared a similar medium water for 40 minutes.
as follows: (2) Filter.
(1) Prepare a veal infusion agar. (3) Dissolve 3, 4, and 5 in sterile (2).
(2) Add 1.0% glucose to (1). (4) Draw one volume of human blood
(3) Adjust to pH = 7.4. into a flask containing 4 volumes
(4) Sterilization not specified. sterile citrated saline(0.5% sodium
(5) Just before tubing add 5.0% sterile, citrate and 0.85% NaCl).
unheated citrated horse plasma. (5) Mix the blood and saline thoroly.
(Fresh rabbit serum may be em- (6) Heat in a water bath at 65C. for 30
ployed instead of plasma.) minutes.
References: Wolbach, Chapman and (7) Remove precipitate by filtering thru
Stevens (1915-16 p. 109), Gates (1919 p. sterilized filter paper.
322). Add 1.0 cc. of (7) to 10.0 cc. of
(8) melted
at 50C.
(3)
1899. Noguchi's Serum Plasma Agar
(9) Slant.
Constituents: Sterilization: Sterilize (2) at 15 pounds
1. Ringer's solution. . . 300.0 cc. pressure in the autoclave
2. Serum rabbit 100.0 cc. Use: Cultivation oi B influenzae (Pfeiffer).
.
3. Plasma, citrated, Variants

rabbit 50.0 cc. (a) Malone prepared a similar medium as
4. Agar (2.0%) 5.0 to 100.0 cc. follows:
Preparation Method of preparation or exact
(1)
(1) Prepare 2.0% agar. composition of nutrient agar not
(2) INIix one part rabbit serum, three parts given.
Ringer's solution (see medium 180), Add 10.0 cc. of pigeons blood to
(2)
one-half part citrated rabbit plasma 90.0 cc. 1.0% sodium citrate
of
and one-half to one part neutral or solution in normal salt solution.
The agar is to
slightly alkaline (1).
(3) Heat (2) for ^ hour at 66 to 70C.
be melted and cooled to 60 to 65C. thru
(4) Filter filter paper.
before mixing with the other Add 1.0 cc. of
(5) (4) to 10.0 cc. of
materials. nutrient agar at a temperature of
Sterilization: Not specified. 50 to 60 C.
(6) Method of sterilization not given. (3) Draw horse blood into flasks con-
(7) Medium is a faint yellow in color taining sterile sodium citrate solution
and transparent. to make the final concentration 1.5%.
(b) Harvey prepared a medium as (4) After have settled, the
corpuscles
follows: plasma is removed and sterile distilled
(1) Add 10.0 cc. of blood to 1.0 cc. water added to make up the original
sterile 10.0% sodium citrate. volume of the blood.
(2) Add 1.0 cc. of (1) to a test tube of (5) Heat sterile (2) to 90C. and add
melted infusion agar at 45C. sterile laked blood up to 1.0 to 2.0%.
(see variant (v) medium 1661 for Sterilization: Method not specified.
preparation) Use: Isolate influenza bacilli.
(3) Rotate the test tubes between the Variants: Harvey prepared a similar
hands to distribute the blood thru medium as follows:
the agar. (1) Mix 5 parts infusion agar at 45C. (see
(c) Harvey gave the following method of variant (v) medium 1661 for prep-
preparation: aration) with 2/lOths part of citrated
(1) Add 10.0 cc. of pigeon's blood to blood at 45C. (one part 10.0%
90.0 cc. of a 1.0% citrated 0.85% citrate in 0.95% sterile salt solution
sterileNaCl solution. to ten parts blood).
(2) Heat 30 minutes at 65 to 70C. (2) Place in a water bath at 80C. until
(3) Filter thru thick filter paper. the agar has become chocolate in
(4) Mix 1.0 cc. of the filtrate from (3) color.
with 10.0 cc. of melted infusion, (3) Slope.
cooled to 45C. with a reaction References: Hirsch and McKinney (1919
slightly alkaline to litmus. (See p. 605), Harvey (1921-22 p. 77).
variant (v) medium 1661 for prep-
1902. Harvey's Glucose Blood Agar
aration of agar.)
(d) Soparkar (Harvey) prepared a similar Constituents:
medium as follows: 1. Distilled water 1000.0 cc.
(1) Mix 10.0 cc. of fresh human blood 2. Ox heart 1000.0 g.

with 40.0 cc. of a 0.5% citrated 3. Peptone 10.0 g.
0.85% NaCl solution. 4. NaoHP04 10.0 g.
(2) Heat in a water bath 30 minutes at 5. Agar 30.0 g.

64 to 68C. 6. Glucose 25.0 g.
(3) Filter thru sterilized paper. 7. Citrated blood

(4) Mix 1.0 cc. of the filtrate from (3) Preparation
with 10.0 melted infusion,
cc. of (1) Mix 1000.0 cc. distilled water with
cooled to 45C. with a reaction 1000.0 g. ox heart.
slightly alkaline to litmus. (See (2) Heat the mixture 20 minutes at a
variant (v) medium 1661 for prep- temperature not exceeding 50C.,
aration of agar.) with constant stirring.
References: Liston (1918-19 p. 418), (3) Raise the temperature to boiling
Malone (1919-20 p. 504), Harvey (1921-22 point.
p. 77). (4) Boil 10 minutes.
(5) Pour the mixture on to a wet, thick,
1901. Hirsch and McKinney's Chocolate
clean cloth.
Agar
Constituents the cloth together with that obtained
1. Infusion agar 1000.0 cc. by squeezing the meat in the cloth.
2. Blood(citrated horse (7) Add peptone 1.0%; di-sodium phos-
1.0 to 2.0%) 10.0 to 20.0 cc. phate 1.0%, prepared agar (see
Preparation medium 1401) 3.0%.

(1) Prepare beef infusion agar. (8) Steam gently 2^ hours to dissolve the
(2) Adjust to a reaction of 0.2%. agar.
(9) Allow to cool to 60C. e.xact amount of soda solution, or
(10) Clarify with white of egg. dilution of soda solution was not
(11) Bring the volume to its orginal specified.)
amount. (5) Pour into plates.
(12) Estimate and make the reaction (6) Smear 1.0 cc. of blood over each plate.
0.6% acid to phenolphthalein. Sterilization: Not specified.
(13) Add to the melted nutrient agar, Use: Cultivation of influenza bacilli.
2.5% glucose. Other investigators employed similar

(14) Steam 30 minutes. (Note: It is media for various purposes.
better to rely on one steaming for Variants
sterilization than to sterilize by (a) Waksman studied the metabolism of
steaming on three successive days.) actinomycetes on a medium prepared
(15) Distribute in quantities of 4.0 cc. as follows:
into test tubes. (1) Boil 500.0 g. veal with 1000.0 cc.
(16) Keep until required for use in the tap water for 10 minutes.
ice chest. (2) Filter.
(17) Prepare sterile 10.0 cc. centrifuge (3) Add 10.0 g. Bacto peptone, 5.0 g.
tubes each containing 2.0 cc. sterile NaCl, and 25.0 g. agar and dissolve.
2.0% sodium citrate. (Heat).
(18) Have in readiness corks or rubber (4) Adjust to +1.0 (pH = 7.6-7.8).
bungs, contained in alcohol to fit the (5) Flask and sterilize.
centrifuge tubes. (6) Cool to 45-50C. and add 10.0%
(19) Fill up the centrifuge tubes with (100.0 cc.) sterile whole rabbit
human blood sterilely aspirated. blood. Shake.
(20) Replace the wool plugs of the centri- (7) Pour, under sterile conditions, into
fuge tubes by corks after burning sterile test tubes or Petri dishes.
off the alcohol. (8) Slant tubes.
(21) Centrifuge. (b) Twort and Twort prepared a similar
(22) Prepare with sterile precautions: medium as follows:
Centrifuged blood fluid at 45C. (1) Prepare infusion agar and infusion
(21) 75; melted nutrient agar (13) broth.
contained in the test tubes at 45C. (2) Sterilize the broth in a flask.
4. (3) Add 5.0% rabbit blood, withdrawn
(23) Roll the test tubes between the from the vein of the ear with a
hands to mix. sterile syringe, to (2).
(24) Test sterility by incubating 48 hours. (4) Incubate (3) at 37 for one hour,
Sterilization: See step (14) under prep- shaking repeatedly to prevent the
aration. forming of too much clot.
Use: Cultivation of gonococci. (5) Melt infusion agar tubes and cool
Reference: Harvey (1921-22 p. 82). to 50C.
(6) Add about 20 drops of the fluid
1903. Grassberger's Blood Agar
from (4) to each tube of (5).
Constituents (7) Slant.
1. Infusion agar. (c) Harvey gave the following method
2. Blood. of preparation
Preparation: (1) Wash and scrub the finger to be
(1) Prepare beef infusion agar. used well with hot soap and water.
(2) Add 10.0% NaOH until blue litmus (2) Drop on to the finger absolute
paper is no longer blue. alcohol followed by ether to remove
(3) Distribute in 20.0 cc. lots. the alcohol.
(4) Add 20 drops diluted 10.0% soda (3) Congest the pulp of the finger by
solution to each flask so as to obtain a winding a bandage round the base.
normal agar (10.0 cc. N/1 soda solu- (4) Prick the congested pulp with a
tion, for 1000.0 cc. agar). (The sterile needle.
(5) Take up a drop of the blood which (9) Stopper tubes air tight with sterile
exudes in a sterile platinum loop. cork stoppers.
(6) Smear on the surface of an agar Sterilization: Sterilize in the autoclave.
slope. (Serum may be used in the Use: Cultivation of gonococci. The
same way.) author reported that the addition of 1.0%
(7) Cover the test tube with an India- glycerol or 1.0% glucose inhibited growth
rubber cap. just as did the absence of peptone.
(8) Test sterility before use by incuba- Reference: Ruediger (1919 p. 377).
tion 48 hours.
1905. Dieudonne's Alkaline Blood Agar
(d) Harvey also prepared a medium as
(Tanner)
follows
Add 0.25 cc. blood to 5.0 cc. melted
Constituents
(1)
agar infusion in a test tube. (See 1. Infusion agar 700.0 cc.
variant (v) medium 1661 for prep- 2. Blood, beef 150.0 cc.
aration.) 3. KOH(N/1) 150.0 cc.
Boil one minute. Preparation

(2)
Slope. (The precipitate settles to (1) Mix equal volumes of beef blood and
(3)
the bottom.) normal KOH solution.
(e) Harvey prepared a similar medium as (2) Steam for 30 minutes.
follows: (3) Mix 3 volumes of (2) with 7 volumes of
Add neutral (to litmus) infusion agar.

(1) directly, blood drawn
from off
a vein or from the heart with a (4) Pour into plates.
sterile syringe to melted infusion (5) Dry the plates at 60C. for 30 minutes,
agar in a test tube at 45C. (See and at room temperatures for 24
variant (v) medium 1661.) hours before use.
(2) Roll the test tube between the Sterilization: Not specified.
palms to mix. Use: Cultivation of Microspira cholerae.

Slope. Reference: Tanner (1919 p. 70).
(3)
(4) Test sterility by incubation 48 1906. Sherwood and Downs' Glucose Blood
hours. Agar
References: Grassberger (1897 p. 462),
Waksman Constituents
(1919 p. 210),Twort and Twort
(1921 p. 88), Harvey (1921-22 p. 72).
2. Glucose 20.0 g.
1904. Ruediger's Blood Agar 3. Blood (whole) 50.0 cc.
Preparation
Constituents
(1) Prepare meat infusion agar with a re-
1. Distilled water 1000.0 cc. action of +0.2% to phenolphthalein.
2. Veal, minced 500.0 g. Add 20.0 g. glucose and 50.0 cc. of
(2)
3. Peptone, Bacto 10.0 g. whole blood to 1000.0 cc. of (1).
4. Bacto agar 15.0 g. Sterilization: Not specified.
5. NaCl 5.0 g. Use: To determine hemolytic ability of
6. Blood, human 100.0 cc. streptococci.
Preparation Reference: Sherwood and Downs (1919 p.
(1) Boil 2 in 1 for one hour. 135).
(2) Cool and strain thru cheese cloth.
(3) Neutralize to phenolphthalein with 1907. Hall's Testicular Infusion Blood Agar
N/1 NaOH. (Stitt)
(4) Dissolve 3, 4 and 5 in (3). Constituents

(5) Tube in 10.0 cc. lots. 1. Distilled water 1000.0 cc.
(6) Heat human blood to 56C. for 30 2. Testicles, beef 500.0 g.
minutes. 3. Peptone (2.0%) 20.0 g.
(7) Add 1.0 cc. of (6) to 10.0 cc. of sterile 4. Glucose (0.5%) 5.0 g.
(5). 5. NaH2P04 (0.2 to
(8) Slant and solidify. 0.3%) 2.0 to 3.0 g.
CULTURE MEDIA FOR CULTIVATION' OF MICROORGANISMS 605
6. Agar (2.5%) 25.0 g. Preparation

7. Blood, human (0.5 to (1) Boil 500.0 g. human blood clot with
2.5%) 5.0 to 25.0 cc. a liter of water.
(1) Remove connective tissue from
all (3) Add 0.5%, NaCl and 1.0% peptone to
beef testicles and put thru a meat the filtrate.
grinder. (4) Distribute as desired (tube).
(2) Cover (1) with twice its weight of (5) Add several drops of blood to each
distilled water. tube of sterile (4) when ready for use-
(3) Infuse on ice over night. Sterilization : Method of sterilization of (4)
(4) Heat to 50C. in a double boiler on not given.
the following morning. Use: Cultivation of amoeba.
(5) Allow to stand for an hour and then Variants: Klimmer infused placenta in-
raise to the boiling point. stead of blood clot.
(6) Allow to stand for another hour to Reference: Klimmer (1923 p. 229).
permit the settling of the solid
particles.
1909. Harvey's Trypsinized Blood Agar
(7) Decant the liquor. Constituents
(8) Add 2.0% peptone, 0.5% glucose, 1. Infusion agar.
0.2 to 0.3% NaHaPOi and 2.5% 2. Trypsin.
granular agar. 3. Blood, human.

(9) Heat over a free flame until solution Preparation
is complete, stirring constantly. (1) Add 0.25 g. of anhydrous Na2C03 to
(10) Adjust the reaction to pH = 7.4 to 100.0 cc. of a 5.0%o trypsinized 0.85%
7.8, using phenol red as an indicator. sterile salt solution under aseptic
(11) Tube. conditions.
(12) Check the reaction after steriliza- (2) Distribute in quantities of 10.0 cc.
tion. in sterile test tubes.
(13) Add human blood in the proportion (3) Add to each test tube 2.0 cc. sterile
of 0.5 to 2.5%, to the tubes while human blood.
they are liquid (just before the
still (4) Incubate 8 days or longer with occa-
agar solidifies). sional shaking.
by heating at 15
Sterilization: Sterilize (1) (5) Add one part of (4) at 45''C. to nine
pounds pressure for 15 minutes. parts of melted infusion agar at 45 ^C.
Use: Cultivation of gonococci. (see variant (v) medium 1661 for
Variants: The author gave the following preparation).
variants: (6) Distribute into test tubes or plates.
(a) Defibrinated rabbits blood (1.0 to Sterilization: Method not given.
5.0%) may be added instead of Use: Cultivation of B. influenzae.
human blood. Reference: Harvey (1921-22 p. 77).
(b) The blood may be added before
This gives a
1910. Harvey's Hydrolyzed Blood Agar
sterilization. "choc-
olate" colored medium. Constituents
Reference: Stitt (1923 p. 46). 1. Infusion agar 350.0 cc.
2. Water 50.0 cc.
3. Blood 10.0 cc.
1908. Cutler's Blood Clot Infusion Agar
(Klinuner)
4. H2SO4 (normal) 10.0 cc.
Preparation
Constituents (1) Mix one part of blood, one part nor-
1. Water 1000.0 cc. mal H2SO4 with 5 parts water.
2. Blood clot 500.0 g. (2) Boil.
3. Peptone (1.0%) 10.0 g. (3) Store for use.
4. NaCl (0.5%) 5.0 g. (4) Make faintly alkaline to litmus at the
5. Blood time of use.
(5) Mix one part of (4) at 45C. with five Use: Cultivation of hemoglobinophilic
parts melted infusion agar at 45C. bacteria.
(See variant (v) medium 1661 for Reference: Kristensen (1922 p. 231).
preparation.)
1912. Brown and Orcutt's Hemoglobin
Infusion Agar
Use: Isolation of B' influenzae.
Variants: Author reported that Brilliant Constituents
green which inhibits the gram + bacteria 1. Veal infusion agar.
may be added. 2. Hemoglobin.
Reference: Harvey (1921-22 p. 76). Preparation
(1) Prepare veal infusion agar.
1911. Kristensen's Hemoglobin Infusion (2) Defibrinate blood and wash re-
Agar peatedly with sterile physiological

salt solution.
Constituents (3) Lake the washed corpuscles with dis-
1. Water 25,000.0 cc. tilled water and remove the cor-
2. Meat 25,000.0 g. puscles stroma by centrifugation.

3. Peptone 1.0% (Centrifuge the laked blood cor-
4. NaCl 0.5% puscles until the supernatant hemo-
5. Hemoglobin 1.0% globin solution no longer gives a
Preparation clouding reaction with salt.)
(1) Mince 25,000.0 g. beef (4) Add the hemoglobin solution thus
(2) Add 25 liters of water and allow to obtained to melted agar (amounts not
stand in the cold until the next day. specified) and pour in plates.
(3) Boil for a short time. Sterilization: Not specified.
(4) Press the fluid from the meat. Use: To determine food requirements for
(5) Add about 16 liters of water to the Bacillus pyogenes.
meat, heat to boiling once more and Reference: Brown and Orcutt (1920 p. 223).
press the juice again from the meat.
1913. Esch's Alkaline Hemoglobin Infusion
(6) Mix the fluid from (4) and (5).
Agar (Tanner)
(7) Add 1.0% peptone and 0.5% NaCl.
(8) Adjust to pH about 7.8 to 7.9.
of Constituents
(9) Dissolve 2.0% agar in (8). (Agar 1. Infusion agar 85.0 cc.
may be omitted giving a liquid 2. Hemoglobin (horse) (Merck).. 5.0 g.
medium.) 3. KOH (N/2) 30.0 cc.
(10) Filter thru cotton wool and distri- Preparation
bute in 600.0 cc. flasks. (1) Dissolve 5.0 g. of Merck's horse hemo-
(11) The pH of the sterile agar should globin in 30.0 cc. of half normal KOH.
be about 7.2. (2) Steam for 30 minutes.
(12) Allow a flask containing defibrinated (3) Mix 15.0 cc. of (2) with 85.0 cc. of
blood to stand several hours to per- neutral (litmus) infusion agar.
mit the corpuscles to sink. (4) Pour in plates.
(13) Pour off the supernatant fluid and (5) Dry at room temperatures with covers
replace with distilled water. removed.
(14) Distribute the hemoglobin solution Sterilization: Not specified.
thus obtained in 20.0 cc. quantities. Use: Cultivation of Microspira cholerae.
(15) Store in the ice box until ready for Reference: Tanner (1919 p. 70).
use.
1914. Sherwood and Downs' Basal Serum
(16) Add 20.0 cc. of (15) to 600.0 cc. of
Agar
sterile melted (11) cooled to 40 to
50C. Constituents:
(17) Pour into plates. 1. Infusion agar 1000.0 cc.
Sterilization: Sterilize (10) in the steamer 2. Serum 20.0 cc.
on each of 3 successive days. 3. Andrade's Indicator
Preparation Use: Cultivation of gonococci. Other in-

and adjust vestigators used similar media for the
(1) Prepare meat infusion agar
to a reaction of +0.2% to phenol- cultivation of a large variety of organisms.
phthalein. Variants: The following authors prepared
(2) Dilute serum with distilled water 1:3. media as indicated below:
Add 6.0 cc. of (2) to each 100.0 cc. of (a) Joos used the following medium for
(3)
the diagnosis of diphtheria. He re-
Add under aseptic conditions a suffi- ported that diphtheria colonies may
(4)
cient amount of a sterile solution of appear after 4-5 hours as small grey
one of the added nutrients so that it white colonies, of moist appearance
be present in 2.0% strength in which can easily be recognized.
sterile (3).
Streptococci and staphylococci were
Add Andrade's indicator. inhibited. Serum need not be col-
(5)
Distribute directly into sterile test lected under aseptic conditions.
(6)
tubes in 8.0 cc. lots and slant and (1) Extract 500.0 g. beef (several days
inoculate as in Russell's medium. old) with 1000.0 cc. water.
contamination. Filter and dissolve 20.0 g. peptone
(7) Incubate to determine
(2)

and 5.0 g. NaCl in (1).
Method of sterilization of solutions of (3) Make alkaline with N/1 NaOH

added nutrients not given. by adding (about 7.0 to 8.0 cc. of
Use: To determine fermentation of carbo- normal solution per liter) to neu-
tral medium.
hydrates by streptococci. Authors re-
ported that this medium supported the (4) To 300.0 cc. of blood serum add
pneumococci and quite a num- 50.0 cc. of N/1 NaOH solution and
growth of
150.0 cc. of (3). (Distilled water
ber of the delicate growing streptococci.
Added nutrients: The authors added 2.0% may be used instead of (3).
Place the mixture in a flat bot-
of any desired carbohydrate. (5)
and Downs (1919 tomed flask and place the flask in

Reference: Sherwood
a water bath at about 60 to 70 ""C.
p. 135).
for 2 to 3 hours.
1915. Harvey's Placenta Blood Serum Agar Raise the temperature to 100C., or
(6)
better, place in a steamer for 30
Constituents:
Infusion agar. to 45 minutes.
1.
Then add an equal amount
2. Serum, Placental blood. (7)
Mix equal parts of sterile (500.0 cc.) of (3) and 20.0 g. of

Preparation: (1)
sterilized placental blood at 45C. agar agar.
or
Dissolve the agar as quickly as
with infusion agar at 45C., 1.0% acid to (8)
possible.
phenolphthalein. (See variant (v) me-
(9) When solution is complete, filter
dium 1661 for preparation.)
in hot condition and sterilize for
15 minutes at 100 to 110C. in an
autoclave.
(10) Pour into sterile Petri dishes.
1916. Veillon's Serum Agar (b) Behrens cultivated Trypanosoma
Brucei on the following medium:
Constituents
(1) Digest chopped
beef in 250.0 cc.
1. Infusion agar
100.0 cc.
100.0 cc. water over night in the cold, or for
2. Serum (human)
one hour at 55C.
Preparation
agar and cool to (2) Strain (1), boil the extract and
(1) Prepare infusion
filter.
40 C.
(3) Dialyze the filtrate in a large col-
(2)Heat serum at 38 to 40C.
of (1) and
lodium sac against running dis-
(3) Mix equal parts
(2).
tilled water for 24 to 48 hours.
(4) Dilute (3) to 1000.0 cc. (e) Harvey.

(5) Dissolve 20.0 g. peptone, 5.0 g. (1) Mix two parts melted infusion
NaCl, 0.1 g. CaCls, 10.0 cc. N/1 agar at 45 C. (see variant (v)
Na2C03 solution, and 20.0 g. agar medium 1661) with one part sterile
in (4). human serum at 45 C.
(6) Distribute in test tubes in 1.0 cc. (f) Harvey cultivated meningococci on a
lots. medium prepared as follows:
(7) Sterilize in autoclave by heating Collect ox or sheep blood at the
(1)
to 105-108 for 15 minutes. slaughter house in a sterile blood
Centrifuge
(8) defibrinated rabbit jar.
blood and draw off serum.
(2) Leave the jar at the slaughter
(9) Dilute serum with 0.5% NaCl
house to avoid the shaking up con-
solution to original blood volume.
sequent on transportation.
(10) Shortly before use melt the desired
(3) Transfer the separated serum
tubes of agar, cool to 60C. and
24 hours later with a sterile pipette
add two volumes of (9) to each
to a sterile flask.
tube.
Mix well and solidify in slanting (4) Transport to laboratory.
(11)
positions. (5) Add ether to 0.5%.
(c) Ferry and Noble cultivated Diplococ- (6) Cork tightly.
cus pneumoniae, Streptococcus viri- (7) Leave 24 hours.
dans and Streptococcus hemolyticus (8) Replace the cork with a sterile
on a medium prepared as wool plug.
(1) Add 500.0 g. of finely chopped lean (9) Keep the flask and contents in a
veal to 1000.0 cc. of water. water bath for 3 hours at 50 C. to
(2) Macerate and allow to stand over drive off the ether.
night. (10) Mix 25 parts (9) at 45C. with 100
(3) Strain thru cheese cloth and bring parts melted infusion agar (see
to a boil. variant (v) medium 1661).
(4) Filter. (g) Jones cultivated an organism re-
(5) Add 20.0 g. peptone (this may be sembling Bacillus actinoides from
omitted) 5.0 g. NaCl and 30.0 g. pneumonic rat lungs on a medium
of finely chopped agar. prepared as follows
(6) Boil and adjust the reaction to the (1) Prepare veal infusion agar,
neutral point, using phenolphthal- (2) Slant.
ein as an indicator. (3) Add 0.5 cc. of calf serum water
(7) Filter. (or defibrinated horse blood) to the
(8) Tube in 3.0 cc. quantities and water of condensation.
sterilize the fractional method in (4) Remove small pieces of lung tissue
the steamer. from an infected rat under aseptic
(9) When ready for use, melt the agar, conditions.
cool to 45C. and add 2.0 cc. of (5) Push the pieces of tissue down in
sterile normal horse serum to each the tube into the liquid.
tube. (6) Seal the tubes with sealing wax.
(d) Ball. (h) Fitch cultivated Bact. abortus Bang
(1) Add 500.0 g. lean chopped veal to on a medium prepared as follows:
1000.0 cc. water, 20.0 g. peptone, (1) Prepare beef infusion broth.
5.0 g. NaCl and 30.0 g. finely (2) Dissolve 20.0 g. agar in (1).
chopped agar. (3) Adjust to pH from 6.8 to 7.2.
(2) Adjust reaction. (4) Tube.
(3) Sterilize by fractional method. (5) Method of sterilization not given.
(4) Cool to 45C. (6) At time of use melt agar and add
(5) Add 2.0 cc. of sterile normal horse (10.0%) naturally sterile horse
serum. serum.
(7) Cool and solidify in slanting (3) Prepare 2.5% agar in the usual way,
position. adding 0.5% NaCl, 1.0% glucose,
(8) Inoculate tubes and place in a 2.0% nutrose and 2.0% Chapoteaut's
10.0% atmosphere of CO2. peptone. The reaction is slightly
(i) Kinsella, Brown and Garcia used the alkaline.
following medium for the isolation of (4) Distribute in 100.0 cc. lots.
gonococci: (5) To 3 parts sterile (4) add 1 part sterile
(1) Prepare 1.6% beef or veal infusion beef serum.
agar. (6) Mix well and pour into plates. The
(2) Adjust to pH = 7.6. reaction is slightly alkaline.
(3) Distribute into 100.0 cc. quanti- Sterilization: Method of sterilization of (4)
ties inErlenmeyer flasks and not given. Sterilize beef serum in
sterilize (method not given). 50.0 cc. lots by placing it at 60''C. for one
(4) When ready for use melt (3) and hour on 4 successive days.
add to each flask containing Use: Cultivation of meningococci. Author
100.0 cc. of hot agar (90-100C.) reported that after 18-24 hours at 37C.
30.0 cc. of (sterile) beef seriun. meningococci colonies were circular, light
(5) Mix thoroly, distributing the small transparent, grayish-blue and about
particles of coagulated serum thru 2-3 mm. in diameter.
the medium. Variants
(6) Pour into sterile Petri dishes, (a) Scott gave the following method of
cover with porous terracotta lids preparation:
and place in incubator over night (1) Mince the placenta and prepare a
(18 hours). boiled extract as with meat (exact
(7) Replace terra-cotta lids by glass method not given).
lids and the medium is read for (2) Add 2.0% nutrose, 1.0% glucose,
inoculation. 1.5% peptone (Chapoteaut) and
Authors reported that gonococci 2.5% agar. Dissolve by steaming.
reached maximum growth in 48 hours. (3) Adjust the reaction to a -1-8 (Eyre).
The type of serum used had little or (4) Flask in 75.0 cc. lots.
no influence on the value of the (5) Sterilize (method not given).
medium. Plates should never dry (6) When ready for use melt and cool
under the terra-cotta lids longer than to 50 C.
24 hours. (7) Add 25.0 cc. of sterile (filtered) ox
References: Veillon (1898 p. 22), Joos serum to each flask.
(1899 p. 303), Behrens (1914 p. 29), Ferry (8) Mix and pour at once into plates.
and Davis (1918 p. 295), Ball (1919 p. 82), (b) Klimmer specified the use of 2.0%
Harvey (1921-22 pp. 79, 80, 82), Jones agar instead of 2.5%.
(1922 p. 363), Fitch (1922 p. 234), Kinsella, References: Kutscher (1908 p. 287), Scott
Brown and Garcia (1923 p. 1). (1915-17 p. 466), Harvey (1921-22 p. 79),
Klimmer (1923 pp. 202, 225).
1917. Kutscher's Serum Placenta Agar
1918. Bezanfon's Serum Placenta Agar
Constituents:
2. Placenta 500.0 g. 1. Water 1000.0 cc.
3. Agar 25.0 g. 2. Placenta 500.0 g.
4. NaCl 5.0 g. 3. Agar 25.0 g.
5 Glucose 10.0 g. 4. Glucose 10.0 g.
6. Xutrose 20.0 g. 5. Peptone 20.0 g.
7. Peptone (Chapoteaut) 20.0 g. 6. NaCl 5.0 g.
8. Serum (Beef) 335.0 cc. 7. Serum 300.0 cc.
(1) Cut fresh placenta into small pieces. (1) Soak 500.0 g. of finely chopped pla-
(2) Weigh the pieces and juice and add a centa in 1000.0 cc. of water in the
double weight of water. ice box over night.
(2) Filter. (3) Add 2 and 3 to (2).

(3) Dissolve 2.5 g. agar, 0.5 g. NaCl, Sterilization: Not specified.
1.0 g. glucose and 2.0 g. peptone in Use: Isolation of meningococci. Authors
each 100.0 cc. of the filtrate. reported that after 24 hours meningo-
(4) Heat beef serum at 60C. for one hour cocci produced a colony 0.5-1.5 mm. in
on each of 4 successive days. diameter, easily recognizable from influ-
(5) To three parts (3) add one part (4). enza bacilli, streptococci and others.
(6) Mix well. Reference: Robey, Saylor, Meleney, Ray
(7) Distribute in sterile containers. and Landmann (1918 p. 324).
Sterilization: Sterilization of (3) not spec-
ified.
1921. Noguchi's Ringer Solution Serum
Use: General culture medium. Agar
Reference: Bezangon (1920 p. 119). Constituents
1. Ringer solution 45.0 cc.
1919. Shmamine's Nucleic Acid Serum Agar
2. Rabbit serum 15.0 cc.
Constituents: 3. Agar 2.0% lO.O cc.
1. 3.0% agar with 0.5% Preparation
glucose 100.0 cc. (1) Prepare 2.0% agar.
2. Serum 100.0 cc. (2) Mix 4.5 parts Ringer's (see me-
3. Physiological salt solu- dium 180) solution and 1.5 parts rab-
tion 10.0 cc. bit serum with 1.0 part 2.0% agar.
4. Sodium salt of nucleic Sterilization: Not specified.
acid 0.5 to 1.0 g. Use: Cultivation of Leptospira ictero-
Preparation haemorrhagiae. Medium was covered
(1) Dissolve 0.5 to 1.0 g. of the sodium with a sterile layer of paraffin oil after
salt of nucleic acid in 10.0 cc. physio- inoculation.
logical salt solution. Variants
(2) Add sterile (1) to 100.0 cc. sterile (a) Noguchi prepared a similar medium
clear transparent serum (or just as follows:
10.0 cc. of sterile physiological salt (1) Prepare a 2.0% agar.
solution may be added). (2) Mix 1.5 parts rabbit serum, 4.5 parts
(3) Distribute into sterile test tubes. Ringer's solution and 1.0 part 2.0%
(4) Prepare a 3.0% infusion agar contain- agar.
ing 0.5% glucose. (3) Allow (2) to solidify.
(5) Melt (4) and cool to 50C. (4) Add to solidified (3) a mixture of
(6) Mix equal parts of sterile (5) and 1.5 parts rabbit serum and 4.5 parts
sterile (3). Ringer's solution.
Sterilization: Sterilize (1) for 15 minutes in (5) Cover with a layer of sterile par-
boiling water bath. Sterilize (3) by affin oil.
heating for one hour on each of 3 suc- (b) Harvey cultivated S. icterohaemor-
cessive days at 60C. Method of steri- rhagiae on a medium prepared as
lization of (5) not given. follows:
Use : Isolation of spirochetes. (1) Dissolve 0.88 g. NaCl, 0.025 g.
Reference: Shmamine (1912 p. 317). KCl, 0.02 g. CaCl2 and 0.015 g.
NasCOa in 100.0 cc. distilled water.
1920. Robey, et al., Glucose Serum Agar
(2) Mix 1.5 parts of sterile rabbit serum
Constituents at 45C., 4.5 parts (1) at 45C. and
1. Veal infusion agar 1000.0 cc. 1 part of melted infusion agar at
2. Glucose 5.0 g. 45C. (See variant (v) medium

3. Serum, horse 50.0 g. 1661.)
Preparation (3) Cover with a thin layer of paraffin
(1) Prepare veal infusion agar. oil.
(2) Adjust reaction to 0.3 to 0.5% acid to References: Noguchi (1918 p. 606), Harvey
phenolphthalein. (1921-22 p. 80).
1922. Todd's Lactose Serum Agar 4. NaCl (0.5%) 5.0 g.

5. Cystine (0.1%) 1.0 g.
Constituents 6. Glucose (1.0%) 10.0 g.
1. Sugar free agar 1000.0 cc,
7. Serum horse (5.0%) 50.0
. . cc.
2. Lactose 20.0 g.
Preparation
3. Neutral red (1.0% watery
(1) Prepare beef infusion.
solution basic stain) 0.4 cc.
Serum, beef 200.0 cc.
4.
Preparation
(4) Keep this as a stock agar.
(1) Prepare sugar free agar. Add 0.1% cystine (or cystein hydro-
(5)
(2) Dissolve 2 and 3 in 1. (Dye strength chloride) and 1.0% glucose to (4)
such that medium is colored.)
when ready for use.
(3) Tube in 7.0 to 8.0 cc. lots.
(6) Place in the Arnold for 15 minutes
(4) Just before use add 2.0 cc. of sterile in flowing steam to melt the agar,
beef serum to each tube of sterile (3). and sterilize the cystine.
Sterilization: Sterilize for three successive Cool to 45 to 50C. and add 5.0%
(7)
days in Arnold. (Time not specified.) sterile horse serum.
Use: Isolation and differentiation of strep- (8) Tube in sterile tubes.
tococci. Author reported that Strept, Slant.
(9)
pyogenes formed deep red colonies with Incubate for 24 hours to test
(10)
pink border. Strept. viridans (Schott- sterility.
miiller) formed red colonies surrounded
by large area of milky-like haze; Strept. (3) not given. Cystine sterilized in step
mucosus (Schottmiiller) pink centered
(6) above.
colonies with white border, edge irregu-
Use: Cultivation of Bacterium tularense,
lar with milky-like haze. Pneumococci gonococci and diphtheria bacilli.
formed red centered colonies with narrow Reference: Stitt (1923 p. 46).
white border, smooth edge sharply de-
fined. Other sugars and dyes were tried 1925. Elser and Huntoon's Basal Ascitic
but this combination gave best results. Fluid Agar
Reference: Todd (1910 p. 74). Constituents
1923. Harvey's Telluric Acid Serum Agar
2. Lean beef 500.0 g.
1. Infusion agar 4. NaCl 5.0 g.
2. Serum (sheep) 50.0 cc. 5. Agar agar 25.0 g.
3. Telluric acid (1.0% solution). 9.0 cc. 6. Litmus solution (Kubel &
Preparation: (1) Mix 50 parts sterilized Tiemann) 15.0 cc.
sheep serum at 45C., 9 parts of a sterile 7. Ascitic fluid 335.0 cc.
1.0% telluric acid solution at 45C., and Preparation
1000 parts of melted infusion agar, cooled (1) Prepare basic litmus infusion agar
to 45 C., neutral red to litmus (see from 1, 2, 3, 4, 5, 6 and one of the
variant (v) medium 1661 for prepara- added nutrients in the same manner
tion).* exactly as given in medium 1677.
Sterilization: Not specified. Use 25.0 g. agar instead of 16.0 g.
Use: Enrichment medium for B. diph- agar.
theriae. (2) To the finished product which is
Reference: Harvey (1921-22 p. 92). cooled to 55C. add I its volume of
sterile ascitic fluid. The degree of
1924. Francis' Cystine Serum Agar (Stitt)
alkalinity of the ascitic fluid must be
Constituents previously determined and taken into
1. Beef infusion 1000.0 cc. consideration when correcting the
2. Peptone (1.0%) 10.0 g. reaction of the agar.
3. Agar (1.0 or 1.5%) . . 10.0 or 15.0 g. Sterilization: Not specified.
Use: Cultivation of meningococci, pseudo- (v) for preparation) containing 1.0%

meningococci and gonococci. Authors glucose at 45C. Reaction of the
reported that the medium was especially- agar to be faintly alkaline to litmus,
suited to freshly isolated cultures. (d) Harvey cultivated meningococci on a
Added nutrients: The author added 1.0% mixture of seven parts sterile ascitic
of one of the following: fluid at 45 C., three parts of a sterile
glucose sucrose standard litmus solution at 45 C., two
galactose mannitol parts of a sterile 10.0% solution of
levulose inulin any desired carbohydrate at 45 C.,
lactose dextrin 0.15 part of a normal NaOH solution
maltose dulcitol at 45 C., and 21 parts of melted infu-
Variants sion agar at 45C. (See variant (v)
(a) Plotz, Olitsky and Baehr used the medium 1661 for preparation.) The
following medium to study fermenta- litmus solution was prepared as
tion by organisms causing typhus follows:
exanthematicus: (1) Place in a well stoppered glass
(1) Prepare 3.0% agar. bottle, solid commercial litmus 1;
(2) Add 2.0% of one of the added nu- 95.0% alcohol 6.
trients to (1). (2) Shake one daily for seven days.
(3) Add volume of ascitic fluid (spe- (3) Reject the alcohol.
cific gravity to be 1013 or over). (4) Add fresh 95.0% alcohol.
(4) Reaction of medium to be between (5) Shake well once daily.
1.0 and 1.5%. (6) Repeat the addition of fresh alco-
(5) Tinge with Kahlbaum's litmus. hol until the alcohol becomes only
(6) Method of sterilization not given. lightly tinged with violet on shak-
The added nutrients employed ing up with the litmus.
were: (7) Reject the alcohol.
glucose arabinose (8) Dry the alcohol insoluble residue.
lactose inulin (9) Make a saturated solution of the
sucrose raffinose dried residue in distilled water.
maltose galactose (10) Filter.
mannitol dextrin (11) Dilute a portion of this saturated
(b) Harvey prepared a medium as solution with distilled water until
follows: its tint is a pure blue.
(1) Mix one part nutrose with 15 parts (12) Add to this pure blue solution very
ascitic fluid and 35 parts distilled dilute sulphuric acid till the blue
water. color is turned to wine red.
(2) Raise slowly to boiling tempera- (13) Add to this wine red solution the
ture with frequent shaking to pre- saturated solution obtained from
vent burning. the alcohol insoluble residue until
(3) Boil or steam 30 minutes. the blue color returns.
(4) Clarify by the addition of egg and (14) Use as a sensitive litmus solution.
filterthru thick filter paper. Sterile 10.0% sugar at 45C., 2;
(5) Add one part (4) at 45 C. to two sterile N/1 sodium hydroxide at
parts melted infusion agar cooled 45C., 0.15; melted nutrient agar
to 45C. (see variant (v) medium at 45C., 21.
1661 for preparation). (e) Harvey cultivated B. acnes on a
(6) Steam 30 minutes. medium composed of 2 parts sterile
(7) Distribute into test tubes. oleic 20 parts sterile ascitic
acid,
(8) Sterilize. fluid, part of a sterile 1.0%
0.8
(c) Harvey cultivated meningococci on a neutral red and 100 parts of sterile
mixture of one part ascitic fluid at infusion agar (see medium 1661,
45C. with three parts melted infu- variant (v) for preparation).
sion agar, (see medium 1661, variant (f) Torrey and Buckell used the follow-
ing medium to determine fermenta- (5) Allow the undissolved portion to

tion of gonococci: settle out and pour off the super-
(1) Prepare sugar free infusion broth.

natant fluid.
(2) Add 15.0 g. peptone and 6.6 g. agar (6) Add more water to the residue and
to (1) and dissolve. grind again. Combine the two
(3) Adjust to reaction pH = 7.0. portions.
(4) Add brom-thymol-blue in suffi- (7) Rinse the mortar with the remainder
cient amount to give fairly deep of the 25.0 cc. of water.
color. (8) Filter thru paper.

Tube in 5.0 cc. lots. (9) Heat sterile (8) to 50C. and add
(5)
(6) Sterilize in autoclave. to (2).
To each tube (6) add 1.0 cc.

of (10) Mix (9) thoroly and insert in a sterile
(7)
from fermentable
ascitic fluid free siphon.
substances by prolonged storage (11) Dispense in Petri dishes.
in ice box. (12) Incubate the plates to test sterility.
(8) Expose a 12.0% solution of any de- Sterilization: Method of sterilization of
sired carbohydrate to flowing agar, serum or glucose not given. Steri-
steam at 100C. for 12 minutes. lize the potassium tellurite solution by

Add 1.0 cc. of (8) to each tube filtration thru a Mandler.
(9)
of (7). Use : Isolation of diphtheria bacilli. Diph-
(10) Incubate 3 days at 37C. to deter- theria colonies white, or white about the
mine sterility. periphery with grey centers. Staphylo-
Author reported that with glucose, cocci are black.
medium below surface growth was Reference: Gilbert and Humphreys (1926
changed from bluish green to yel- p. 149).
low in 18 to 48 hours. In case of Agar
1927. Scholtz's Ascitic Fluid
unf ermented sugars color remained
unchanged or a bluish tinge de- Constituents
veloped. 1. Infusion agar.
References: Plotz, Olitsky and Baehr 2. Ascitic fluid.

(1915 p. 10), Elser and Huntoon (1909 Preparation
p. 407), Harvey (1921-22 pp. 83, 84, 87), (1) Prepare infusion agar.
Torrey and Buckell (1922 p. 145). (2) Mix (1) with ascitic fluid (pericardic
or hydrocelenic fluid may be used)
1926. Gilbert and Humphrey's Tellurite in the ratio of 3 to 1.
Serum Agar Sterilization: Not specified.
Constituents Use: Cultivation of gonococci.

1. Beef infusion agar (1.5%). 1000.0 cc. Variants
2. Serum, horse (5.0%) 50.0 cc. (a) Swartz and Davis used the following
3. Glucose (1.0%) 10.0 g. medium to cultivate gonococci.
4. Potassium tellurite 1% Have the medium at body tempera-
solution (0.58% on test) ture when inoculating. Following
(1%) 10.0 cc. inoculation hold the tubes horizon-
Preparation tally so that the agar slant is upper-
(1) Melt sterile beef infusion agar and most. Holding the tubes by the
cool to 50 C. butt, pass the tubes thru the flame
(2) Add 50.0 cc. of sterile horse serum, three times longitudinally and cork
10.0 cc. of a sterile 20% glucose quickly.
solution, heated to 50C. (1) Prepare a 2.0% beef (or veal) infu-
Weigh out 250 mgm. KiTeOs on a sion agar in the usual manner.
(3)
chemical balance. Grind to a very (2) Adjust (1) to pH 7.6 (pH 7.4 after
finepowder. autoclaving) and sterilize in the
(4) Add about 10.0 cc. of water. Mix autoclave.
well. (3) Mix one part sterile ascitic fluid
with two parts sterile melted (2). (d) Mulsow used the following medium
(Pleuritic or hydrocele fluid may for the isolation of gonococci:
be used instead of ascitic fluid.) (1) Infuse one pound of ground lean
(4) Replace the cotton stoppers with beef in 500.0 cc. of distilled water
sterile corks. over night in the ice box.
(b) Harvey prepared a similar medium (2) Dissolve 10.0 g. peptone in the
as follows: juice while cool.
(1) Mix one part ascitic fluid with two Add 500.0 cc. of a
(3) 3.0% agar solu-
parts infusion agar (see variant tion, melted and cooled to 60C
,
(v) medium (1661) for prepara- to (2).
tion). Adjust to +0.9 to phenolphthalein.
(4)
(2) Sterilize in the water bath 30 min- Before the agar has cooled suffi-
(5)
utes at 56C. on five successive ciently to harden, it is heated in
days. the autoclave at 15 pounds pressure
(3) Test sterility before use by incu- for 25 minutes.
bation 48 hours. (6) Filter thru moistened cotton and
(c) Schwartz (Stitt) gave the following canton flannel.
method of preparation:
(7) Distribute in 100.0 cc. lots in
(1) Prepare beef or veal infusion agar flasks.
in the ordinary manner. When ready for use, sterile (7) is
(8)
(2) Adjust (1) to pH = 7.6 phenol- melted and cooled to 60C. and
sulphonephthalein being used as added to 50.0 cc. of ascitic fluid.
an indicator in the hot agar. (0.5 g. ofany carbohydrate (levu-
(3) Cool to 50"C. lose or maltose) may be added
(4) Add the whites of three fresh eggs along with 1 cc. of a 0.04% solution
to each liter of agar. of brom cresol purple.)
(5) Start with a low flame, boil for (9) Pour in sterile plates.
10 minutes. (10) Final reaction -1.2 to phenol-
(6) Strain thru cloth and filter thru phthalein or pH = 6.8.
paper. References: Scholtz (1899 p. 5), Schwartz
(7) Tube in 5.0 to 6.0 cc. quantities and Davis (1920 p. 1125), Harvey (1921-
and autoclave at 10 pounds pres- 22 p. 83), Stitt (1923 p. 45), Mulsow
sure on 3 successive days. (1925 p. 421).
(8) Sterilization reduces the pH to 7.4.
Add sterile ascitic,
1928. Kiefer's Ascitic Fluid Agar (Abel)
(9) pleuritic or
hydrocele fluid to each tube. One Constituents
part fluid to two parts agar. 1- Water lOOO.O cc.
(10) Seal the tubes with sterile rubber 2. Meat 500.O g.
stoppers and slant. 3. Agar (3.5%) 35.0 g.
(11) Cork the tubes and store in the 4. Peptone (5.0%) 5O.O g.
incubator. 5. Glycerol (2.0%) 20.0 g.
(12) Discard all contaminated tubes. 6. NaCl (0.5%) 5.0 g.
(13) Inoculate when the medium is at 7. Ascitic fluid 1000.0 cc.
body temperature. Preparation
(14) After inoculation hold the tube (1) Chop 500.0 g. of fat free meat and
horizontal by the butt with the add to a liter of water at 50C.
agar slant up. Pass longitudi- (2) Keep at 50C. for 30 minutes and then
nally thru the Bunsen flame about boil for 30 to 45 minutes.
three or four times and cork (3) Filter or strain the fluid from the
quickly. This reduces the pres- meat.
sure within the tube. (4) Make up the fluid to 1 liter.
(15) The agar tubes should have about (5) Dissolve 3, 4, 5 and 6 in (4).
0.5 cc. of water of condensation in (6) Neutralize.
the lower angle of the slant. (7) Cool to 50C.
615
fluid free from bile and 0.5 cc. of a

(8) Mix (7) with an equal volume of
1:3000 dilution of iodin-green (Gr ab-
ascitic fluid.
ler) are added to each tube of sterile
(9) Distribute in tubes or plates.
melted (11) just before pouring.
gonococci. Author (12) Final reaction pH = 7.2.
Use: Cultivation of
ascitic fluid be Sterilization: Autoclave (10) at about
reported that if the
strongly alkaline mix with unneutralized
Use: Isolation of gonococci. Authors re-
or strongly acid agar. The final medium
ported that hours gonococci
after 48
should be slightly alkaline.
colonies were semi-translucent and had
raised or even indented edges. They
dium as follows:
pure neutral glycerol stood out from the medium. Centers
(1) Mix equal parts
and ascitic fluid, (pleuritic fluid, somewhat thickened. If used in slants
ovarian fluid, milk, use 40.0 g. agar.
hydrocele fluid,
Variants: The authors omitted the iodin-
urine, etc., may be used instead).
on culture test. green.
(2) Leave until sterile
infusion agar (see Reference: Torry and Buckell (1922 p. 125).
(3) Add (2) to sterile
variant (v) medium 1661 for prepa- Whey Ascitic Fluid Agar
1930. Watabiki's
ration).
Harvey Constituents
References: Abel (1912 p. 162),
(1921-22 p. 83).
1. Whey 200.0 cc.
2. Ascitic fluid (5.0%) agar
1929. Torrey and Buckell's Urine Ascitic 3. Nutrose (0.3%) agar 400.0 cc.
Fluid Agar 4. Urea 2.0 g.
Preparation
Constituents
2000.0 cc. (1) Warm 200.0 cc. of cow's milk to 60C.
1. Distilled water
1250.0 g. (2) Add 5.0% ascitic agar drop by drop.
2. Veal, fat-free
20.0 g. (Composition or method of prepara-
3. Peptone (Difco)
40.0 cc. tion not given) the milk being shaken
4. Urine, fresh
100 to cause precipitation of the casein.
5. NaCl g-
6. Glvcerol 40.0 cc.
36.0 g. (4) To filtrate add 10.0% caustic soda to
7. Flaked agar
1000.0 cc. slightly alkaline.
8. Ascitic fluid
9. lodin-green (5) Add urea (2.0 g.).
Preparation: (6) Mix sterile (5) with 0.3% nutrose agar.
chopped lean veal in (Composition or method of prepara-
(1) Place finely
tion not given.) One part fluid (5)
water and bring slowly to a boil.
20 minutes with to 2 parts agar at 45C.
(2) Allow to simmer
(7) Slant or plate as desired.
occasional stirring.
Incubate to insure sterility.
flannel.
(3) Strain thru cotton
(8)
Sterilization: Sterilize by heating at
(5)
(4) Cool and remove
the fat.
Place in double boiler over a satu- 60C. for 30 minutes on each of three
(5)
successive days.
rated brine bath and raise the
temperature to about 60C. Use: Cultivation of gonococci. Author
min- reported that the medium was not as
(6) Add 3, 4, 5, 6 and 7 and boil 45
satisfactory as blood agar or blood broth.
utes to dissolve materials.
Adjust reaction to pH = 6.9, using Ordinary broth or peptone water may be
(7)
10.0% Na2C03. mixed with the fluid or liquid medium.
Boil 30 minutes longer.
Reference: Watabiki (1916 p. 734).
(8)
(9) Remove from brine and add distilled 1931. Esch's Maltose Ascitic Fluid Agar
water to 2 liters.
Filter thru canton flannel and tube
Constituents
(10)
1. Infusion agar
750.0 cc.
in 10.0 cc. amounts.
2. Peptone. Witte (1.0%) 7.5 g.
(11) In preparing plates 5.0 cc. of ascitic
3. Maltose 10. o g. (3) Dissolve 3, 4 and 5 in 250.0 cc. of

4. Ascitic fluid 250.0 cc. water by heating slightly.
Preparation
(4) Mix (3) and (2).
(1) Prepare infusion agar, containing (5) Neutralize to litmus or adjust to
1.0% Witte's peptone. 1.0 to 1.5 acid to phenolphthalein.
(2) Mix 75 parts (2) with 25 parts ascitic
(6) Boil for an hour.
fluid.
(7) Readjust the reaction if necessary.
(3) Add 1% maltose to (2).
(9) The reaction of the sterile agar when
Use: Cultivation of meningococci.
ready for use should be 1.8%.
Reference: Esch (1909 p. 152).
(10) Melt sterile (9) and add 30.0 cc. of
1932. Steinschneider's sterile beef bile and 1.0 cc. of a solu-
Hydrocele
Fluid Agar tion of 1.0 g. malachite green I
(Hochst) in 60.0 cc. distilled water.
Constituents
It is preferable to mix 20.0 cc. of
1. Infusion agar
agar
300.0 cc. with 0.05, 0.1, 0.15, 0.2, etc., cc. of a
2. Hydrocele fluid 100.0 cc. 2.0% malachite green solution and
Preparation
pour into plates. Inoculate with
(1) Mix three parts sterile infusion agar
colon and typhoid bacilli and use
with one part sterile liquid hydrocele
that dilution of malachite green that
fluid.
inhibits the colon organisms but
Pour into not
(2) sterile Petri dishes.
the typhoid organisms. Add a
Sterilization: Method not given. corresponding amount of malachite
green and bile to the 100.0 cc. of agar.
Reference: Steinschneider (1890 p. 533). Sterilization: Method of sterilization of
(8)
1933. Heiman's Pleuritic Serum Agar not given.
Use: Cultivation of colon-typhoid group.
Constituents
1. Infusion agar (2.0%) 200.0 cc.
2. serum
Pleuritic 100.0 cc '
1935. Lentz and Tietz's Malachite Green
3. Peptone (1.0%) 2.0 g Nutrose Bile Agar (Klimmer)
4. NaCl (0.5%)
1.0 g. Constituents
Preparation: (1) Add one part sterilized 1. Meat infusion 2000.0 cc.
liquid pleuritic (chest) serum to 2.0% 2- Agar 60^0 g.
agar containing 1.0% peptone and 5% 3. Peptone 20 g
NaCl.
4- NaCl io:og:
Sterilization: Sterilize on 9 successive days
5. Nutrose 20.O g.
at 65C.
6. Bile, beef 30.0 cc.
7. Malachite green
Reference: Heiman (1896 p. 888).
Preparation
1934. Lentz and Tietz's Malachite Green (1) Soak 60.0 g. of agar in 2000.0 cc. of
Bile Agar (Klimmer) meat water.
(2) Boil for three hours.
Constituents
(3) Dissolve 3, 4 and 5 in 250.0 cc. of
1. Meat infusion 2000.0 cc. water by heating slightly.
2- Agar 60.0 g. (4) Mix (3) and (2).
3. Peptone 20.0 g. (5) Neutralize to litmus or adjust to
4. NaCl 10.0
g'
1.0 to 1.5 acid to phenolphthalein.
5- Bile 30.0 cc. (6) Boil for an hour.
6. Malachite Green I (Hochst) (7) Readjust the reaction if necessary.
Preparation
(1) Soak 60.0 g. of agar in 2000.0 cc. of
(9) The reaction of the sterile agar when
meat water.
ready for use should be a -3.5%.
(2) Boil for 3 hours.
(10) Melt sterile (9) and add 3.0 cc. of
and 1.0 cc. of a solu- (13) Add 20.0 cc. of a freshly prepared
sterile beef bile
solution of 0.1 g. Crystal violet B
tion of 1.0 g. of malachite green I
(Hochst) in 60.0 cc. distilled water. Hochst in 100.0 cc. sterile warm dis-
tilled water.
It is preferable to mix 20.0 g. of agar
with 0.05, 0.1, 0.15, 0.2, etc., cc. of a (14) Pour into plates.
2.0% malachite green solution and Sterilization: Not specified.

Use: Differentiation of colon-typhoid
pour into plates. Inoculate with
colon and typhoid bacilli and use group. Authors reported that coli colo-
that dilution of malachite green nies were red, deep red and wine red.
that inhibits the colon organism but Bad. typhi colonies were small, of bluish
not the typhoid organism. Add a color and glossy.
corresponding amount of malachite Variants: The following authors prepared

green and bile to the 100.0 cc. of the medium as indicated:
agar. (a) Frost used a sugar free infusion broth

Sterilizatiou : Method of sterilization of (8) and specified that the medium be
sterilized for 20 minutes on 3 suc-
not given.
cessive days.
Use: Cultivation of colon-typhoid group.
Reference: Klimmer (1923 p. 212). (b) Harris (Heinemann).
(1) Dissolve 20.0 g. nutrose and 40.0 g.
1936. V. Drigalski and Conradi's Crystal agar in 2000.0 cc. glucose free infu-
Violet Nutrose Agar sion broth.
(2) Neutralize (1) to phenolphthalein.
Constituents
1. Water 2000.0 cc. (3) Autoclave at 120 for 5 minutes.
3.0 lbs. (4) Clarify with the white of eggs and
2. Beef
20.0 g. filter.
3. Peptone (Witte)
(5) Add 30.0 g. lactose, 260.0 cc. of
4. Nutrose 20.0 g.
NaCl 100 g- litmus solution and 20.0 cc. of a
5.
6. Agar 60.0 g. 0.1% aqueous solution of Crystal
violet.
7. Litmus solution
Lactose 30.0 g. (6) Tube.
8.
(7) Sterilize in the Arnold,
9. Crystal violet (B. Hochst)
Preparation (c) Wesbrook.
with Add 680.0 g. finely chopped lean
(1) Allow 2 liters of water to stand
(1)
beef to 1000.0 cc. water and place
3 pounds of finely chopped beef over
night. in cold for 24 hours.
Filter off the meat water and boil (2) Express the juice and make up to
(2)
one hour. one liter.
(3) Coagulate albumin either by vigor-
(3) Filter.
Add 3, 4, 5 and boil one hour. ous boiling 10 minutes or by heat-
(4)
Filter. ing at 120C. in the autoclave.
(5)
60.0 g. of fine agar, boil 3 hours Filter.
(6) Add (4)
(or autoclave one hour) and make (5) Dissolve 10.0 g. Witte's peptone,
slightly alkaline to litmus. 10.0 g. nutrose and 5.0 g. NaCl in
(7) Filter and boil 3 minutes. (4).

Heat in autoclave at 120C. for
(8) Boil 260.0 g. of litmus solution (pre- (6)
pared according to Kubel and Tie- 30 minutes or boil vigorously for
mann, method or reference not 15 minutes.

Render slightly alkaline to litmus
given) for 10 minutes and add 30.0 g. (7)
c.p. lactose.
paper.
Filter.
(9) Boil (8) for 15 minutes. (8)
(10) Mix hot (9) and (7). (9) Add 30.0 g. agar to (8).
(10) Heat in autoclave at 120C. for
(11) Again adjust to slight alkalinity.
Add 4.0 cc. of a hot, sterile 10.0% 30 minutes, or over flame until
(12)
water free soda solution. agar is dissolved.
(11) Render slightly alkaline to litmus as an elective medium for typhoid

while hot if necessary. bacilli:
(12) Filter thru glass wool into a sterile (1) Obtain 7 or 8 liters of blood from
vessel. the slaughter house.
(13) To 130.0 cc. of litmus solution (2) Pour off the serum from the clot.
(Kubel and Tiemann) add 15.0 g. (3) Add a double amount of water to
c.p. lactose. the clot.
(14) Boil (13) for 10 minutes. Boil the mixture.
(4) Stir constantly
(15) Mix (14) and (12) while hot. but take care not to break up the
(16) Render slightly alkaline to litmus clot too much or filtration will be
paper if necessary. too diflficult. Boil for 5 to 10
(17) To add 2.0 cc. of hot sterile
(16) minutes.
solution of 10.0% sodium hydrate
(5) Filter thru a towel, then thru cot-
in distilled water, and 10.0 cc. of ton or glass wool.
fresh solution of Hochst's crystal Add peptone
(6) nutrose, in the usual
violet (0.1 g. to 100.0 cc. of sterile amount (exact amount not speci-
water). fied) and 3.0% agar.
(18) Pour in sterile Petri dishes.
Glass (7) When the agar has been completely
covers of Petri dishes may be dis- dissolved, add the serum that had
carded and use porous clay covers. been previously poured from the
(d) Reitz used the following medium to
blood and that had been allowed
study the bacteriology of butter and to stand undisturbed.
butter examination: (8) Boil for a few minutes.
(1) Chop 3 pounds of horse meat to (9) Place in a flask with a patented
small pieces. stopper and heat for one hour in
(2) Pour 2 water over (1) and
liters of streaming steam.
allow to stand until the next day (10) Allow to cool slowly so that the
(in ice box).
sediment and turbidity may settle
(3) Press the water from the meat. to the bottom. Use only the top
(4) Add 2.0% Witte peptone, 2.0% clear medium.
nutrose and 0.5% NaCl to the meat (11) If caffeine is desired in the medium
water and boil an hour. prepare a solution of caffeine in
(5) Filter thru a linen towel and add water so that 1.0 cc. of solution
3.0% agar. contains 0.1 g. caffeine. Sterilize
(6) Boil in the steamer for 3 hours and and add 6.0 cc. of this solution to
filter thru linen in the steamer.
100.0 cc. of the agar.
(7) Prepare 300.0 cc. of a 15.0% Kahl- (f) Roddy.
baum's litmus solution. Mix 750.0 g. of finely
(1) minced beef
(8) Add 30.0 g. lactose to (7) and boil with 1000.0 cc. of water.
for 15 minutes. Place in a shallow dish in the ice
(2)
(9) Mix hot (8) and hot (6). ice box over night.
(10) Add 10.0% soda solution until the (3) Skim off the fat.
reaction is slightly alkaline to (4) Boil for one hour.
phenolphthalein. (5) Filter.
(11) To (10) add 6.0 cc. of a sterile warm (6) Bring this volume to 1000.0 cc.
10.0% soda solution and 20.0 cc. (7) Add 10,0 g. Witte's peptone, 10.0 g.
of a fresh solution of 0.1 g. crystal nutrose and 5.0 g. CaCl2 to (6).
violet (Hochst c.p.) in 100.0 g.
(8) Boil.
sterile distilled water. Add
(9) 30.0 g. agar and boil until
(12) Distribute in 200.0 cc. lots in solution is complete.
sterile Erlenmeyer flasks. Make faintly alkaline to litmus.
(10)
(13) Final sterilization not specified, (11) Autoclave for one hour.
(e) Schmitz used the following medium (12) Filter thru paper.
619
(21) Pour large plates for use, with a

(13) Autoclave for 15 minutes.
Kahlbaum's lit- layer of medium about 4 mm.
(14) Add 150.0 cc. of
thick.
mus solution and 15.0 g. lactose û .
^gs (22) Place the plates so made, in the

inverted position in the incubator
(15) Sterilize in the Arnold steamer for
^ ,^ ^ .
without their covers and resting
15 minutes.
0.1% solution on a support.
(16) Add 10.0 cc. of a hot
(h) Klimmer reported that tropon could
of crystal violet (B. Hochst).
be used instead of nutrose, that man-
nitol or maltose might be used in-
(l)"nnce finely fat free beef.
stead of lactose, and a 1 0% Eich-
2 Add 15.0 cc. to 2000.0 cc. water.
at a loff's solution may be substituted for
3 Heat the mixture 20 minutes
meat infusion.^ (Eich off is a skmi
over a free flame.
^^^ preparation obtained from a
food factory.)
(4) Skim off fat floating on the surface.
References: v. Dngalski and Conradi
5 Raise the temperature to boiling
^
. (1902 p. 291), Frost (1903 p. 342), Heine-
nânn (1905 p. 130), Westbrook (1905
^fi^ Ron 10 minutes
p. 319), Reitz (1906 721), Abel (1912
? ur the mlTand fluid on to a p^
^^ PP- 128, 129) Ko^^^^^V
wet, thick, clean cloth.
415), Schmitz (1915 p- TtTr^H
307) Roddy
v
(8) Filter the fluid collected thru p.
(1917 p. 44), Tanner (919 p^ 62) Ball
thick, well-wetted, filter paper.
(1919 p. 81), Gi U-er (1919 p 387), Be.an-
(9) Add to the filtrate, peptone 20.0 g.,
sodium chloride ?on (1920 p. 342), Levine (1921 p. 116),
nutrose 20 g ,
Abbott (1921 p. 523), Harvey (1921-22

?0 0g and see that the mixture is
alkaline to litmus. P- 90), Klimmer (1923 p. 208).
(10) Steam 45 minutes. j^^y^ Galli-Valerio's Congo Red Nutrose

(11) Add 60.0 g. prepared fiber agar *
^^^j.
(see medium 1401).
2 hours or heat Constituents:
(12) Steam gently onnn n cc.

Water 2000.0
45 minutes at 118C. to bring the
1.
2. Meat, horse 1000.0 g.

agar thoroly into solution.
3. Peptone
(13) Make faintly alkaline to
litmus. ^^-^
Nutrose Û.u g.
well-wetted, 4.
(14) Filter, while hot, thru
NaCl lU. g.
paper by placing filter 5.
thick filter
6. Agar W.u g.
funnel, stand and receptacle for
Lactose - ^
filtrate in the steam sterilizer and 7.
ti'^
8. Congo red (1.0% soln.) 30.0 cc.
steaming until filtration is com-
plete.
Preparation:
(D Boil 1000.0 g. of horse meat with
(15) Boil 300.0 cc. litmus solution
10
minutes and dissolve in it 30.0 g. 2 liters of water for an hour,

Filter.
chemically pure lactose. (2) ,,
c^
Add 3, 4 and 5 to the filtrate
^
andj u ;i
boil
(16) Boil the lactose litmus solution (3)
ôv an hour.
15 minutes.
and (4) Filter.
(17) Mix hot lactose litmus solution u
,
* f
Add 60.0 g. of agar and heat for an
the melted nutrose agar. (5)
(18) Make faintly alkaline if

necessary hour m the autoclave
by the addition of 10.0% sodium (6) Add soda solution unti the reaction
is slightly alkaline to
litmus.
hydroxide
Boil again for an hour and filter hot
(19) Add 4.0 cc. hot 10.0% sodium (7)
in the sterilizer
hydroxide and 20.0 cc. hot sterile , , ,
1% c.p. crystal violet. (8) Distribute m , ,

100.0 cc. lots and add
1-5 g- lactose and 30.0 cc. of 1.0%
MICROORGANISMS
water solution of congo red to each
(14) Dry by inverting plates in the incu-
flask.
bator for 20 to 30 minutes.
Sterilization: Method not given. Sterilization: Not specified.
Use; Water analysis. Detection of B. coli.
Use: To isolate cholera vibrio.
Author reported that B. coli colonies were Authors
reported that cholera vibrio colonies
intensive black (bluish black). B. had
typhi, a dark colored center. Liebig's
B. paratyphi A and B, B. dysentery, meat
extract may be substituted for
Shiga Flexner Y, B. enteritidis, B. infusion.
Reference : Teague and Travis (1916 p. 602)
cholerae did not show this black or
dark
color. B. acidi lactici and B. lactis- 1939. Gassner's Nutrose Agar
aerogenes gave the same reaction
as Constituents
B. coli.
Reference: Galli-Valerio (1916 p. 136). 2. Peptone jq.O g.
3. Nutrose 10.0 g.
1938. Teague and Travis' Eosin Bismark 4- Agar 30 og'
Brown Nutrose Agar SNaCl 50g.
Preparation
Constituents:
(1) Prepare meat infusion.
1. Distilled water 2000.0 cc. (2) Dissolve 2, 3, 4 and 5 in 1.
2. Chopped beef 2.0 lbs. (3) Adjust to slightly alkaline to litmus.
3. Peptone (Witte) 20 e Sterilization: Not specified.
4- NaCl lO.oJ; Use: Cultivation of colon-typhoid group.
5. Nutrose 5 g. Reference: Gassner (1916-17 p. 315).
6. Sucrose 20.0 g.
7. Eosin 3.0% bluish solution 40.0 cc, 1940. Loeffler's Malachite Green Nutrose
8. Bismark brown 1.0% solu- Agar (Roddy)
tion 80.0 cc. Constituents:
9- Agar 15 g. 1. Distilled water 500.0 cc.
Preparation
(1) Soak beef in water in ice chest over 3- Agar 30.0 g.
night.
4. Nutrose lo.O g.
(2) Squeeze out fluid.
5. Malachite green
(3) Heat liquid in Arnold sterilizer and (2.0% soln.) 20.0 to 30.0 cc.
filterthru filter paper.
Preparation
(4) Neutralize with NaOH and heat (1) Prepare infusion broth.
again
(2) Mix equal volumes of (1) and dis-
(5) Cool and inoculate with B. coli and tilled water.
incubate for 2 or 3 days.
(6) Add 3, 4 and 9 to (5).
(4) Add 7.5 cc. of a normal HCl solution."
(7) Clear with egg. Filter.
(5) Dissolve 30.0 g. of shredded agar in
(8) Add nutrose.
(4) by boiling.
(9) Prepare a 3.0% stock solution of
(6) Neutralize by the addition of so-
bluish eosin in distilled water. dium hydrate.
Store in the dark.
(7) Add 5.0 cc. of normal NajCOs solu-
(10) Prepare a 1.0% solution of bismark
tion.
brown in water containing 10.0%
(8) Steam for 2 hours.
alcohol (not soluble to 1.0% in (9) Add 100.0 cc. of a 10.0% nutrose
pure water).
solution.
(11) To 50.0 cc. of (8) add 1.0% sucrose (10) When ready for use, liquefy sterile
(0.5 g.), 1.0 cc. of (9) and 2.0 cc
(9) and add between 2.0 and 3.0 cc.
of (10).
of a 2.0% aqueous solution of mala-
(12) Shake well.
chite green (Hochst 120) to each
(13) Pour into sterile Petri dishes. 10.0 cc. of medium.
621
(b) (1) Mix 15 parts lactose and 1 part

(9) in streaming
Congo red.
steam for 15 minutes on each of 2 suc-
(2) To 100.0 CC. of the sterile melted
cessive days.
agar,add 1.5 g. (1).
(3) Pour the red medium into sterile
Reference: Roddy (1917 p. 44).
Petri dishes.
1941. Schmitz's Nutrose Blood Clot (4) If caffeine is desired in the me-
Infusion Agar dium prepare a solution of caf-
feine in water so that 1.0 cc. of

Constituents
1000.0 cc. solution contains 0.1 g. caffeine.
1. Water
Sterilize and add 6.0 cc. of this
2. Blood clot 500.0 g.
solution to 100.0 cc. of the agar.
3. Peptone
Nutrose Reference: Schmitz (1915 pp. 307, 315).
4.
Agar (3.0%) 30.0 g. Milk Agar
5. 1942. Harvey's
Preparation
Obtain 7 or 8 liters of blood from the Constituents
(1)
1. Infusion agar.
slaughter house.
2. Milk.
(2) Pour off the serum from the clot.
Add a double amount of water to Preparation: (1) Add 3.0 cc. of sterilized
(3)
the clot.
milk to a tube of melted infusion agar (see
Boil the mixture. Stir constantly
variant (v) medium 1661 for prepara-
(4)
but take care not to break up the tion).
clot too much or filtration will be Sterilization: Not specified.
too difficult. Boil for 5 to 10 Use: General culture medium.

Variants: Harvey gave the following
minutes.
Filter thru a towel, then thru cot- variants
(5) plain
(a) Use litmus milk instead of
ton or glass wool.
milk.
(6) Add peptone nutrose, in the usual
amount not specified) (b) Mix equalparts of sterilized milk at
amount (exact
and 3.0% agar. 45C. and melted infusion agar at
When the agar has been completely 45C. (See variant (v) medium 1661
(7)
dissolved, add the serum that had for preparation).
been previously poured from the Reference: Harvey (1921-22 p. 95).
blood and that has been allowed to 1943. Lubenau's Caffeine Whey Agar
stand undisturbed.
Boil for a few minutes. Constituents
(8)
(9) Place in a flask with a patented 1.
Beef 500.0 g.
stopper and heat for one hour in 2.
3. NaCl 100 g-
streaming steam.
Peptone 60.0 g.
(10) Allow to cool slowly so that the 4.
Agar 40.0 to 60.0 g.
sediment and turbidity may settle 5.
to the bottom. Use only the top 6. Litmus whey 900.0 cc.
7. Caffeine 6%, solution
medium.
clear
Boil 500.0 g. of finely chopped lean
Use: Selective medium for typhoid bacilli. (1)
Variants: The author gave the following beef with one liter distilled water for
variants: 30 minutes.
the medium (2) Filter and make up to one liter.
(a) If caffeine is desired in
prepare a solution of caffeine in water (3) Add and 5 to (2) and dissolve by
3, 4,
so that 1.0 cc. of solution contains

boiling in a salt water bath.
0.1 g. caffeine. Sterilize. Add (4) Neutralize to litmus.

6.0 cc. of this solution to 100.0 cc. of (5) Boil and filter.
(6) Add 900.0 cc. of a sterile litmus whey,

the agar.
(preparation not given) to hot sterile Variants Schloffer reported that urine may
:
^ collected in sterile flasks and then

n\ M,v +K . 1 ^ 11 . ,
S
(8) no n
Tâ 110.0
Add
"^ '
cc. off a sterile T -^ "l"L
6.0% caf-
^'^''"^ * ^' ^ 80=C. to sterilize.
References: Schloffer (1893 p. 657), Ghon-
feme solution to (7). Mix well. Schlagenhaufer (1893 p. 619)^'
(9) Pour in plates.
Sterilization: Method of sterilization of (5) ^^'^^- dicker's Glycerol Sputum Agar
^"*giên. Constituents:
Use Detection of typhoid bacteria.
: 1. infusion agar (2.0%) 200 cc
Authors reported that typhoid colonies 2. Sputum inn n
were colorless.
3. Glycerol :.:::::;:::::::
Reference: Lubenau (1907 p. 248).
" so cc'
Preparation: "
1944. Harvey's Whey ^^^ Prepare neutral 2.0% infusion agar.

Infusion Agar
^'^
Constituents: "^ZH Tu ''T"'
^'^ "?^ ' P'^*
*"^^^^^^" ^ bronchitus
1. Infusion agar.
ll^t
^- ^'^7- Add 2.0%
^ (3) glycerol.
m
(1) MiTlOOO
Mix n
1000.0 cc. off f . milk
fresh -H
u
with
^^^ ^"^ ''^
solidify.
^^' "^^'^ *^^ly ^^d allow to
'' Sterilization: 'sterilize tuberculous or bron-

(9^ K^ fJr'oi^^^-
''^'^''^ ^^""^^ ^'" ^"^ ^''''' "'^ ^^^^
3 Fit ^ 3 suc-
cessive days in the steamer. Method of
Jzi AT i^^\i.
Make the fiu . . , ,.
(4) filtrate neutral to litmus sterilization of (1) not given
with sodium hydroxide. Use: Cultivation of tubercle bacilli.
(5 Steam 2 hours. Reference: Ficker (1900 p. 508).
(b) Histimate and adjust the reaction.
(7) Filter. 1947. Szaboky's Sputum Lung Infusion Agar
(8) Mix one part sterile with two parts(7) Constituents
sterile infusion agar (see variant (v)
1. Water .. ^
200 cc
medium 1661 for preparation).
Sterilization: Sterilize (7) at 100C.
2. Lung infusion agar.'.'
.'
'
^ '
50 cc'
3. Sputum 50 l
Use: Cultivation of gonococci. 4 NaCl 5
Reference: Harvey (1921-22 p. 95). 5^ Glucose '.[[[[]][][[[[[[][][] o.5 g.'
Glycerol
1945. Schloffer's Urine Infusion Agar ^ 12.0 g.
^ 7. Peptone 10?^
Constituents:
Preparation:
1. Infusion agar (2.0%) 200.0 cc. (1) See medium 1746 for preparation of
^2- Urine 100.0 cc. lung infusion agar.
Preparation:
(2) Boil slowly for one hour.
(1) Prepare a 2.0% infusion agar.
(3) Filter while hot
(2) Obtam urine under aseptic con- (4) Adjust to neutral or |% acid.
Sterilization Sterilize on 3 successive days
/o^ aI^^^^^' .,
(3) Mix
two parts sterile agar with one in streaming steam
:
^'^' Cultivation of tubercle bacilli.

r^>??'!'l'f'''-
(4)Distribute mto sterile
.,
plates, tubes Author reported that growth appeared in
or flasks
two days with neutral or |% acid medium.
Sterihzation: Method of sterilization of in- Reference: Szaboky (1906 p. 657)
fusion agar not given.
Use: Cultivation of diphtheria bacilli. 1^48. Costa and Boyer's Gum Infusion Agar
Author reported that in pure culture Constituents:
colonies were grayish-white to white, 1. Water
quite luxuriant and rather dry. Ghon-
2. Beef 500 g
Schlagenhaufer used a similar medium for 3. NaCl 5
e
the isolation of gonococci. 4' ..'.'.'. ...'..'.'.'. '
."
[
Peptone. '. [ ] ] [ 2o!o g.
623
30.0 g. (4) Melt sterilized agar and cool to 45C.

5. Agar
10.0 g. Remove 3-4 loops (exact amount not
6. Gum tragacanth (5)
given) of egg yolk thru the portion
Preparation
chopped of covering of the yolk that had been
(1) Macerate 500.0 g. of finely
sterilized and add to tube of (4).
beef in 1200.0 cc. water at 37 for
5 or 6 hours. (6) Mix thoroly and plate or slant.
cloth. Sterilization: Method not given.
(2) Press thru a linen
Use: Culture and diagnosis of diphtheria.
(3) Add 3 and 4 and
boil.
Also used to cultivate tubercle bacilli.
Filter on a wet filter paper.
(4)
Neutralize with soda using litmus Variants: Bezan5on prepared a similar
(5)
and then add 7.0 cc. of normal soda medium as follows:
Place an egg in boiling water for
solution to a liter of medium. (1)
tragacanth in a several minutes. This coagulates the

(6) Pulverize the gum
white.
large dry glass mortar by means of
(2) Sterilize one end of the shell and make
a pestile.
a small opening into the egg.
(7) Carefully mix (6) with (5), so as not
to obtain lumps or a gum. Mix the (3) Remove the yolk from the egg by
asparating with a sterile pipette.
bouillon thoroly after each addi-
tion of (6). (4) Add the yolk (amount not specified)
to sterile melted agar.
(8) Add the mixture to a 1.5 or 2 liter
flask and add the agar. (5) Mix thoroly.
Heat at 115 for 30 minutes to dis- (6) Slant.

(9)
References: Capaldi (1896 p. 801), Heine-
solve the agar.
mann (1905 p. 130), Tanner (1919 p. 58),
Bezan9on(1920p. 121).
(11) Distribute.
Sterilize at 115 for 300 Pea
Sterilization: 1949a. Tulloch's Ox Heart Infusion
minutes. Flour Agar
Use: Isolation of gonococcus. Cultivation
and salmonella. Constituents
of pasteurella
Water 600.0 cc.
1.
Variants
Peptone (Bacto) 10.0 g
(a) To prepare a semi-solid agar use only 2.
3.KC1 0.38g
3.0 g. agar instead of 30.0 g.
CaClj 0.44 g
(b) van Saceghem recommended the use 4.
gum traga- 5. NaHCOs 018g

of gum arable instead of
5.0
Na-iHPO (crystalline) g
canth in this medium. When gum 6. 4
Agar 25.0 g
arable is used he obtained vigorous
7.
Heart, ox 500.0 g.
growth of pasteurella and salmonella 8.
also.
9. Pea extract
Preparation
References: Costa and Boyer (1922 p. 857),
(1) Dissolve 2, 3, 4, 5 and 6 in 1 by steam-
van Saceghem (1923 p. 968).
ing (500.0 cc. of Douglas' trypsinized
1949. Capaldi's Egg Yolk Agar broth and 100.0 cc. of Cole's trypa-
mine may be used instead of 600.0 cc.
Constituents:
of Bacto peptone solution).
1. Nutrient agar.
(2) Soften 25.0 g. of fiber agar by soaking
2. Egg yolk.
in 0.25% acetic acid for 15 minutes.
Preparation
Wash the agar free from acid and
(1) Break an egg, separate the white
squeeze thru lint to get rid of as
from the yolk and place the yolk in a
sterile Petri dish.
much water as possible.
(3) Heat (1) and (2) on a water bath for
20 to 30 minutes. Then boil over a
(3) Place a glowing glass rod on a spot
free flame for 15 minutes to complete
on the covering of the egg yolk.
the solution of the agar.
(This sterilizes the covering of the
naturally sterile yolk.) (4) Adjust to pH 7.6 and keep at 56C.
(5) Add 500.0 cc. of water to 500.0 g. of (5) Evaporate to the volume by boiling.
fresh ox heart and immerse in a (6) Allow to stand two hours in the
56C. water bath shaking the con- steamer.
tainer frequently. (7) Filter the thick liquid thru a hot
(6) When the extract has reached a water funnel.
temperature of 39 to 42C. place on (8) Distribute into sterile test tubes.
a 37C. water bath and allow to ex- Sterilization: Sterilize in the steamer.
tract for 2 hours. Use: Cultivation of diphtheria, cholera
(7) Strain thru butter muslin. influenza and other pathogenic forms.
(8) Add (7) to (4) in five 100.0 cc. lots Variants: Besson prepared a similar me-
allowing 5 minutes between each dium as follows:
addition. Keep the agar at 56C. (1) Collect egg yolk under aseptic con-
(9) Heat (8) after all the infusion has ditions.
been added on a water bath for (2) Mix one volume of (1) with 3 volumes
20 minutes. of sterile water.
(10) Adjust to pH 7.6. (3) Melt tubes of nutrient agar.
(11) Cool to 56C. and add the whites of (4) Add each tube of
2.0 cc. of (2) to (3)
2 eggs. under aseptic conditions.
(12) Heat on a water bath for 20 minutes. (5) Slant.
(13) Pass thru butter muslin and filter References: Nastiukof! (1893 ^33 and 34),
thru English Chardin paper at 55C. Besson (1920 p. 55).
(14) Adjust to pH 7.6.
1951, Bezanfon and Griffon's Glycerol Egg
(15) Transfer, for storage into bottles of
Yolk Agar
which con-
200.0 cc. capacity, each of
Constituents
tains 10.0 to 15.0 cc. of pea extract.
2. Glycerol 6.0 g.
100 C. It is necessary to have the
3. Egg yolk 50.0 cc.
steamer at this temperature before plac-
Preparation
ing the medium in the sterilizer. The
medium must not be over-heated, es-
(2) Add 6 parts of glycerol to 100 parts (1)
pecially after the addition of the heart
(3) Tube.
extract, hence all glassware, butter mus-
(4) Melt sterile (3) and cool to 50C.
lin, paper, etc. are to be Steamed before
(5) To each tube add one-half the volume
use.
of sterile egg yolk under aseptic con-
Use: Culture medium for gonococci.
ditions.
Reference: Tulloch (1922 p. 348).
(6) Mix thoroly and slant to cool.
1950. Nastukoff's Egg Yolk Nutrient Agar Sterilization: Method of sterilization of
(Recktsamer) (3)not given.

Constituents:
References: Bezangon and Griffon (1903
p. 603), Besson (1920 p. 55).
2. Egg yolk 100.0 g.
3. NaOH(10%) 0.5 cc. 1952. Pergola's Tellurite Egg Yolk Agar
4. Nutrient agar Constituents
Preparation 1. Nutrient agar (1.7%) 100.0 cc.
(1) Separate the yolk of egg from the 2. Potassium tellurite (1.0%
white by Bunge's method (allow the soln.) 2.0 cc.
whites to roll from a blotting paper 3. Egg yolk 1
containing the egg). Preparation Details of method of prepara-

:
(2) Add 0.5 cc. of a 10% NaOH solution tion not given in the abstract.
and 100.0 cc. of egg yolk to 1000.0 cc. Sterilization: Method not given.
distilled water. Use: Diagnosis of diphtheria.
(3) Prepare nutrient agar. Reference: Pergola (1918 p. 101) taken
(4) Mix equal parts of (2) and (3). from (1919 p. 57).
1953. Lipschiitz's Egg Albumin Agar 3. Egg albumin (10.0% solution) 400.0 cc,
Same as medium 4. Egg yolk (10.0% solution). . . 100.0 cc.

953 but the bouillon is
Preparation
solidified by the addition of 1.0% agar.
(1) Mi.x 5 parts bouillon with 4 parts of a
1954. Krumwiede, Pratt and Grund's Egg 10.0% solution of egg white in sterile
Albumin Agar water and one part of a 10.0% egg
Constituents: yolk emulsion in sterile water. If the
1. Water 150.0 cc. eggs are not obtained under aseptic
2. Egg albumin 150.0 cc. conditions, the solutions may be
3. NaoCOs 3.6 g. sterilized by heating at 55 C.
4. Nutrient agar (3.0%) 700.0 cc. Add 4.0 cc. of (1) to the surface of
(2)
Preparation solidified agar and allow to stand for
(1) Mi.x equal parts of 1 and 2 and add 3. 24 hours.
(May be filtered thru thin layer of Sterilization: Not specified.
cotton to remove any thick parts of Use: General culture medium.
egg.) Reference: Besson (1920 p. 55).
(2) Steam (1) in Arnold sterilizer for
20 minutes. 1957. Scales' Whole Egg Agar
(3) Prepare 3.0% nutrient agar neutral Constituents
to litmus. 1. Nutrient agar (1.5%) 70.0 cc.
(4) IVIix (2) and (3) while (3) is boiling hot. 2. Whole egg 20.0 cc.
(5) Pour medium thick plates, allow them Preparation:
to stand open for 20 to 30 minutes. (1) Strain fresh eggs thru four thick-
Sterilization: Method not given. nesses of cheese cloth.
Use: Isolation of cholera vibrio. Authors (2) Prepare 1.5% nutrient agar.
reported that by transmitted light (3) Thoroly mix 20.0 cc. of (1) with
cholera colonies appeared deep in the 70.0 cc. of melted (2).
agar with hazy appearance. Medium in- Sterilization: Sterilize at 12 pounds for
ferior to a whole egg medium. 15 minutes.
Variants: Sacquepee (Bezangon) gave the Use: To study variation in morphology of
following method of preparation: B. coll.
(1) Add
200.0 cc. of sterile water to the Variants: The author added 1.0% glucose.
whites of two eggs. Reference: Scales (1921 p. 595).
(2) Heat to 50C.
(3) Make alkaline by adding 0.5 cc. of 1958. Stitt's Glycerol Egg Agar
10.0% soda solution for each 100.0 cc. Constituents:
of egg solution. 1. Glycerol agar.
(4) Sterilize in the autoclave. 2. Egg.
(5) Prepare 3.0% agar neutral to litmus. Preparation
(6) Add 9.0 cc. of 10.0% soda solution to (1) Take the yolk and white of one egg
a liter of (5). and mix thoroly with an equal amount
(7) Add 200.0
cc. of (4) to 1000.0 cc. of (6). of glycerol agar, at a temperature of
(8) Sterilize in the autoclave at 115C. 45 to 55 C.
References: Krumwiede, Pratt and Grund (2) Tube.
(1912 p. 137), Bezan?on (1920 p. 121). (3) Inspissate in a rice cooker as for
1955. Oberstadt's Egg Albumin Agar serum tubes.
(4) Since egg medium has a tendency to
Same as medium 952 but the bouillon is
be dry it is well to add 1.0 cc. glycerin
solidified by the addition of agar.
bouillon to each tube before steri-
1956. Besredka and Jupille's Egg Agar lization.
(Besson) Sterilization: Sterilize in the same manner
Constituents as for blood serum.
1. Nutrient agar Use: Cultivation of tubercle bacilli.
2. Bouillon 500.0 cc. Reference: Stitt (1923 p. 38).
1959. Robertson's Alkaline Egg Agar (Park, Preparation:

Williams and Krumwiede) (1) Remove the brain under aseptic con-
Constituents: ditions from a dead new-born human
1. Water 500.0 cc. foetus, and place in a sterile covered
jar.
2. Egg
3. NaOH(normal solution) .... 6.0 cc. (2) Add sterile ascitic fluid to (1) and
4. Nutrient agar
shake the mixture for an hour in a
Preparation shaking machine.
Place in the ice box and allow to stand
(1) Mix the yolk of one egg, the whites
(3)
of two eggs, 500.0 cc. of water and for several hours.
6.0 cc. of N/1 NaOH. (4) Mix the clear opalescent fluid with
Mix one part (1) with five parts agar. an equal amount of 3.0% agar.
(2)
Use: Cultivation of cholera organisms. Use: Cultivation of gonococci. The use

Reference Park, Williams and Krimiwiede
:
of glucose agar increases the vigor of
(1924 p. 126). growth.
Reference: Pettersson (1920 p. 1385).
1960. Cantani jun's Sperm Agar
Constituents 1962. Smith and Taylor's Fetus Agar

1. Nutrient agar. Constituents
2. Sperm. 1. Nutrient agar.
Preparation 2. Fetus tissue.
(1) Remove the testicles from a bull Preparation
under aseptic conditions. (1) Prepare nutrient agar.
(2) Extract the sperm by pressing the (2) Slant. The tube should contain a
Funiculus sperm aliens between two small quantity of water of condensa-
fingers and remove the sperm, thus tion or add a little bouillon.
pressed out with a sterile platinum (3) Add to each slant \ g. of tissue or an
loop. equivalent amount of stomach or
(3) Streak the sperm on sterile agar other fluid or intestinal contents of
slants. the fetus.
(4) Incubate to prove sterility. (4) Seal the tubes with sealing wax.
Use: Cultivation of influenza bacilli, tuber- Use: Isolation of Vibrio felus.
cle bacilli and others. Reference: Smith and Taylor (1919 p. 301).
Variants: A similar medium was prepared 1963. Noguchi's Ascitic Fluid Tissue Agar
as follows:
Constituents
(2) Free testicular material of its capsule.
2. Ascitic fluid 100.0 cc.
(3) Wash the outer surface with alcohol
3. Tissue (rabbit testicle or
and ether.
kidney)
(4) When dry cut cross sections with a
Preparation
sterile knife.
(1) Prepare 2.0% nutrient agar.
(5) Remove some of the liquid from the
(2) Adjust (1) to slightly alkaline.
cross sections by means of a strong
(3) Place small pieces of fresh tissue
platinum loop.
(preferably rabbit testicle or kidney,
(6) Streak the liquid on sterile solidified
but human placenta, sheep testicle or
agar slants.
other sterile organs will suffice) into
(7) Incubate 10 hours to prove sterility.
a tube measuring 2 by 20 cm.
References: Cantani jun (1897 p. 601).
(4) Mix 2 parts melted (2) with one part
1961. Pettersson's Brain Ascitic Fluid Agar ascitic (or hydrocele) fluid and add
Constituents 15.0 cc. to each tube of (3).
1. Nutrient agar. (5) After solidification add a layer of
2. Ascitic fluid. sterile paraffin to prevent evap-
3. Brain. oration.
(6) Incubate to test sterility. morphic streptococci (causing polio-

Sterilization: Not specified. myelitis) on a medium prepared as
Use: Isolation of Treponema pallidum follows
Inoculate by forcing small bits of pal- (1) Prepare 1.5% nutrient agar.
lidum-containing tissue far into the butt (2) Mix two parts (1) with one part
of the tube. Other investigators culti- ascitic fluid.
vated a variety of organisms on similar (3) Method of sterilization not given.
media. (4) Distribute in test tubes of 0.8 cc.
Variants in diameter to a depth of 13 centi-
(a) Zinsser, Hopkins and Gilbert pre- meters.
pared a similar medium as follows (5) Add to each tube a piece of fresh
for the cultivation of large quantities sterile rabbit kidney.
of Treponema pallidum. (6) Add a layer of sterile mineral oil.
(1) Prepare serum agar. (d) Loewe and Strauss cultivated organ-
(2) Melt (1) and pour to the height of isms causing epidemic encephalitis
about one inch into the bottom of on a medium prepared as follows:
a 200.0 cc. flask. (1) Mix one part sterile 2.0% nutrient
(3) Drop in sterile bits of tissue. agar with four or five parts ascitic
(4) Inoculate. fluid. Ascitic fluid should be ster-
(5) When the agar has solidified, cover ile, contain no bile or fibrin, and
with a mixture of either salt solu- have a high specific gravity.

tion and heated ascitic fluid, or (2) Place kidney into tube and cover
slightly acid broth and ascitic fluid. with (1).
Fill to the neck of the flask. (3) Inoculate.
(6) Add a few bits of sterile tissue. (4) Pour on or cover(2) with auto-
(7) Cover with liquid paraffin oil. claved petrolatum and incubate.
Authors reported that large quanti- (e) Stitt cultivated treponemata on a
ties of Treponema pallidum may be medium prepared as follows
found within periods of from two (1) Prepare a 2.0% nutrient agar.
to four weeks. It is not necessary (2) Adjust (1) so that it is slightly
to add the agar. The tissues may alkaline.
be heated in the autoclave. (3) Mix two parts (2) with one part
(b) Fle.xner, Noguchi and Amoss culti- ascitic or hydrocele fluid.
vated organisms from poliomyelitic (4) Fill 2 by 20 cm. test tubes by the
tissue on a medium prepared as addition of 15.0 cc. of (3).
follows: (5) Place a fragment of fresh sterile
(1) Prepare 1.0% nutrient agar and rabbit kidney or testicle in each
bouillon. tube. The tissue should be in the
(2) Introduce a fragment of kidney into bottom of the tube.
an Erlenmeyer or Florence flask of (6) After solidification add sterile para-
100.0 cc. capacity. The inoculum ffin oilso that it covers the solid
is placed on the kidney. medium to a depth of 3 cm.
(3) Mix equal volumes agar and
of (7) Inoculate in the bottom of the tube
ascitic fluid. (.\gar to be melted by means of a capillary pipette.
and cooled at 40C.) References: Noguchi (1912 p. 91), Flexner,
(4) Pour 15.0 cc. of (3) over the kidney Noguchi and Amoss (1915 p. 92), Zinsser,
(giving a solid layer 1 cm. deep). Hopkins and Gilbert (1915 p. 215), Rose-
(5) When solidified add 50.0 cc. of an now and Towne (1917 p. 177), Loewe and
equal mixture of sterile ascitic fluid Strauss (1920 p. 253), Stitt (1923 p. 53).
and bouillon.
Cover with a layer of sterile para-
1964. Gozony's Kidney Agar
(6)
ffin to yield a layer about 1 cm. Constituents:
deep. 1. Water 500.0 cc.
(7) Incubate at 37C, 2. Bouillon 500.0 cc.
(c) Rosenow and Towne cultivated pole- 3. Peptone 20.0 g.
4. Agar 5.0 g. Preparation :
5. Kidney (dog) (1) Dissolve 100.0 g. of gelatin in a liter
6. NaoCOsdOô) 5.0 cc. of ordinary bouillon.

Preparation : (2) Make alkaline.
(1) Prepare bouillon. (3) Add 0.5% agar and dissolve by
(2) Mix equal parts bouillon and water. boiling.
(3) Dissolve 2.0% peptone and 0.5% (4) Cool to 55C. and add the white of
agar in (2). an egg. Mix well.
(4) Add 5.0 cc. of a 10.0%, NaoCOs solu- (5) Boil 20 minutes at 115C.
tion to give a slightly alkaline (6) Distribute as desired.
reaction. Sterilization: Sterilize for 20 minutes at
(5) Distribute in tubes. 113 to 114C.
(6) Cool sterile (5) to 45C. and to each Use: General culture medium.
tube add a small piece of sterile dog Variants: Besson (Tanner) prepared the
kidney under aseptic conditions. medium as indicated:
Sterilization: Sterilize (5) in the autoclave. (1) Dissolve 80.0 g. of gelatin in bouillon.
Use: Cultivation of Author re-

flagellates. (2) Neutralize.
ported that the flagellates grown in this (3) Dissolve 5.0 g. of agar in (2). 50.0 g.
medium were larger than those grown in of gelatin and 8.0 g. agar may be used
blood culture. instead of 80.0 g. of gelatin and 5.0 g.
Reference: G6zony (1920 p. 566). of agar.
Agar (4)Sterilize. Do not heat over 115C.

1965. Duval's Trypsinized Tissue
References: Thoinot and Masselin (1902
Constituents :
p. 36), Tanner (1919 p. 56).
1. Nutrient agar.
2. Tissue. 1967. Fremlin's Phosphate Gelatin Agar

3. Trypsin (1.0% solution).
Constituents:
Preparation :
1. Water 1000.0 cc.
(1) Pour nutrient agar in sterile Petri
2. Agar 15.0 g.
dishes and allow to solidify.
(2) Cut an excised leprous nodule into
4. Bouillon gelatin 50.0 g.
thin slices, two to four millimeters in
Preparation :
breadth and 0.5 to 1.0 mm. in thick-

(1) Exact composition or method of
ness and distribute over the surface
preparation of bouillon gelatin not
of the plate.
given.
(3) Bathe the medium with a 1.0% tryp- and 4
(2) Dissolve 2, 3 in 1.
sin solution, taking care not to sub-
merge the pieces of leprous tissue.
Use: To study nitrification by nitroso bac-
Add sufficient fluid to moisten thoroly
teria. Author reported that sub-cultures
the surface of the medium.
from this medium to other plates did not
(4) Incubate in a moist chamber at 37C.
show nitrification, proving that this is not
for a week to 10 days. Remove the
a suitable medium for continued activity.
plates from time to time and add more
trypsin as evaporation necessitates.
Sterilization: Not specified. 1968. Vierling's Fat Agar
Use: Cultivation of B. leprae. The author
Constituents :
reported that the colonies were grayish 100.0 cc.

1. Nutrient agar
white, but after several days they as- 10.0 g.
2. Lanolin
sumed a distinct orange yellow tint.
Preparation :
Reference: Duval (1911 p. 369).

1966. Thoinot and Masselin's Gelatin Agar (2) Prepare an emulsion of 1.0 g. lanolin
Constituents :
in 10.0 cc. nutrient agar.
1. Bouillon 1000.0 cc. Sterilization: Not specified.
2. Gelatin 100.0 g. Use: To study lipase production by Myco-

3. Agar (0.5%) 5.0 g. bacteria. Author reported that good
growth occurred. Crystals of salts of (2) Mascerate equal weights of fresh

fatty acids could not be demonstrated. lean beef heart.
Variants: The author gave the following (3) Take up each portion of (2) with
variants 9 volumes of saline solution.
(a) Used beef fat instead of lanolin.
(4) Treat each of these suspensions in
(b) Vierling reported that on the follow- one of the following ways:
ing medium the fat was saponified (a) Keep in the ice box over night
causing the agar to become turbid and steam in an Arnold sterilizer
and the crystalline salts of fatty acids for one hour.
to form. The layer of fat was not
(b) Keep in the ice box over night and
destroyed in any place.
filter thru a Berkefeld candle.
(1) Pour a thin layer of beef fat into
(c) Keep at 55C. over night and heat
Petri dishes.
in an Arnold sterilizer for one
(2) When the fat has solidified pour a
hour.
layer of glucose agar that has been
(d) Keep over night and
at 55C.
cooled to 45C. over the layer
filter thru a Berkefeld candle.
of fat.
(e) Extract by boiling one hour and
Reference: Vierling (1920 p. 205).
then sterilize in an Arnold steri-
1969. Abe's Meat Water Infusion Agar lizer for one hour.
Constituents: (f) Extract by boiling for one hour.
1. Water 1000.0 cc. (5) Take 1.0 cc. of one of the extracts
2. Nutrient agar (2.0%) obtained and add to 5.0 cc. of sterile
3. Beef 500.0 g. nutrient agar.
Preparation Kligler reported that heat destroyed
(1) Chop 500.0 g. of fat free beef in a the growth stimulating material in
meat chopping machine. the extract. Best growth in (4) (b),
(2) Mix (1) and 1000.0 cc. of water, and no stimulated effects in (4) (e)
allow to stand for 18 to 24 hours in and (f).
an ice box. (b) Kligler also used the following media

(3) Filter the liquid thru filter paper to study the effects of growth acces-
and then thru a Chamberland filter. sory substances on pathogenic bac-
(4) Distribute this sterile reddish liquid teria:
into sterile test tubes. (1) Prepare nutrient agar.
(5) Melt tubes of 2.0% sterile nutrient (2) Obtain beef heart, rabbit tissues,
agar. rabbit organs, cat organs, cat tis-
(6) Heat (4) to 45-50C. sues or mucosa of various organs
(7) To each 5.0 cc. of (5) add 2.0 cc. of (6). as free from blood as possible under
(S) Mix thoroly. aseptic conditions.
(9) Pour into sterile Petri dishes. (3) Wash (2) with saline solution to
Sterilization: Method of sterilization of remove all visible traces of blood.
nutrient agar not given. The infusion is (4) Weigh and chop into small bits and
sterilized by filtration, see step (3) above. suspend in 9 times the weight of
Use: Cultivation Author re-
of gonococci. saline solution.
ported that gonococci colonies appeared (5) Shake thoroly and place in the ice
after 18 hours at 37C. as light gray small box over night.
typical colonies. Kligler used a similar (6) Centrifuge and filter thru a Berke-
medium to study effect of extracts on feld candle.
growth of pathogenic organisms. (7) Test sterility before using.
Variants (8) Tube (1) in 5.0 cc. lots.
(a) Kligler prepared media as given (9) Add amounts of (7) to each tube of
below to study effect of extracts on varying from 0.01 cc. to 1.0 cc.
(8)
growth of pathogenic organisms: References: Abe (1907 p. 707), Kligler
(1) Prepare nutrient agar. (1919 pp. 32, 43).
1970. Esch's Hydrolyzed Meat Agar (2) To 150.0 cc. of defibrinated beef blood
(Kohlisch and Otto) add an equal part of N/1 KOH.
(3) To 7 parts melted (1) add 3 parts
Constituents (2), mix well and pour into plates.
1. Nutrient agar (4) Dry the plates for several days at
2. Beef 500.0 g.
37C. or for 4 minutes at 60C.
3. NaOH (normal) 250.0 cc.
Sterilization: Sterilization of agar not
Preparation specified. The laked blood alkali mixture
(1) Dissolve 500.0 g. of beef in 250.0 cc. maj^ be sterilized in the autoclave.
of N/1 NaOH solution in an aluminum Use: Enrichment medium for cholera
kettle by heating. Stir constantly. Author reported that cholera
vibrio.
Solution is complete after from about vibriogrew very luxuriantly on this
10 to 15 minutes. medium. B. coli was inhibited.
(2) When cool filter thru a towel. Variants
(3) Mix 3 parts sterile (2) with 7 parts (a) Sineff and Drosdowitsch gave the
neutral nutrient agar. following method of preparation:
(1) Collect beef blood in a sterile
(5) at 60 for 30 minutes.
Dry enamel container and defibrinate it
(6) Allow the plates to stand
open at with a sterile egg beater.
room temperature for 24 hours. (2) Strain thru a fine tin sieve.
Sterilization: Sterilize in streaming steam Mix equal parts of the filtrate from
(3)
for one hour. Method of sterilization of (2) and a normal solution (5.61%)
agar not given. of KOH.
Use: Elective medium for cholera vibrio. (4) Sterilize for one-half hour.
Variants (5) Mix 7 parts sterile agar and 3
(a) The author used pike instead of beef. parts (4) (sterile alkaline defibri-
(b) Stitt prepared the medium as follows: nated blood).
(1) Treat 500.0 g. chopped beef with (6) Pour into sterile Petri dishes.
250.0 cc. of normal NaOH until dis-
(7) Dry at 37C. for 24 hours.
integrated. (b) Hofer and Hovorka prepared a sim-
(2) Filter thru cloth. ilar medium as follows:
(3) Sterilize (method not given). (1) To 3.0% melted nutrient neutral
(4) Add about one part of (3) to about agar add 4.0 cc. of defibrinated
2.5 or 2 parts of nutrient agar. beef blood, and 16.0 cc. of N/1
(5) Dry the plates. KOH.
References: Kohlisch and Otto (1915 p.
(2) Boil.
435), Stitt (1923 p. 50). (3) Distribute into 10.0 cc. lots.
(4) Prepare a 0.1% solution of crystal
1971. Wellman's Placenta Infusion Agar
violet in distilled water.
Add Wellman's Placenta Infusion (see
(5) To each 10.0 cc. of (3) add 0.5 cc.
medium 1377) to 2.0% nutrient agar. of (4).
(6) Pour into Petri dishes.
1972. Orcutt and Howe's Fat Blood Agar
(7) Place the plates in the incubator
Same as medium 960 but solidified by the for 24 hours with covers partly re-
addition of agar. moved and then at room tempera-
tures for 12 hours with covers on
1973. Dieudonne's Alkaline Blood Agar
before use.
Constituents (c) Lentz prepared the medium as
1. Nutrient agar 700.0 cc. follows:
2. Blood, defibrinated beef 150.0 cc. (1) Mix fresh defibrinated beef blood
3. KOH N/1 150.0 cc. with equal amount of N/1 alkali.

Preparation (2) Heat 30 minutes.
(1) Prepare nutrient agar and neutralize (3) Dry in Faust-Heim drying appa-
to litmus. ratus.
(4) Grind in mortar. Use: Enrichment medium for cholera

(5) Preserve in glass stoppered bottle. vibrio.
(6) For use dissolve 3.0 g. powder in Variants :
30.0 cc. distilled water. Add to (a) Klimmer prepared the medium as
70.0 cc. neutral agar. follows:
(d) Roddy gave the following method of (1) Mix 150.0 cc. of fresh defibrinated
preparation: beef blood, with 150.0 cc. of a 12.0%
(1) Obtain ox blood from the slaughter soda solution.
house. (2) Shake well.
(2) Collect the blood in small sterile (3) Allow to stand for 1 to 6 days.
bottles containing glass beads. (4) Steam for 60 to 90 minutes.
(3) Shake until the blood is defi- (5) Mix 30 parts (4) with 70 parts
brinated. sterile neutral agar.
(4) Mi.x equal volumes of (3) and (b) Park, Williams and Krumwiede gave
normal sodium hydrate solution. the following method of preparation
(5) Sterilize (3) in a steam sterilizer (1) Mix equal parts defibrinated beef
for 15 minutes on each of 2 succes- blood and 12.0% Crystalline soda
sive days. solution.
(6) Add 1 part of (5) to 7 parts sterile (2) Steam in the Arnold for 30 minutes.
nutrient agar. (3) Prepare 3.0% agar neutral to litmus
(7) Tube. (about pH 6.6).
(8) Slant. (4) Mix 3 parts (2) with 7 parts (3).
(9) Do not have the cotton plugs Sterilization not specified.
tighter than necessary. (5) Pour into Petri dishes (15.0 cc. in
(e) Stitt used normal NaOH instead a 10 cm. dish).
of KOH. (6) Allow to harden, uncovered but
References: Dieudonne (1909 p. 108), Sineff protected by paper.
and Drosdowitsch (1909 p. 429), Hofer (7) Plates can be used after drying
and Hovorka (1913 p. Ill), Lentz (1915 30 minutes.
p. 425), Roddy (1917 p. 45), Ball (1919 References: Pilon (1911 p. 331), Klimmer
p. 83), Klimmer (1923 p. 218), Stitt (1923 (1923 p. 218), Park, Williams and Krum-
p. 50), Park, Williams and Krumwiede wiede (1924 p. 130).
(1924 p. 130).
1975. Fildes' Pepsinized Blood Agar
1974. Pilon's Alkaline Blood Agar Same as 961 but using nutrient agar in-
stead of bouillon.
Constituents:
1. Nutrient agar (4.0%) 70.0 cc. 1976. Carpano's Hemolysed Blood Agar
2. Blood, defibrinated 15.0 cc. Constituents
3. NaaCOs (crystalline 12.0% solution) 1. Nutrient agar (2.5%) 1000.0 cc.
Preparation 2. Hemolysed defibrinated blood
(1) Mix defibrinated blood and a 12.0% Preparation
crystalline Na2C03 solution in equal (1) Prepare 2.5% nutrient agar (peptone
parts. broth with 2.5% agar).
(2) Prepare 4.0% nutrient agar. (2) Neutralize to phenolphthalein.
(3) Neutralize (2). (3) Adjust the reaction by the addition
(4) Add 3 parts non-sterilized to (1), of 4.0% normal HCl.
7 parts melted (3)and mix thoroly. (4) Add sterile, defibrinated blood natu-
(5) Pour into Petri dishes and allow the rally hemolysed (details of prepara-
plates to solidify. tion not given in the abstract) to the
Sterilization: If the plates are to be used solidified agar.
at once sterilization is not necessary due Sterilization: Not given.
to high alkali content. If the plates are Use: Cultivation of gonococci.
to be kept several days, draw blood under Reference: Carpano (1919 p. 599) from
aseptic conditions and use sterilized agar. (1920 p. 176).
1977. Mandelbaum's Lactose Blood Agar (3) To 95.0 cc. of (2), add 5.0 cc. of a2.2%
solution of neutral sodium oleate.
Constituents Add defibrinated or citrated blood to
(4)
1. Nutrient agar. while it is still hot (temperature
(3)
2. Lactose. amount of blood not
or specified).
3. Rosolic acid. Sterilization: Not specified.
4. Blood, defibrinated human. Use: Cultivation of Pfeiffer's bacilli.
Preparation Reference: Stitt (1923 p. 43).
(1) To a tube containing 8-10.0 cc. nu-
trient agar is added 0.3 g. lactose. 1980. Esch's Ascitic Fluid Blood Agar
(2) Heat to 100 C. Constituents
(3) Cool to 50C. 1. Nutrient agar 60.0 cc.
(4) Add 0.3 cc. 1.0% alcoholic rosolic 2. Blood, defibrinated sheep 20.0 cc.
acid solution. 3. Ascitic fluid 10.0 cc.
human
(5) Add 1.0 cc. defibrinated sterile 4. Maltose 1.0 g.
blood (Probably animal blood may 5. Bouillon 3.0 cc.
be used). Preparation
(6) Mix and pour into Petri dishes. (1) Prepare nutrient agar containing
(7) Stand 24 hours, or dry | hour at 37C. 1.0% Witte's peptone.
Use: Isolation of colon typhoid group. (3) Mix 60.0 cc. of (1) that has been
Reference: Mandelbaum (1912 p. 306). cooled to 50C. with 20.0 cc. sterile
defibrinated sheep blood, 10.0 cc.
1978. Thompson's Glucose Plasma Agar
ascitic fluid and 1.0 g. of maltose dis-
Constituents solved in 3.0 cc. of bouillon.
1. Nutrient agar (2.5%) 1000.0 cc. (4) Pour into sterile Petri dishes.
2. NaCl 9.0 g. Sterilization: Not specified.
3. CaCh 0.25 g. Use: Cultivation and isolation of meningo-
4. KCl 0.42 g. cocci. Author reported that typical
5. Glucose (2.5%) 25.0 g. colonies developed after 8 hours.
6. Plasma (human) References: Esch (1909 p. 153), Klimmer
Preparation (1923 p. 225).
(1) Prepare nutrient agar (2.5%) in the
1981. Listen's Trypsinized Casein Blood
ordinary way using 1.0% Witte pep-
Agar
tone (Omit the NaCl).
(2) Adjust to +6 acid. Constituents
(3) Add 2, 3, 4 and 5 to (1). 1. Distilled water 100.0 cc.
(4) Tube in 4.0 cc lots. 2. Blood 20.0 cc.
(5) Melt sterile (4) in boiling water and 3. Trypsinized casein 100.0 cc.
cool to 50C. Then add 1.0 cc. of 4. Agar (3.0%) 400.0 cc.
human plasma to each tube. Mix and Preparation
slant. (1) A 200.0 cc. capacity flask, containing
Sterilization: Method of sterilization of (4) a few glass beads and 100.0 cc. dis-
not given. tilled water, is sterilized and kept
Use: Cultivation of gonococci. ready.
Reference: Thompson (1917 p. 869). (2) 20.0 cc. of human (or rabbit) blood,
removed with due precautions to
1979. Avery's Oleate Blood Agar (Stitt)
avoid contamination, are added to
Constituents this sterile distilled water. The

1. Nutrient agar 95.0 cc. flask is then at once vigorously
2. Sodium oleate (2.0% soln.). . . 5.0 cc. shaken to prevent the formation of
3. Blood, defibrinated or citrated large fibrin masses and the blood
Preparation soon lakes.
1 Prepare nutrient agar. (3) 5.0 cc. of an alcoholic pancreatic ex-
(2) Adjust (1) to pH = 7.2 to 7.5. tract are then added to the flask.
this extract being prepared locally the tube is sloped, and the agar
from goat's pancreas, according to allowed to cool.
the method recommended by Cole (12) Agar slopes thus prepared are trans-
and Onslow (Lancet (1916, see parent and have a slightly more
medium 1130.) Pig's pancreas was golden color than ordinary agar.
not used for various reasons. Sterilization: Final sterilization not spe-
(4) Next 5.0 cc. of the enterokinase solu- cified.
tion are added with a view to hasten Use: Cultivation of B. m/uensae (Pfeiffer).
the trypsinization process. This The author reported that agar slopes con-
enterokinase solution is a very dilute taining the digested blood gave almost
watery extract of the duodenal as good results as those containing in
mucous membrane of goats and sheep addition casein digest (stock broth of
which has been macerated in chloro- Cole Onslow). Many involution
and
form water. forms, however, were developed on this
(5) Lastly, 1.5 cc. of pure chloroform simpler medium, but the addition of
are added to the flask as a pre- sodium phosphate eliminated these. A
servative and it is plugged with an culture of B. influenzae inoculated on
India rubber cork to prevent the this medium showed a perceptible growth
evaporation of the chloroform. after six hours incubation at 37C., and
(6) The flask is then placed in the incu- after 24 hours incubation a luxuriant
bator at 37C., after having shaken growth was obtained. This growth had a
it well in order to mix the contents characteristic translucency. Individual
thoroly. isolated colonies on this medium meas-
(7) On the second day the flask will be ured from 1.0 mm. to 2.0 mm. in 24 hours.
found to contain a well-settled sedi- Reference: Liston (1918-19 p. 419).
ment with a clear supernatant red-
1982. Bernstein's Basal Blood Agar
dish fluid. The flask is shaken again
to resuspend the sediment and put Constituents
back into the incubator. The shak- 1. Nutrient agar 150.0 cc.
ingis repeated on the third day, but 2. Blood, beef 10.0 cc.
after that the flask is allowed to Preparation
stand undisturbed in the incubator (1) Prepare nutrient agar with 1.0% of
until the eighth day. one of the added nutrients.
(8) On the eighth day the flask is care- (2) Draw 400.0 cc. of beef blood directly
fully removed so as not to disturb into sterile Erlenmeyer flasks of
the sediment and the clear super- 500.0 cc. capacity containing 35.0 cc.
natant fluid is removed with aseptic of a 1.0% solution of ammonium
precautions. In case the sediment oxalate in distilled water.
has not settled firmly at the bottom, (3) Shake for one or two minutes.
as much of the clear fluid should be (4) Add volume formalin and
0.5 cc. of 40
removed as possible and the re- allow the flask to stand undisturbed
mainder filtered thru sterile filter for an hour.
paper, making arrangements to (5) Distribute the blood into sterile
maintain sterility. Erlenmeyer flasks and dilute with
(9) Broth prepared from casein after twice its volume of sterile (0.9%)
the method recommended by Cole saline solution.
and Onslow (see medium 1130) is (6) Allow the blood to stand for 24 hours
kept ready on hand. to 48 hours at room temperatures
(10) Tubes, each containing about 4.0 cc. before use.
of 3.0% agar, are kept ready steri- (7) Seal the flasks and keep on ice until
lized. Theagar is melted and cooled ready for use.
to a temperature of about 45C. (8) To 15.0 cc. of sterile melted (1), add
(11) One cc. of the stock broth and 1.0 cc. 1.0 cc. of (6).
of the blood fluid are well mixed; (9) Pour into sterile plates.
Sterilization: Method not given. cautions to a ground glass stoppered

Use: To study fermentation and hemolysis. bottle and add 30.0 cc. of methylated
Author reported that typhoid colonies ether.
did not produce hemolysis on lactose (6) Stopper firmly and shake well.
plates. Colon colonies did produce (7) Incubate over night at 37C.
hemolysis on lactose plates. On raffi- (8) When required for use, 3.0 cc. of (7)
nose, typhoid colonies showed umbili- are pipetted to 100.0 cc. of melted
cated colonies with lines radiating from and cooled to 50C. nutrient agar.
the center. Colon colonies did not show (9) Keep the blood agar at 50C. for one
these lines. On maltose typhi were hour before pouring into plates, if to
deeply pigmented, almost black; colon be used at once. Or pour into plates
colonies were white. On dextrin ty- and incubate at 37C. over night.
phoid colonies caused precipitation of the This drives off the ether, and if the
medium and were black. Colon colonies plates are incubated over night, tests
caused hemolysis and were white. sterility.
Variants: The author added 1.0% of any Sterilization: Not specified.
desired carbohydrate to nutrient agar. Use: Isolation and preservation of menin-

Reference: Bernstein (1909 p. 2). gococci. Also used for the cultivation of
B. influenzae and anaerobes.
1983. Wordley's Oxalated Blood Agar Wilson and Darling (1918
Reference:
Constituents : p. 105).

1985. Wilson and Darling's Lactose Blood
2. Blood (human oxalated) 10.0 cc.
Agar
Preparation: (1) Prepare nutrient agar.
(2) Melt agar and pour into
sterile Petri Constituents :
dishes in 15.0 cc. lots. 1. Distilled water 10.0 cc.
(3) Add 1.0 cc. of human oxalated blood 2. Nutrient agar

to each Petri dish. 3. Blood, sheep 1000.0 cc.
Sterilization: Not specified. 4. Sodium citrate 10.0 g.
Use: To study hemolysis by streptococci. 5. Lactose 0.5 to 1.0%

Reference: Wordley's (1921 p. 66). 6. Crystal violet (0.1%,
soln.)
1984. Wilson and Darllng^'s Laked Blood 7. Litmus solution (Kubel
Agar
and Tiemann)
Constituents : Preparation:
1. Water 10.0 cc. (1) Dissolve 10.0 g. of
sodium citrate in
2. Nutrient agar 10.0 cc. of water. Place this in a
Blood, sheep 1000.0 cc. bottle with volume graduation
3.
4. Sodium citrate 10.0 g. marks.

Preparation : (2) Take (1) to the slaughter house and
sodium citrate in when a sheep is slaughtered, after
10.0 g. of water. Place this in a the first gush of blood, collect the
bottle with volume graduation marks. blood in the bottle until a liter of
Take (1) to the slaughter house and fluid has been obtained.
(2)
.when a sheep is slaughtered, after (3) Shake the bottle vigorously during
the first gush of blood, collect the the process.
blood in the bottle until a liter of (4) Add 1.25 cc. of formalin to the liter
fluid has been obtained. of citrated blood and mix thoroly.

Shake the bottle vigorously during (5) Transfer the blood with aseptic pre-
(3)
the process. cautions to a ground glass stoppered
1.25 cc. of formalin to the liter bottle and add 30.0 cc. of methylated
(4) Add
of citrated blood and mix thoroly. ether.
(5) Transfer the blood with aseptic pre- (6) Stopper firmly and shake well.
(7) Incubate over night at 37C. Sterilization: Method of sterilization of

(8) To 100.0 cc. of sterile melted and agar not given.
cooled to 50C. agar containing 0.5 Use: Cultivation of pneumococci, menin-
to 1.0% lactose, add 3.0 cc. of (7), gococci and gonococci.
1.0 cc. of a 0.1% crystal violet solu- Variants
tionand 12.0 cc. of Kubel and Tie- (a) Stitt prepared an opaque medium
mann's litmus solution. as follows:
(9) Pour into plates. (1) Prepare 2.0 to 3.0% nutrient agar.
(10) Incubate over night at 37 to drive (2) The reaction of (1) should be -0.3
off the ether. to phenolphthalein.
Sterilization: Method of sterilization not (3) To 100.0 cc. of (2) add 20.0 cc. of a
given. mixture of equal parts blood and
Use: Cultivation of colon-typhoid group. sodium citrate solution (1.0% in
Reference: Wilson and Darling (1918 normal saline).
p. 105). (4) Pour into 10 plates.
(b) Stitt prepared a transparent medium
1986. Stitt's Glycerol Blood Agar (Chocolate
as follows
Agar)
Constituents: (2) Mix equal parts 1.0% sodium ci-
1. Glycerol agar 100.0 cc. trate solution and blood.
2. Citrated blood (3) Add 20 to 30 drops of (2) to 100.0 cc.
Preparation of (1).
(1) Prepare glycerol agar. (4) Pour into 10 plates.
(2) Melt (1) and while the tubes are at References: Besson (1920 p. 31), Stitt
a temperature of about 90 C. add (1923 p. 44).
about 4.0 or 5.0% citrated blood
1988. Bezangon, Griffon and LeSourd's
(0.5 cc. to 10.0 cc. of agar in a tube).
Blood Agar
(3) Mix thoroly avoiding bubbles.
(4) Pour in sterile plates. Constituents:
Sterilization: Not specified. 1. Nutrient agar.
Use: Cultivation of Pfeiffer's bacilli. 2. Blood, rabbit.
Author reported that this medium was Preparation
less satisfactory for isolation of Pfeiffer's (1) Prepare nutrient agar.
bacillus than plain blood agar (due to its (2) Cool sterile melted agar tubes to 50C.
brown color). (3) Add fresh rabbit blood, drawn under
Variants: Stitt employed nutrient agar aseptic conditions. (Amount not
instead of glycerol agar. given.)
Reference: Stitt (1923 p. 43). (4) Mix the blood and agar.
(5) Slant and cool.
1987. Besson's Citrated Blood Agar
Constituents not given.
1. Nutrient agar. Use: Cultivation of the Ducrey bacillus,
2. Blood, citrated. A large variety of organisms were culti-
Preparation vated on the same or similar media by a
(1) Collect 400.0 cc. beef blood in a number of investigators.
sterile flask containing citrate solu- Variants
tion and 0.1 cc. of commercial formol. (a) Thoinot and Masselin streaked agar
(2) Shake thoroly. slants or agar plates with whole or
(3) Store for several days at room tem- defibrinated blood, using a sterile
perature. pipette.
(4) Dilute (3) with three volumes dE (b) Davis specified that one volume of
sterile physiological salt solution. freshly drawn rabbit blood be added
(5) Add one part (4) to 15 parts sterile to two volumes of 2.0% nutrient agar.
agar. (c) Czaplewski specified the use of pigeon
blood and mixed with nutrient agar (3) Add

a drop of infected blood to the
cooled to 45 to 60C. The medium tubes before solidification and dis-
was flaky and of a brownish color and tribute by stirring the tubes.
used for the cultivation of influenza (4) Incubate at 15 to 37C. Paraffin
bacilli. may be poured over the medium but
(d) Miiller cultivated fowl diphtheria growth may be obtained without it.
bacilli on a medium prepared as Authors reported that the erythro-
follows: cytes settled to the bottom of the
(1) Prepare nutrient agar. agar before solidification, giving it an
(2) Liquify (1) and place into sterile opaque and deeper color.
400.0 cc. Erlenmeyer flasks in (g) WoUstein cultivated Pfeiffer's bacil-
250.0 cc. lots. lus on the following media:
(3) Allow about 25.0 cc. of blood to (1) Prepare nutrient agar.
pass directly from the jugular vein (2) Adjust the agar to pH = 7.5.
of a goat into the melted agar or (3) Boil fresh rabbit blood for two
gelatin which has been cooled to minutes in a water bath.
about 45C. (Dog and chicken (4) Centrifuge.
blood gave good results also.) (5) Add 0.5 cc. of the resulting clear
Pour the blood medium into sterile pale pink or yellow fluid to 20.0 cc.
(4)
Petri dishes. of the broth or two or three drops
(e) Savini and Savini-Castano cultivated to 5.0 cc. of the melted agar.
influenza bacilli on a hematin agar (h) Levinthal (Klimmer) cultivated in-
prepared as follows: fluenza bacilli on a medium prepared
(1) Allow about 100.0 cc. of blood to as follows. Klimmer reported that
coagulate. best results were obtained using a
(2) Pour off the serum. medium 24 hours old.

Melt 1000.0 cc. of neutral 2.0 or
(3) Cut the blood clot into fine pieces (1)
and place into a flask containing 3.0% agar and cool to 70C.
100 to 150.0 cc. of N/1 NaOH or (2) Mix 50.0 cc. of fresh human or dog
N/10 soda solution. blood with (1). The blood may be

Boil for a long time over a free flame defibrinated and stored on ice, if
(4)
until all is well dissolved. desired.
and sterilize (method not (3) Heat (2) to boiling over a wire gauze
(5) Filter
given). until the agar begins to raise in the
Prepare nutrient agar. neck of the flask.

(6)
Melt sterile (6) and cool to about (4) Repeat the boiling twice more.
(7)
50C. (5) Separate the agar from the coagu-
To each 10.0 cc. of (7) add 1.0 cc. lum by filtering thru sterile cotton
(8)
of (5) and mix well without shaking or gauze filter. Do not heat again
violently. in steam.
Agar is alkaline to litmus.
(9) Allow the agar to solidify over night (6)
in a cool place. (7) Tube

or pour in plates.
References: Bezangon, Griffon and Le-
Authors reported that influenza
Sourd (1901 Thoinot and Masselin
bacilli developed upon this medium p. 3),
Davis (1903 p. 405), Cza-
when another organism grew side by (1902 p. 36),
Staphylococcus aureus plewski (1902 p. 668), Heinemann (1905
side with it.
p. 128), (1906 p. 519), Savini and
MuUer
gave best results for this purpose,
Savini-Castano (1911 p. 493), Ito and
(f) Ito and Matsuzaki cultivated Spiro-
chaeta icterohaemorrhagiae on the Matsuzaki (1916 p. 558), Roddy (1917
medium p. 45), Wollstein (1919 p. 556), Ball (1919
following
p. 82), Bezangon (1920 p. 120), Kristensen
(1922 p. 223), Klimmer (1923 pp. 225,
(2) Mix one part normal guinea pig
Stitt (1923 p. 44), Park, Williams
blood or human blood with one or 227),
two parts melted agar at 50C. and Krumwiede (1924 p. 125).

1989. Fleming's Brilliant Green Blood Agar Preparation

Constituents (1) Add 0.25 cc. of Allen and Hanbury's
1. Water 9.0 cc.
trypsin compound to a series of tubes
Blood containing 4.75 cc. of sterile bouillon.

2. 1.0 cc.
3. Nutrient agar (2) Incubate for 24 hours.
4. Brilliant Green 1 to 500,000
(3) Discard tubes showing contami-
nation.
Preparation
(4) Add 1.0 cc. of blood from a vein
(1) Boil 1.0 cc. of blood with 9.0 cc. water.
(Time not specified.) puncture to each tube.
(5) Incubate the mixture for 3 or 4 days.
(2) Add some of the clear supernatant
liquid of to nutrient agar. (6) Mix 5.0 cc. of the finished product
(1)
with about 30.0 cc. of nutrient agar,
(Amount of liquid not specified).
as in the preparation of ordinary
(3) Add brilliant green in the ratio of 1
to 500,000.
blood agar.
Use: Isolation of influenza bacillus.
Use: Isolation of B. influenzae. Author
Author reported that medium may be
reported that pneumococci, staphylococci
prepared in larger quantities. This
and streptococci were inhibited. B. influ-
enzae apparently was not inhibited.
amount is sufficient for a diagnosis, how-
ever. Do not allow the trypsin to act
Reference: Fleming (1919 p. 139).
longer than one week. Citrated blood
1990. Hachla and Holobut's Alkaline Blood may be used. Pneumococci, streptococci
Agar and other gram positive organisms were
Constituents inhibited. Staphylococci, however, were
1. Nutrient agar 700.0 cc. favored. Gram negative cocci of the
2. Blood, beef 200.0 cc. catarrhalis group and bacilli of the coli
3. KOH N/1 100.0 to 150.0 cc. form group were not inhibited, but were
Preparation not favored as were the influenza.
(1) Prepare neutral nutrient agar. Reference: Matthews (1918 p. 104).
(2) Mix 200.0 cc. of beef blood with
1992. Bieling's Glucose Blood Agar
100.0 cc. or 150.0 cc. of normal KOH.
(3) Steam (2) for 30 minutes. Constituents:
(4) Mix 7 parts liquid (1) with 3 parts (3). 1. Nutrient agar
(5) Pour into sterile plates. 2. Agar 150.0 cc.
(6) Dry the plates for 24 hours at 37C. 3. Blood, horse 50.0 cc.
and then for 24 more hours at room 4. Glucose 2.0% 3.0 g.
temperature. Preparation
Sterilization : Method not given. (1) Mix one part horse blood with two
Use: Enrichment medium for cholera vi- parts water.

brio. Authors reported that better re- (2) Prepare nutrient agar.
sults were obtained using 1 part blood to (3) Add 2.0% glucose to (2).
0.75 normal KOH solution. Hog and (4) Melt the glucose agar and cool to
horse blood gave as good results as did 60C.
beef blood. (5) Mix equal parts (1) and (4).
Reference: Hachla and Holobut (1909 Sterilization: Not specified.

p. 299). Use: Differentiation of streptococci and
pneumococci. Author reported that
1991. Matthews' Trypsinized Blood Agar hemolytic streptococci did not change the
Constituents: color of the medium. Colonies flat, about
1. Bouillon 4.75 cc. 2 mm. in diameter, with a central point
2. Trypsin (Allen and Han- and a border. Colonies could easily be
bury's) 0.25 cc. picked from the agar with a platinum
3. Blood 1.0 cc. loop aft?r 24 to 48 hours. Streptococcus
4. Nutrient agar 30.0 cc. mitior colonies were about the same size,
dark brown or brownish violet, dry and (3) Mix the tubes without shaking.
hard to remove from the agar. Pneumo- (4) Cool in a slanting position.
cocci colonies were larger than strepto- (c) Bezangon and Griffon (Besson) gave
cocci, about 5 mm. in diameter, colonies the following method of preparation:
were dark brown or brownish, forming a (1) Add 5.0% glycerol to nutrient agar.
smooth, coherent mucilaginous mem- (2) Liquify sterile (1) and cool to 45C.
brane. A light yellow decolorization of (3) Add about 1.0 cc. of fresh rabbit
the place when pneumococci
medium took blood. (A 1.0% solution of com-
growth was luxuriant. If agar and blood mercial hemoglobin may replace
were mixed at 50C. a clear ruby red the blood.)
transparent medium was obtained. (4) Mix well without shaking.
Heating above this temperature gave a (5) Slant and cool.
darker medium. When mixed at 60C. (d) Cantani (Besson) prepared a medium
the medium was not transparent in thick as follows:
[layers. (1) Mix equal parts of sterile blood and
References: Bieling (1921 p. 262), Klimmer sterile glycerol.
(1923 p. 226). (2) Allow to stand several hours.

(3) Add 0.5 to 0.75 cc. of (2) to tubes of
1993. Mandelbaum and Heinemann's Glyc-
liquified sterile agar.
erol Blood Agar (KoUe and Wasser- (e) Stitt cultivated pneumococci, strep-
mann and meningococci
tococci, gonococci
Constituents on a medium prepared by smearing
1. Glycerol agar. the surface of a glycerol agar slant
2. Blood human. with a platinum loop of blood ob-
Preparation: (1) Streak glycerol agar with tained from the lobe of a well cleaned
human blood. ear.
Use: Cultivation of diphtheria bacilli. References: KoUe and Wasserman (1912
Author reported that diphtheria colonies p. 414), Sick (1912 p. Ill), Tanner (1919
were light with yellowish brown ring. p. 69), Besson (1920 pp. 53, 54), Stitt
Pseudo diphtheria colonies red. Similar (1923 p. 47).
media were used to cultivate a large
1994. Wasserman's Nutrose Blood Agar
number of organisms by a number of
(Abel)
investigators.
Variants Constituents:
(a) Sick prepared a medium as follows 1. Water 30.0 to 40.0 cc,
for clinical purposes: 2. Blood (hog) 15.0 cc.
(1) Draw blood under aseptic condi- 3. Glycerol 2.0 or 3.0 cc.
tions directly from a vein into a 4. Nutrose 0.8 g.
sterile Erlenmeyer flask containing 5. Agar (2.0% peptone)
glass beads, using a sterile needle Preparation
and sterile tube. (1) Mix 15.0 cc. of hog blood, 30.0 to
(2) Shake the flask to defibrinate the 40.0 cc. of water, 2.0 to 3.0 cc. of
blood. glycerol and 0.8 g. nutrose.
(3) Prepare 3.0% agar containing 2.0 (2) Shake the mixture constantly and
to 3.0% glycerol. boil for 15 minutes.
(4) Mix one part (2) with 4 or 5 parts (3) Repeat the boiling and shake on the
sterile (3), cooled to 50C. following day for 15 minutes.
(5) Distribute into sterile test tubes. (4) When desired for use heat to 50 to
(b) Besson (Tanner) gave the following 60C. and mix with an equal quantity
method of preparation: of sterile 2.0% peptone agar.
(1) Melt tubes of glycerol agar and cool (5) Pour into plates.
to 40C. Sterilization: Not specified.
(2) Add 1.0 CO. of blood from a rabbit's Use: Cultivation of gonococci.
artery. Reference: Abel (1912 p. 162).
1995. Savini and Savini-Castano's Bacteria Variants

Blood Agar (a) Savini and Savini-Castano reported
influenza bacilli developed only upon
Same as medium 977 but use nutrient
the following medium when other
agar instead of bouillon.
organisms were growing side by side
1996. Bieling's Optochin Hydrochloride with it. Staphylococcus aureus gave
Blood Agar best results for this purpose.
(1) Prepare a solution of 5.0% hemo-
Constituents: globin in N/10 NaOH or soda solu-
tion. Boil until the solution is
2. Optochin hydrochloride 0.1 g.
complete.
3. Nutrient agar (3.0%) and (Method
(2) Filter sterilize (1).
4. Blood, horse 60.0 cc.
of sterilization not given.)
Preparation Prepare nutrient agar.
(3)
(1) Dissolve 0.1 optochin hydrochloride
g.
(4) Melt sterile (3) and cool to about
in 10.0 cc. water (should
distilled
50C.
not be kept more than 3 days).
(5) To each 10.0 cc. of (4) add 1.0 cc.
(2) Prepare 3.0% nutrient agar. of (2) and mix well without shaking
(3) Mix 1.0 cc. of (1) with 150.0 cc. of (2). violently.
(4) Mix 60.0 cc. horse blood with 90.0 cc. Allow the agar to solidify over
(6)
distilled water.
night in a cool place.
(5) Heat (4) for one hour at 60C.
(b) Thalhimer cultivated Bac. influenzae
(6) Melt (3) and cool to 60C. on the following medium. He re-
(7) Mix equal parts (6) and (5), both
ported that a slightly better medium
being at 60 ''C.
was obtained if a few cc. of hydrogen
peroxide were added before filtering.
Use: Differentiation of streptococci and Growth was even more luxuriant than
pneumococci. Author reported that
on ordinary blood agar.
pneumococci showed no growth. Hemo- amorphous powdered
(1) Dissolve
lytic streptococci colonies flat, and did
hemoglobin (Merck & Co., and
not change the color of the medium.
Eimer and Amend) in 100.0 cc. of
May be easily removed from the medium. water until a deep mahogany brown
Streptococcus mitior colonies were dry
color is obtained. (About 10.0 g.
and brown and hard to remove from the hemoglobin.)
medium. Reichel porcelain
(2) Filter thru a
References: Bieling (1921 p. 264), Klim-
filter.
mer (1923 p. 226).
(3) Add enough of (2) to sterile fluid
agar to obtain the same intensity
1997. Heim's Hemoglobin Agar
of color as ordinary blood agar.
Constituents: (4) Tube and slant.
1 Distilled water 90.0 cc. References: Heim (1907 p. 1587), Savini
2. Hemoglobin 10.0 g.
and Savini-Castano (1911 p. 493), Thal-
3. KOH (10%,) 10.0 cc. himer (1914 p. 189).
4. Nutrient agar
Preparation 1998. Esch's Alkaline Hemoglobin Agar
(1) Mix 1, 2 and 3.
(2) Mix 1.0 cc. sterile (1) with 7.0 cc. Constituents
nutrient agar. 1. Distilled water.
(3) Pour into plates or slant. 2. Hemoglobin (Merck's).

Sterilization: Sterilize (1) in streaming 3. Neutral agar.
steam. Preparation:
Use: Cultivation of pneumococci. Also (1) Triturate hemoglobin.
used by other investigators for the culti- (2) Add 5.0 g. hemoglobin to 15.0 cc. N/1
vation of influenza bacilli. NaOH + 15.0 cc. distilled water.
(3) Mix sterile (2) with 85.0 cc. neutral 2. Soda (18.0% solution) 10.0 cc.
agar. 3. NaCl (0.85% solution) 10.0 cc.
Sterilization: Sterilize (2) in streaming 4. Hemoglobin extract (Pfeuffer) 3.0 g.
steam. Preparation
Use: Isolation of cholera vibrio. (1) Place 80.0 cc. of melted neutral nu-
Variants trient 3.0% agar in an Erlenmeyer
(a) Costa gave the following method of flask.
preparation (2) Add 10.0 cc. of 18.0% soda solution
(1) Pulverize crystalline blood in a and boil this mixture for about 10
mortar. minutes.
(2) Add distilled water slowly to (1). (3) Cool to 50C.
This forms sort of a sticky mixture. (4) Dissolve 3.0 g. hemoglobin extract
(3) Add 100.0 cc. of a normal KOH (Pfeuffer) in 10.0 cc. of 0.85% NaCl
solution to (2). solution.
(4) Heat in the autoclave for 30 (5) Mix (4) and (3) thoroly.
minutes. (6) Pour into 7 Petri dishes and allow to
(5) Filter. stand with covers removed until
(6) Sterilize by heating in the auto- solidification has taken place.
clave at 100C. for several hours. (7) Place the dishes in an incubator with
(7) Mix volumes of (6) with 7 volumes
3 cover removed for 20 to 30 minutes to
of sterile melted agar under aseptic remove the water of condensation.
conditions. Sterilization: Not specified.
(8) Pour into sterile Petri dishes. Use: Selective medium for cholera vibrio.
(b) Kohlisch and Otto prepared the Author reported that medium was trans-
medium as follows: parent and dark brown. Some cholera-
(1) Rub 15.0 g. of Merck's horse haemo- like vibrio and other organisms were
globin in a mortar. inhibited on this medium.
(2) Mix 15.0 cc. of N/1 NaOH with Variants: Klimmer prepared the medium
(1), and add 15.0 cc. distilled in the following manner:
water. (1) Dissolve 3.5 g. of Pfeuffer's hemo-
(3) Sterilize for one hour in the globin extract in 10.0 cc. of physio-
steamer. logical salt solution.
(4) To 15.0 cc. of (3) add 850.0 cc. of (2) Add 10.0 cc. of a 5.5% solution of
neutral agar. water free soda and 2.0 cc. of KOH
(5) Mix thoroly. solution (strength not given) to (1).
(6) Pour in plates. (3) Steam for 15 minutes.
(7) Dry the plates at room tempera- (4) Cool to 50C.
tures for one hour. (5) Add (4) to 80.0 cc. of 3.0% melted
(c) Klimmer used the following method nutrient agar, neutral to litmus and
in preparing the medium: cooled to 80 or 90C.
(1) Dissolve 50.0 g. of Merck's hemo- (6) Mix thoroly.
globin in 300.0 cc. of half normal (7) Pour in plates.
KOH by heating in a steamer for (8) Dry thoroly in the incubator.
an hour. References: Kabeshima (1913 p. 203),
(2) To hot (1) add 1700.0 cc. of melted Klimmer (1923 p. 220).
hot neutral agar.
Pour into
(3) plates. 2000. Besson's Glycerol Hemoglobin Agar
References: Esch (1910 p. 559), Costa (1912 (Tanner)
p. 846), Kohlisch and Otto (1915 p. 438), Constituents:
Klimmer (1923 p. 218). 1. Glycerol agar.
2. Hemoglobin.
1999. Kabeshima's Alkaline Hemoglobin
Preparation
Agar
(1) Melt tubes of glycerol agar and cool
Constituents to 40C.
1. Nutrient agar (3.0%) 80.0 cc. (2) Add 1.0 cc. of a solution of hemoglobin
to each tube, (strength of hemo- Sterilization: Method not given.

globin solution not given). Use: Cultivation of gonococci. Authors
(3) Mix the tubes without shaking. reported that growth was equally as good
Cool in a slanting position. in non-dialyzed serum medium.
(4)
Sterilization: Not specified. Reference: Finger, Ghon and Schlagen-
Use: General culture medium. haufer (1894 p. 14).

2003. Miiller's Serum Agar
2001. Kabeshima's Hemoglobin Extract
Constituents:
Agar
1. Nutrient agar.
Constituents: 2. Serum, goat.
1. Nutrient agar (3.07o) 1000-0 cc Preparation
2. Hemoglobin extract 50.0 g. (1) Prepare nutrient agar.
3. Saline solution 1000.0 cc. (2) Melt (1) and cool to about 60C.
Preparation (3) Mix equal parts of (2) and goat serum
(1) Add 0.1 cc. of an 18.0% NajCOa solu- that has been obtained under aseptic
tion to 1000.0 cc. of neutrient neutral conditions and heated to 60 C.
agar. (4) Pour into sterile plates or sterile
(2) Boil 10 minutes. tubes.

(3) Cool to about 15C. and add 1000.0 cc. Sterilization: Method of sterilization of
of hemoglobin extract prepared by agar not given.

dissolving 50.0 g. of Shinoda's hemo- Use Cultivation of fowl diphtheria bacilli.
:
globin extract in 1000.0 cc. of 0.9% Similar media were used for the cultiva-
salt solution. tion of various organisms by a number of
(4) Distribute in petri dishes and remove investigators.
the bubbles by flaming with a Bunsen Variants
Burner. (a) Miihlens and Hartmann cultivated
(5) When cool remove the covers and Spirochaeta dentium and Bacillus
place in the incubator for 30 minutes. fusiformis on a medium prepared as
(6) Use when dry. follows:
Sterilization: Not specified. (1) Prepare nutrient agar with a
Use: Cultivation of cholera vibrio. slightly alkaline or neutral re-
Reference: Kabeshima (1916 #225) taken action.
from (1917 p. 395). (2) Boil half filled tubes of (1) for
about 30 minutes in a water bath to
2002. Finger, Ghon and Schlagenhaufer's free the agar of oxygen.
Dialyzed Serum Agar (3) Heat horse serum for about 30
Constituents
minutes in a water bath at a tem-
perature from 58 to 60C.
1. Nutrient agar.
(4) Cool (2) and (3) quickly to 45C.
2. Blood Serum (Dialyzed).
Preparation (5) Mix two parts of the agar with one
human blood serum in part of the serum.
(1) Place sterile
(6) Inoculate while the medium is still
parchment sack.
sterile
in the sack in a liquid state.
(2) Suspend the blood serum
(7) Solidify by placing the tubes in
in a sterile cylinder containing sterile
cold water.
water.
48 hours and (b) Carrel mixed serum with one-fifth
(3) Change the water every
its volume of 2.0% agar and used the
continue the dialysis until the water
medium for the cultivation of tissue.
is colored by the albumin that
(c) Besson prepared the medium as
dialyzes thru the parchment.
follows:
(4) Mix the dialyzed blood serum with
Melt tubes of sterile nutrient agar.
agar in the same manner as in the (1)
Cool to 45 to 50C.
preparation of blood serum agar. (2)
(3) Add one-half or one-third the vol- 2005. Muhlen's and Hoffman's Glucose
ume serum to each tube.
of sterile Serum Agar (Stitt)
(4) Mix
thoroly by rolling the tube be-
Constituents
tween the palms of the hands.
(5) Slant or pour into a Petri dish.
2. Glucose (0.5%) 0.5 g.
(d) Dopter and Sacquepee gave the fol-
3. Serum (Horse) 100.0 cc.
lowing method of preparation:
Preparation
(1) Add 0.5 to 1.0 cc. of serum to an
(1) Fill sterile test tubes one-third full
agar slant.
with horse serum.
(2) Incline so that the entire surface is
(2) Add an equal amount of a 3.0% agar
covered with the serum.
containing 0.5% glucose which has
(3) Allow to stand for 12 to 24 hours.
been melted and cooled to 50C. to
(e) Klimmer mixed one part gelatin agar
sterile (1).
with one-half part serum at a tem-
(3) Keep at 55 for two hours.
perature of 45C.
Sterilization: Sterilize (1) on 3 successive
References: Muller (1906 p. 520), Muhlens
days at 55C.
and Hartmann (1906 p. 86), Carrel (1912
Use: Cultivation of treponemata. The
Besson (1920 p. 53), Dopter and
p. 393),
medium was inoculated while still liquid
Sacquepee (1921 p. 138), Klimmer (1923
and incubated under anaerobic condi-
p. 200), Kligler and Robertson (1922
tions.
p. 315).
2004. Joos' Alkaline Serum Agar (Klimmer)

2006. Cantanis' Glycerol Serum Agar
Constituents (Besson)
2. Serum 300.0 cc. 1. Nutrient agar.
3. NaOH (normal) 50.0 cc. 2. Glycerol.
4. Bouillon 500.0 cc. 3. Serum.
5. Agar 20.0 g. Preparation
Preparation Mix equal
(1) parts of sterile serum and
(1) Heat 1, 2 and 3 for 2 to 3 hours in a
sterile glycerol.
water bath at 60 to 70C. Allow to stand several hours.
(2)
(2) Steam for 30 to 45 minutes.
Add 0.5 to 0.75 of
(3) (2) to tubes of
(3) Prepare bouillon using 2.0% peptone liquified sterile agar.
and 1.5% NaCl. Sterilization: Method not given.
(4) Adjust the reaction of (3) to alkaline. Use: General culture medium.
(5) Dissolve 20.0 g. agar in (4). Reference: Besson (1913 p. 54).
(6) Mix (5) and (2).
(7) Filter. 2007. Kodama's Fuchsin Sulphite Serum

Sterilization: Method not given. Agar
Use : General culture medium. Constituents
Variants: Klein (Klimmer) gave the fol- 1. Water 40.0 cc.
lowing method of preparation for a me- 2. Nutrient agar (3.0%) 100 cc.
dium for the cultivation of diphtheria 3. Fuchsin (saturated alcoholic
bacilli. solution) 0.5 cc.
(1) Mix 9 parts serum with 1 part normal
4. NasSOa (10.0% solution) .... 2.5 cc.
NaOH and incubate at 37C. for 5. Starch, potato 3.0 g.
2 days. 6. Serum, beef 10.0 cc.
(2) Neutralize by the addition of HCl. Preparation
(3) Mix 1 part (2) with 4 parts nutrient (1) Prepare nutrient 3.0% agar, neutral
agar. to litmus.
(4) Sterilize at 105C. for 1 hour. (2) Add 1.0 cc. 10.0% soda solution,
Reference: Klimmer (1923 p. 222). 0.5 cc. of a saturated alcoholic fuchsin
solution, 2.5 cc. of a freshly prepared (2) Add 2 or 3.0 cc. of glycerol to (1).
sodium sulfite solution and 3.0 g. of (3) Add 0.8 or 0.9 g. nutrose to (2).
potato starch (Merck) stirred up in (4) Heat over a free flame to boiling,
and steam in a
5.0 cc. water, to (1) shaking constantly. The turbid fluid
steamer for 30 minutes. becomes clear.
(3) To a 50.0 cc. of beef serum diluted (5) Liquefy 2.0% peptone agar and cool
with 5 parts water, add 0.5 cc. of a to about 50C.
10.0% NaOH solution and boil for (6) Mix equal parts (5) and sterile (4).
30 minutes in the steamer. (7) Pour into sterile Petri dishes.
(4) Mix two parts (2) with one part (1). Sterilization: Heat (4) until sterile. With
(5) Distribute into sterile test tubes. fresh serum about 20 minutes is required.
(6) Pour sterile (5) into sterile Petri With old serum a longer time is necessary
dishes, seeing that the starch is well and it is often best to heat on several suc-
distributed. Dry the plates before cessive days. Sterilization of agar not
use. given.
on three successive
Sterilization: Sterilize Use: Cultivation of gonococci.
days for 30 minutes each day in the Variants: Klimmer used alkaline nutrient
steamer. agar.
Use: Elective medium for cholera vibrio. References: Wasserman (1898 p. 300),
Author reported that after 3 to 9 hours Klimmer (1923 p. 200).

the cholera colonies were red. There
2009. Kligler's Nasal Secretion Serum Agar
were two types, one a flat colony with
irregular edges and the other, smaller, Constituents
raised and definitely defined edge. Bad. 1. Glucose agar.
coli, Bad. dysenteriae Shiga-Kruse, 2. Serum, sheep.
Bad. dysenteriae Flexner, anthrax bacil- 3. Nasal secretion.
lus, Staphylococcus pyogenes aureus, Preparation

etc., all formed colorless colonies. The (1) Prepare glucose agar.
cholera-like vibrio all gave one of the (2) Obtain nasal secretions by blowing
two indicated types of red colonies. the nose into sterile gauze.
Wheat starch gave the same results but (3) Place into saline solution or alcohol
an hour later. Ordinary water soluble and keep 3 days.

starch (Merck) gave only the small well (4) Divide the saline mixture into two
defined edge type of colony. Medium equal parts.

prepared with dextrin did not give as (5) Filter one of (4) thru a Berkefeld and
good results as using potato starch. autoclave the other part.
Variants: Kodama used 3.0 g. dextrin in- (6) Desiccate the alcoholic extract to
stead of starch. dryness and then take up in saline
Reference: Kodama (1922 p. 433). solution.
(7) Distribute (1) in 10.0 cc. lots.
2008. Wasserman's Nutrose Serum Agar Add sheep serum to each tube
1.0 cc.
(8)
Constituents one of
of (7), also 1.0 cc. or 0.5 cc. of
1. Water 30.0 to 35.0 cc. the various extracts (6) and one of (5).
2. Serum, hog 15.0 cc. (9) Pour into plates.
3. Nutrose 0.8 to 0.9 g. Sterilization: Method of sterilization of
4. Nutrient agar glucose agar not given.

5. Peptone Use: Show influence of nasal secretion ex-
6. Glycerol tract on growth of meningococci. Author
Preparation reported that saline extracts in each case
(1) Place 15.0 cc. of hog serum as free were superior to alcoholic extract. Fil-
from hemoglobin as possible in an tered saline extract more effective than
Erlenmeyer flask and dilute with 30 heated one.
to 35.0 cc. water. Reference: Kligler (1919 p. 39).
2010. Meyer's Tuberculin Agar (4) Rub up the deposit with 10.0% KOH
solution added drop by drop to pre-
Constituents :
vent e.xcess of alkali.
1. 5.0% glycerol bouillon with Add HCl to (4) until a precipitate
(5)
heavy growth of tubercle begins to appear. Add this fraction
bacilli 200.0 cc.
to the clear supernatant fluid
2. Glycerol bouillon (4.0 or
from (3).
5.0%) 200.0 cc.
(6) Add sufficient glycerinated com-
3. Agar (2.0%) 8.0 g.
mercial trypsin to serum (horse
4. Serum (10.0%) 40.0 cc.
generally) to partially neutralize
Preparation : the antitryptic power. (Requires
(1) Tuberculin flasks containing about from 2.0 to 8.0 cc. of trypsin per
250.0 cc. of a5.0% glycerol peptone 100.0 cc. depending on the strength
bouillon, with a heavy growth of
of the trypsin, 2 to 4.0 cc. of Fair-
human bovine tubercle bacilli,
or child & Co. and Digestive Ferments
about 6 to 8 weeks old are steamed in Co. trypsin being sufficient and 5.0
a Board of Health sterilizer for two to 8.0 cc. of Allen and Hanbury's
hours. trypsin).
(2) Cool and filter thru fine filter paper
(7) Filter (6) thru a porcelain candle of
and centrifuge to eliminate every medium porosity and distribute in
tubercle bacillus. sterile flasks.
(3) Mix the clear liquid with an equal Melt sterile (1) and add 4.0 cc. of (5)
(8)
volume of the same 4.0 or 5.0%
for each 100.0 cc. of agar.
glycerol peptone bouillon and 2.0%
(9) Cool to between 65 and 60C. and
agar isadded and dissolved. add 15.0 cc. of trypsinized serum per
(4) Filter and correct the reaction to 1.5.
100.0 cc. agar.
Distribute in 9.0 cc. quantities in
(5) (10) Mix thoroughly.
test tubes. Tube in sterile tubes.
(11)
(6) Before being slanted 1.0 cc. (or 10.0%) (12) Slant.
of fresh sterile horse or cattle serum Incubate to test sterility.
(13)
is added to each tube of sterile agar.
Sterilization: Sterilize (5) in the autoclave. and (5) not given.
Use: Cultivation of Bacillus para tuber- Use: Cultivation of B. diphtheriae.
culosis.
Reference: Douglas (1922 p. 263).
Reference: Meyer (1913 p. 175).
2012. Czaplewski's Alkaline Serum Glucose

2011. Douglas' Tellurite Trypsinized Serum
Agar
Agar
Constituents :
Constituents :
1000.0 cc. 1. Glucose agar

1. Nutrient agar (2.0%)
Potassium tellurite 2. Serum 300.0 cc.
2.
3. Serum 3. NaOH (normal) 30.0 cc.
4. Trypsin Preparation :
Preparation: (1) Add 30.0 cc. normal NaOH to
Preparenutrient (2.0%) agar. (Pref- 300.0 cc. sheep serum.

(1)
erably a trypsin digest agar, but (2) Sterilize (method not given).
(3) Allow to stand and pour off the
clear
ordinary peptone agar gave almost
identical results). supernatant liquid.
Mix sterile (3) with sterile glucose
(2) Add 1.0% potassium tellurite to (4)
water and dissolve as agar (1.0, 2.0 or 3.0 cc. serum to

100.0 cc. of
much as possible. 10.0 cc. agar).
(3) Allow the insoluble portion of (2) Sterilization: Sterilize (3) in the autoclave.
to settle and pour off the clear Method of sterilization of glucose agar
supernatant layer. not given.
Cultivation of diphtheria bacilli. 2015. Salomon's Basal Ascitic Fluid Agar

Use :
Reference: Czaplewski (1920 p. 828). Constituents

2013. Klein's Alkaline Serum Agar 50.0 cc.
2. Ascitic fluid
Constituents 3. Litmus tincture
1. Nutrient agar 400.0 cc. Preparation
2. Serum 90.0 cc. (1) Prepare 3.0%, nutrient agar.
3. NaOH (15.0%) 10.0 cc. (2) Distribute (1) into 10.0 cc. lots.
Preparation: (3) Prepare a 10.0% solution of
one of
(1) Mix 90 parts serum with 10
parts the added nutrients in litmus tinc-
15.0% NaOH. ture.
(2) Incubate 2 days at 37'=C. (4) Add 1.5 cc. of one of (3) to 10.0 cc. of
(3) Neutralize by the addition of HCl, melted (2) cooled to 58C.

(4) Mix one part (3) with four parts (5) Add 5.0 cc. of ascitic fluid, heated to
nutrient agar. 58C. to each tube of (4).
Sterilization: Heat (4) for 30 minutes Sterilization: Not specified.
at 105C. Use: To study fermentation of strepto-

Use: Cultivation of diphtheria bacillus. cocci. Author gave the following re-
Reference: Klein (1920 p. 297). actions:
A. Strept. pyogenes group.
2014. Frost, Charlton and Little's Milk I. Strept. pyogenes: Acid formation
Serum Agar from soluble amylum. Mannitol
Constituents: and raffinose unchanged.
Distilled water 100.0 cc. II. Strains from blood. Acid forma-
1.
2. Nutrient agar 200.0 cc. tion from glycerol and mannitol.
3. Milk 200.0 cc. B. Strept. mucosus.

4. Serum 100.0 cc. I. Acid from glycerol, arabinose and
mannitol. Raffinose and soluble
Preparation:
(1) Prepare ordinary nutrient agar (1.0 to amylum unchanged.
1.5%) with a reaction of +1.0. II. not attack any of the sugars in
Do
Tube in 1.0 to 3.0 cc. lots. 24 hours and rarely after 48 hours.
(2)
Tube well separated milk in 1.0 to C. Pneumococci formed no acid on carbo-
(3)
3.0 cc. quantities. hydrate litmus ascitic agar.
Mix one part serum (sheep, beef, Similar media were used by other in-
(4)
horse or rabbit) with three parts dis- vestigators to study fermentation of other
tilled water. cocci.
Tube quantities. Added nutrients: The author used one of

(5) (4) in 1.0 to 3.0 cc.
When ready for use melt the sterile the following materials:
(6)
glycerol levulose
agar tubes and cool to 45 to 50C. in
erythritol dulcitol
a water bath.
arabinose lactose
(7) Heat sterile tubes of (3) and (4) in
adonitol raffinose
the water bath and add one tube of
isodulcitol inulin
(3) and one tube of (4) to a tube of
mannose maltose
agar (equal parts agar, milk and
glucose sucrose
serum).
mannitol dextrin
(8) Mix thoroughly.
and in galactose amylum (soluble)
Sterilization: Sterilize (2), (3) (5)
the autoclave.
Variants
diphtheriae. The (a) Symmers and Wilson cultivated
Use: Isolation of B.
meningococci on a similar medium
authors used this medium in preparing
prepared as follows:
"little plate" cultures on slides.
(1) Prepare nutrient
agar containing
Reference: Frost, Charlton and Little
3.0% Chapoteaut's peptone.
(1921 p. 30).
(2) Add 1.0% sugar and some litmus levulose, galactose, mannitol, dul-
solution to (1). citol, sucrose, lactose, inulin with
(3) Steam for 10 minutes on 3 suc- Kubel-Tiemann's litmus and steri-
cessive days to sterilize. lize. (Method not given.)
(4) Add one part of sterile ascitic fluid (2) Add (1) to ascitic agar so that the
to two parts (3). agar content is 1.0%.
(5) Tube and slant. (3) Final sterilization not given.
(6) Incubate 2 days to test sterility. Eastwood reported that meningococci
(b) Gordon (Abel) used a medium pre- gave acid (red colonies) with glucose
pared as follows to study fermenta- and maltose only.
tion of meningococci and gonococci. References: Salomon (1908 p. 2), Symmers
(1) Prepare 10.0% solution of any de- and Wilson (1909 p. 10), Abel (1912
sired carbohydrate, alcohol, etc., in p. 159), V. Przewoski (1912 p. 15), Hancken
Kahlbaum's litmus solution and (1916 p. 368), Eastwood (1916 p. 408).
boil two minutes.
2016, Rosenow's Glucose Ascitic Fluid Agar
(2) When cool add 0.5 cc. of normal soda
solution to each 10.0 cc. of (1). Constituents
(3) Mix 3 parts 3.0% nutrient agar with 1. Nutrient agar (2.0%). 1000.0 cc.

one part ascitic fluid. 2. Glucose 2.0 to 10.0 g.
(4) Add 1.5 cc. of (2) to each 13.5 cc. 3. Ascitic fluid 300.0 cc.
of (3)_. Preparation
(5) Pour into plates. (1) Prepare 2.0% nutrient agar with a
(c) V. Przewoski used the following me- reaction of 0.4 to 0.6% acid to phenol-
dium to determine fermentation phthalein.
ability of diphtheria and pseudo (2) Dissolve 2 in (1).
diphtheria: (3) Tube in 7.0 to 8.0 cc. lots.
(1) Prepare nutrient agar. (4) Boil sterile (3) to drive off o.xygen
(2) To add 5.0
100.0 cc. of liquid (1) cc. and cool to 50C.
litmus solution (Kahlbaum), 2.0 cc. (5) Add 2 to 3.0 cc. heated ascitic fluid
ascitic fluid and 1.0 g. of one of (60C. for 24 hours) to each tube.
the following: (6) Cool to 40C.
fructose mannitol Sterilization: Method of sterilization of
glucose lactose (3) not given. See step (5) above for
mannose inulin sterilization of ascitic fluid.
dulcitol Use: To isolate organism causing rheuma-
(d) Hancken studied the fermentation tism, author added some of rheumatic
ability of meningococci on a medium material to medium and mixed well;
prepared as follows: plunged tube into cold water to "set"
(1) Prepare nutrient agar. and incubated at 37C. Author reported
(2) Dissolve 2.5 g. of any desired carbo- that largest number of colonies developed
hydrate sugar or alcohol in 50.0 cc. between 1.5 cm. from the top and 3.5 cm.
of Kahlbaum's litmus solution. from the bottom. A similar medium was
Boil for a few minutes. used for the cultivation of meningococci
(3) Melt the sterile agar and cool to and gonococci by Tilmant and Carrien.
55C. and add 3.3 cc. ascitic fluid Variant: Tilmant and Carrien cultivated
and 4.0 cc. of (2) to 9 or 10.0 cc. of meningococci and gonococci on a medium
the agar under aseptic conditions. prepared as follows:
(4) Pour into sterile plates. (1) Collect about 100.0 cc. of ascitic fluid
(e) Eastwood studied the fermentation as carefully as possible.
of carbohydrates, alcohols, etc., on (2) Estimate the albumin content and
Lingelsheim's medium prepared as dilute with physiological saline so
follows: that there will be 3.0 g. of albumin
(1) Prepare a 10.0% solution of one per liter.
of the following: maltose, glucose, (3) Add 1.0 cc. of a 10.0% soda solution
for each 200.0 cc. of fluid and sterilize Sterilization: Not specified.
in the autoclave. Use Cultivation of whooping cough bacilli.

:
(4) Add melted glu-

sterile (3) to sterile Author reported that whooping cough
cose agar (or gelatin) in proportion bacilligrew as a glistening opalescent
of 3 to 7.
covering of a grayish shade.
References: Rosenow (1914 p. 61), Tilmant Variants
and Carrien (1917 p. 499) taken from (a) Cantani prepared a similar medium
(1917 p. 222).
as follows:
(1) Mix equal amounts of glycerol and
2017. Ruediger's Inulin Ascitic Fluid Agar the albuminous fluid (urine, pus,
Constituents: milk, egg white, etc.). Place in an
1. Distilled water 200.0 cc. Erlenmeyer flask.
2. Peptone (Witte) 10.0 g. (2) Storing the fluid in glycerol tends

Agar 15.0 g. to sterilize the fluid.
3.
4. Bouillon (sugar free) 800.0 cc. (3) After a time test the sterility of the
5. Inulin 15-0 g. mixture.
6. Litmus (5.0% soln.) 20.0 cc. (4) Prepare nutrient agar.

7. Ascitic fluid 145.0 cc. (5) Mix 6 parts ascitic fluid (with or
Preparation: without glycerol) with one part

sugar free bouil- of (3).
lon by boiling for one hour. Add (6) Add to each tube of sterile (4) 0.5
water to make the volume to 800.0 cc. to 0.75 cc. of (5).
Heat in autoclave for 15 to 20 (7) Incubate 24 hours to test sterility.

(2)
minutes. (b) Cantani (Besson) prepared a medium
Clarify with egg. as follows:
(3)
(4) Filter thru cotton. (1) Mix equal parts of sterile ascitic
Make up volume to 800.0 cc. with fluidand sterile glycerol. .
(5)
distilled water. (2) Allow to stand several hours.
(6) Dissolve 15.0 g. pure inulin in (3) Add 0.5 to 0.75 cc. of (2) to tubes of
200.0 cc. distilled water. liquified sterile agar.
Mix (6) with (5). References: Klimenko (1909 p. 311), Can-

(7)
Add 20.0 cc. of 5.0% solution of tani (1910 p. 472), Besson (1920 p. 54).
(8)
litmus (Merck's highest purity).
Tube in 7.0 to 8.0 cc. lots. 2019. Vellion's Ascitic Fluid Agar
(9)
(10) Just before use, melt sterile agar,
Constituents:
cool to 45 and add 1.0 cc. of heated
1. Nutrient agar (2.0%).
(65) fluid to each tube.
2. Ascitic fluid.
Sterilization: Sterilize (9) autoclave in
Preparation:
under 10 pounds pressure for 15 minutes. Prepare 2.0% nutrient agar with a
(1)
Use: Isolation of pneumococci. Acid pro- slightly alkaline reaction.
duction. Ruediger reported the pneu-
(2) Tube.
mococci colonies were red. Cool melted sterile (2) to about 60C.
(3)
Reference: Ruediger (1906 p. 184).
Add an equal amount or even
(4)
2018. Klimenko's Glycerol Ascitic Fluid amount of ascitic fluid.
Agar (5) Slant.

Constituents
(2) not given.
1. Nutrient agar (3.0 or 4.0%) . 100.0 cc.
2. Glycerol (1.0%) 10.0 cc.
3. Ascitic fluid (human) 100.0 cc. Variants

Preparation (a) Thoinot and Masselin prepared the
nutrient agar con- medium as follows:
(1) Prepare 3.0 or 4.0%
taining 1.0% glycerol. (1) Liquify sterile agar and cool to
Mix equal parts of (1) and human about 50C.
(2)
ascitic fluid (or blood serum). (2) Add a known quantity of sterile
ascitic fluid to (1) under aseptic Preparation:

conditions. Add
,^, ^ (1) 5.0 cc. of normal NaOH to a liter
(b) Besson prepared the medium as of neutral 3.0% agar
^^^ "^^^ ^^^-^ '' ^ ^ 10-0% nutrose solu-
n
(1)l^ÂxT: K
Melt tubes off sterile
. ,
nutrient agar. tion to sterile (1)
(2) Cool to 45 to 50C. When ready
(3) for use liquify the agar
(3) Add one-half or one-third the and cool to 45C.
volume of sterile ascitic fluid to (4) To each 100.0 cc. of (4) add 3 cc of
'^""^^ fil^^^^d ^^ bile, cc. of a
M^ ^r^!f'-, u n-
(4) Mix thoroly by rolling the tube
1.0
0.2% aqueous "Pure Safranin" (Grii-
between the palms of the hands. bier), 3.0 cc. of a 1.0% aqueous
"Pure
5) Slant or pour into a Petri dish. blue double concentrated" (Hoch-
(c) Dopter and Sacquepee prepared the
ster Farbwerke), and 3 to 4 cc
nT7^TnT!''^!T"'^'-
^ " ^-^^^ ^^^^^ ^^l^^îte green
(1)Add 0.5 to 1.0 cc. of. ,
sterile ascitic crystals (Hochst.). Mix thoroly
fluid to agar slants. (5) Pour in plates.
(2) Incline so that the entire surface Sterilization: Method of sterilization of
is covered with the serum.
agar not given. Sterilize the bile by
Allow to stand for 12 to 24 hours.
(3) boiling
References: Vellion (1898 p. 24), Thoinot Use: Enrichment of typhoid group. Klim-
and Masselin (1902 p. 36), Besson (1920 mer reported that colon colonies were
p 53), Bezangon (1920 p. 119), Dopter inhibited. Paratyphoid colonies grew
P- ^^^^' Klimmer luxuriantly as glassy, milk-like colonies
n^^noo 2y?l^^^^^^
(1923 p. 226), Stitt (1923 p. 42). References: Abel (1912 p. 132), KliLe;
(1923 p. 214).
2020. Herrold's Phosphate Ascitic Fluid
Agar 2022. Hasting's Milk Agar
1. Nutrient agar 300.0 cc. ^- ^'"trient agar 1000.0 cc.
2. Na2HP04 '
' 2. xAIilk 100.0 to 120.0 cc.
3. Ascitic fluid 100.0 cc. Preparation:
Preparation: (1) Prepare nutrient agar.
(1) Prepare nutrient agar substituting ^2) Melt (1) and cool to 50C.
Na2HP04 for NaCl. (3) Add 10.0 to 12.0% skimmed milk
(2) Heat ascitic fluid at 56C. for one * (2).
hour. (4)Tube and slant or pour in plates.
(3) Add one part heated ascitic fluid to Not specified.
Sterilization:
three parts melted (1). Use: To demonstrate proteolysis. Hast-
(4) Tube or pour into plates. igs reported that if proteolysis takes
Sterilization: Not specified. place the opaque medium clears. More
Use: Cultivation of streptococci for agglu- advantageous than gelatin to determine
tination and absorption studies. proteolysis for milk agar may be incu-
Reference: Herrold (1922 p. 80). bated at a temperature greater than
20C. Used also to cultivate fowl diph-
2021. LoefHer's Dye Bile Agar (Abel) theria bacilli and B. vulgaricus.
Variants
Constituents
(^^ .^jÛ^^ reported the colonies of fowl
1. Nurient agar (3.0%) 1000.0 cc. diphtheria bacilli appeared after
^."Jtôse 10.0 g. 40 hours when inoculated on the fol-
Z-
/ ^'[^. 20-0 lowing medium:

4. Safranin (0.2% soln.) 10.0 cc. (1) prepare nutrient agar.
5. Pure blue (1.0%
(2) Melt (1) and mix equal parts of
30.0 cc. melted agar and skim milk at
0.
Jr. uv green
Malachite 100 C
^-2^ "^^") 30.0 to 40.0 cc. (3) Pour into sterile tubes or plates.
(b) Stitt cultivated B. bulgaricus on a Preparation

medium prepared by adding 2.0 or (1) Obtain clean sputa and place in
3.0 cc. of plain or litmus milk to a sterile test tubes.
tube of melted nutrient agar. (2) Immerse water bath so

the tubes in a
References: Hastings (1903 p. 384), Miiller that the water level in the bath is
(1906 p. 521), Tanner (1919), Klimmer higher than the sputa in the tube.
(1923 p. 203), Stitt (1923 p. 42). Do not heat over 70C. Usually
60C. is sufficient.
2023. Valleti's Whey Agar (3) Heat the sputa until a homogenous
Constituents thin liquid is formed. Shake or stir
1. Nutrient agar. the sputa occasionally. Do not heat
2. Whey. longer than 1| to 2 hours.
Preparation: (4) Prepare slants or plates from nutrient
(1) Prepare nutrient agar. agar.
(2) Prepare serum from cow's milk by (5) Streak the surface of agar slants or
boiling milk with a few drops of plates with the sterilized sputa, in
acetic acid. the same manner as one streaks agar
(3) Add 2.0 cc. of milk serum to agar with blood to prepare blood agar
(amount not given). slants.
Sterilization: Not specified. Sterilization: See preparation of steriliza-
Use: Cultivation of tubercle bacilli (espe- tion of sputum. Method of sterilization
ciallybovine types). Author reported of agar not given.
that the bovine type developed after Use: Cultivation of influenza bacilli.
about 36 hours. Author reported that influenza colonies
References: Valleti (1913 p. 240), Wigger were generally more turbid and flatter
(1914 p. 3). than on blood agar.
Reference: Fichtner (1904 p. 376).
2024. Klimmer's Casein Agar
Constituents: 2027. Finger, Ghon and Schlagenhaufer's
1. Water 1000.0 cc. Urine Agar
2. Peptone 10.0 g.
Constituents
3. NaCl 0.1 g.
1. Nutrient agar (2.0%).
4. MgS04 0.3 g.
2. Urine.
5. K2HPO4 0.3 g.
Preparation
6. Casein 5.0 g.
(1) Prepare 2.0% nutrient agar contain-
7. Agar (2.0%) 1000.0 cc.
ing 1.0% peptone. Reaction to be
Preparation
neutral or slightly alkaline.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. condi-
(2) Obtain urine under aseptic
(2) Mix equal parts sterile (1) and liquid
tions. The reaction is slightly acid.
2.0% agar.
sterile
two
(3) Add one part sterile (2) to
parts (1). The final reaction is
neutral.
Use: Determine peptonizing ability. The
author reported that peptonizing bac-
agar not given.
teria dissolved the casein giving a light
area.
Variants
that the urine
(a) The authors reported
2025. Mayer's Mucin Agar may be mixed with the agar and then
sterilized. (Method not given.)
Same as medium 969, but substituting
nutrient agar for bouillon.
The urine may be sterilized by heat-
ing at 70 to 80C. for 30 minutes and
2026. Fichtner's Sputum Agar then mixing one part urine with two
Constituents parts sterile agar. The finished
medium reacts neutral or slightly
1. Nutrient agar.
2. Sputum. alkaline.
(b) Hammer specified the use of albumin Di. Digested by means of minced stomach.
urine from a nephritis patient. Not containing animal fluids.
El.
References: Finger, Ghon and Schlagen- Martin and Loiseau's Peptic Digest
haufer (1894 p. 14), Hammer (1895 p. 859). Agar 2035
2028. Eberson's Yeast Infusion Dujarric's Orange Juice Peptic
Agar
Digest Agar 2036
Constituents Stickel and Meyer's Peptic Digest
1- Water 100.0 cc. Agar 2037
Nutrient agar (1.0%, with
2.
Harvey's Basal Peptic Digest Agar. 2037a
2.0% peptone) 100.0 cc. Besredka and Jupille's Egg Stomach
3. Potassium phosphate (0.4%) 0.4 g. Digest Agar 2038
4. Yeast, bakers or brewers 10.0 g. E2. Containing animal fluids.
Preparation
Sellards and Bigelow's Veal Digest
(1) Macerate 10.0 g. of bakers or brewers Blood Agar 2039
yeast in 100.0 cc. of water for 20
Martin's Basal Stomach Digest Veal
minutes.
Infusion Agar 2040
(2) Steam the suspension for 2 hours at a
Harvey's Defibrinated Blood Digest
Agar 2041
(3) Clarify by adding Merck's dialized
Harvey's Pepsin Digest Serum Agar. 2042
iron (5.0% ferric hydroxide) and
Martin's Digest of Ascitic Fluid
filteringthru glass wool.
Agar 2043
(4) Add an equal amount of 1.0% agar
D2. Digested by means of pepsin.
containing 2.0% peptone and 0.4%
Harvey's Pepsinized Blood Agar. 2044
potassium phosphate to (3). . .
Deycke and Voigtlander's Pepsinized

(5) Adjust the reaction to pH 7.6 (pH 7.4
Meat Agar 2045
following sterilization).
Kammen's Pepsinized Yeast Agar
(Klimmer) 2046
Jensen's Milk Digest Agar 2047
15 pounds pressure.
Bacto Nutritive Caseinate Agar
Use: Culture medium for preserving stock
(Dehydrated) 2048
cultures of meningococci.
Bacto Peptonized Milk Agar (Dehy-
Reference: Eberson (1919 p. 852).
drated) 2049
SUBGROUP Co.* Containing tryptic digests only.
II-C. SECTION 7
Di.f Containing digests of casein or milk
Basal or complete media containing agar products.
and a digest or autolysate other than a
El. Additional constituents, if any, in-
commercial digest.
organic.
Ai. Digested by acids or bases.
Cole and Onslow's Trypsinized Ca-
Robinson and Rettger's Lactalbu-
sein Agar 2050
min Agar 2029 Teruuchi and Hida's Trypsinized
Robinson and Rettger's Casein Casein Agar 2051
Edestin Derivatives. Lactal- Norris' Trypsinized Caseinogen
bumin Agar 2030 Agar 2052
Robinson and Rettger's Casein
Norris' Simplified Caseinogen Agar. 2053
Product C Agar 2031 Teague and Deibert's Trypsinized
Robinson and Rettger's Casein
Casein Agar 2054
Product B Agar 2032 E2. Containing additional organic con-
Robinson and Rettger's Edestin
stituents.
Product Agar 2033 Sierakowski's Trypsinized Casein
Deycke and Voigtlander's Albu-
Lactose Agar 2055
minate Agar 2034
Harvey's Trypsinized Caseinogen
Ao. Digested by enzymes.
Blood Agar 2056
Bi.* Enzymes of animal origin employed.
Harvey's Blood Tryptamine Agar. 2057 .
Ci. Containing peptic digests only.

* See C3, page 651.
* See page 651 for Bj.
t See page 651 for Dj.
Teague and Deibert's Serum Tryp- Preparation

sinized Casein Agar 2058 (1) Heat of crude commercial
100.0 g.
milk albumin containing equal
Kulp and Rettger's Trypsinized
Casein Agar 2059 amounts of calcium phosphate and
Kulp and Rettger's Trypsinized protein for 2 to 3 hours with 200.0 cc.
Klim Agar 2060 of 10.0% HCl under reflex condenser.
D2. Not containing digests of casein or (2) Filter and heat residue with 200.0 cc.
milk products. 10.0% HCl until no Biuret is given.
El. Containing digests of mutton or other (3) Heat in open vessel to drive off HCl.
meats. (4) Neutralize with NaOH.
Harvey's Trypsinized Ox Heart (5) Filter.
Agar 2061 (6) Evaporate filtrate to sticky paste.

Norris' Trypsinized Mutton Agar. . 2062 This is lactalbumin. Add paste and
Harvey and Iyengar's Desiccated agar to water. Adjust to faintly
Trypsinized Mutton Bouillon alkaline with litmus. Sterilize.
Agar 2063 (Method not given.)

Harvey and Iyengar's Desiccated (7) Dissolve 20.0 g. of (6) and 15.0 g. agar
Bile Salt Trypsinized Mutton in 1000.0 cc. of water.
Agar 2064 (8) Make faintly alkaline to litmus.
Vardon's Desiccated Aronson's Sterilization: Method not given.
Agar 2065 Use: General culture media for pathogenic

Deycke and Voigtlander's Tryptic forms. Authors reported that 2.0% lact-
Meat Agar 2066 albumin gave best results; a 4.0% medium
Murray and Ayrton's Heart Infu- contained too much NaCl, due to neu-
sion Agar 2067 tralization of acid by NaOH.
Hottinger's Agar (Park, Williams Reference: Robinson and Rettger (1918

and Krumwiede) 2068 p. 220).
Gordon and Hines' Trypagar 2069

2030. Robinson and Rettger's Casein Edes-
E2. Containing other than meat digests. Lactalbumin Agar
tin Derivatives,
Duval and Harris' Tryptic Digest
Agar 2070 Constituents:
Distaso's Trypsinized Serum Agar. 2071 1. Water 1000.0 cc.
Stickel and Meyers' Trypsinized 2. Casein product C 25.0 g.
Blood Clot Agar 2072 3. Edestin product 2.5 g.
C3. Containing both tryptic and peptic 4. Lactalbumin product

digests. (paste) 2.5 g.
Stickel and Meyers' Tryptic and 5. Agar 15.0 g.
Peptic Digest Agar 2073 Preparation

Deycke and Voigtlander's Tryptic (1) Prepare casein product C as follows:
and Peptic Meat Digest Agar 2074 (a) Boil over free flame under a reflux
B2. Enzymes of plant origin employed. condenser for 8 hours, 50.0 g. crude
Kligler's Yeast Autolysate Agar... 2075 casein in 200.0 cc. 10.0% HCl.
van Steenberge's Yeast E.xtract (b) Distill solution by frequently add-
Agar 2076 ing water.
Jotton's Yeast Autolysate Agar 2077 (c) Neutralize residue with NaOH.
Couret and Walker's Autolyzed Tis- (2) Prepare edestin product by:
sue Agar 2078 (a) Heat 40.0 g. pure re-crystallized
edestin over a free flame with
2029. Robinson and Rettger's Lactalbumin 10.0% HCl under a reflux con-
Agar denser, until it no longer gives
Constituents Biuret test.
1. Water 1000.0 cc. (b) Heat for several hours in open dish
2. Lactalbumin 20.0 g. to drive outHCl.
3. Agar 15.0 g. (c) Neutralize with NaOH.
(3) Prepare lactalbumin product as 2. Casein product B 15.0 g.

follows: 3. Ag^r 15.0 g.
(a) Heat for 2 to 3 hours 100.0 g. crude Preparation
commercial milk albumin contain- (1) Boil over a free flame 40.0 g. of crude
ing equal amounts of calcium phos- casein with 200.0 cc. of 9.0% HCl
phate and protein, with 200.0 cc. under a reflux condenser.
of 10.0% HCl. (2) Heat in an open dish until no more
(b) Filter off residue of protein and acid vapor comes off.
heat with 200.0 cc. of 10.0% HCl (3) Evaporate to a thick paste. This is
until no Biuret is obtained. casein product B.
(c) Heat in open vessel to drive off (4) Dissolve 15.0 g. of (3) and 15.0 g. of
HCl. agar in 1000.0 cc. of water.
(d) Neutralize (3) with NaOH and (5) Make faintly alkaline to litmus by
filter.
the addition of litmus.
(e) Use part of filtrate as such and Sterilization: Not specified.
evaporate rest to paste. Use: General culture medium for patho-
(4) Dissolve 25.0 g. casein product C, genic forms.
2.5 g. Edestin product, and 2.5 g. lac- Variants
talbumin product paste, and 15.0 g. The
(a) casein product may be de-
agar in water. colorized with animal charcoal if
(5) Make faintly alkaline to litmus. desired.
Sterilization: Method not given. (b) Author added 5.0 g. of Liebig's meat
Use: General culture medium for patho- extract with 10.0 g. starch to the
genic forms. medium.
Reference: Robinson and Rettger (1918 Reference: Robinson and Rettger (1918
p. 220). p. 220).
2031. Robinson and Rettger's Casein

2033. Robinson and Rettger's Edestin
Product C Agar
Product Agar
Constituents
1- Water 1000.0 cc. Constituents:
2. Casein product C 30.0 g. 1- Water 1000.0 cc.
3. Agar 15.0 g. 2. Edestin product 50.0 g.
Preparation 3. Agar 15.0 g.
(1) Boil 50.0 g. of crude casein over a Preparation
free flame in 200.0 cc. of a 10.0% (1) Prepare edestin product as follows:
HCl solution under a reflux condenser (a) Heat over a free flame under a
for 8 hours. reflux condenser 40.0 g. pure re-
(2) Distill the solution by adding water crystallized edestin and 10.0%
frequently. HCl until a Biuret test is no longer
(3) Neutralize the residue by adding given.
NaOH. (b) Heat in an open dish to remove as
(4) Add 30.0 g. of (3) and 15.0 g. of agar much HCl as possible.
to 1000.0 cc. of water. (c)Decolorize with animal charcoal.
(5) Make slightly alkaline to litmus. (2) Dissolve 50.0 g. of (1) and 15.0 g. of
Sterilization: Method not given. agar in 1000.0 cc. of water,
Use: General culture medium for patho- (3) Neutralize by the addition of NaOH.
genic forms. Sterilization:Method not given.
Reference: Robinson and Rettger (1918 Use: General culture medium for patho-
p. 220). genic forms. The addition of meat ex-
tract improves the medium.
2032. Robinson and Rettger's Casein
Variants: The authors added 5.0 g. Liebig's
Product B Agar
meat extract to the medium.
Constituents: Reference: Robinson and Rettger (1918
1. Water 1000.0 cc. p. 220).
2034. Deycke and Voigtlander's Albuminate (5) Filter.
Agar (6) Distribute in sterile tubes to a depth

of 10 to 12 cm.
Constituents: Sterilization: Sterilize at 100 for 30 min-
Water 3000.0 cc.
1.
utes on 3 days or 115 for 30 minutes.
Use: Isolation of diphtheria bacillus.
3. NaOH (3.0%) 250.0 cc.
4. NaCl 7.5 g.
dium as follows:
(1) Mince finely fat-free veal.
6. Agar Add 500.0 g. of (1) to 1000.0 cc. dis-
(2)
Preparation tilled water or clear tap water, and
(1) Place 200.0 g. of finely chopped fat
heat for 20 minutes over a free flame
free horse meat in 3.0% NaOH solu-
at a temperature not to exceed 50C.,
tion (made by adding 30.0 g. of
or simply keep in a cool place over
fused, pure caustic soda to 1 liter of
night.
water) and grind fine.
Skim off the fat floating on the sur-
(3)
(2) Place in an Erlenmeyer flask and incu- face.
bate at 37C. for 24 to 30 hours until Boil 10 minutes.
(4)
solution is complete. thru wet, thick clean cloth.
(5) Filter
(3) Add HCl to the filtrate until neutral (6) Add 5.0 g. NaCl to the filtrate.
to litmus. Steam 45 minutes.
(7)
(4) Dilute to 3 liters with water. Make up the volume to 1000.0 cc.
(8)
(5) Add 7.5 g. NaCl, 150.0 g. glycerol
and make alkaline wth soda solution. (10) Steam 30 minutes.
Dissolve agar in (5) to solidify.
(6)
(11) Filter while hot thru well wetted
thick filter paper.
Use: Cultivation of parasitic and sapro- Distribute into flasks.
(12)
phytic forms. Add glucose for the culti- Mix 1000.0 cc. of (12) and 1000.0 cc.
(13)
vation of streptococci. Reitz used this of Martin's Stomach Digest Solution
medium in the bacteriological examina- (see medium 998, variant (c)).
tion of butter for diphtheria bacilli. Dissolve 30.0 g. glucose, 4.0 g. KNO3
(14)
Variants: The authors added 1.5 to 2.0%
and 6.0 g. agar in (13).
glucose.
References: Deycke and Voigtlander (1901 References: Martin and Loiseau (1916
p. 624), Reitz (1906 p. 723). Harvey (1921-22 p. 100).
p. 678),
2035. Martin and Loiseau's Peptic Digest

2036. Dujarric's Orange Juice Peptic Digest
Agar
Agar
Constituents
2. Beef 250.0 g. 1. Martin's Stomach Digest
Solution 75.0 cc.
3. Martin's Stomach Digest
2. Orange juice 125.0 cc.
Solution
4. Agar 8.0 g. 3. Agar
5. Glucose 15.0 g. Preparation:
6. KNO3 2.0 g. (1) Filter orange juice thru paper.
(2) Mix 125.0 cc. of (1) with 75.0 cc.
Preparation
Prepare an extract of 250.0 g. of beef Martin's bouillon. (See medium 998)
(1)
in 500.0 cc. water. Method not (3) Add agar to solidify as in the usual
given. way. (Method not given.)
(2) Mix 500.0 cc. of (1) with 500.0 cc. of (4) The reaction is acid. The reaction
Martin's bouillon (see medium 998). may be adjusted to any desired alka-
(3) To a liter of the mixture add 4, 5 and 6. linity by the addition of soda.
(4) After solution is complete, add the Sterilization: Not specified.
white of an egg and heat at 115 for Use: General culture medium.
30 minutes. Reference: Dujarric (1916 p. 843).
2037. Stickel and Meyer's Peptic Digest Carry out step (1) thru (6) as
Agar given above.
Constituents: (7) Sterilize at 10 pounds pressure for
1. Water, tap 4000.0 cc. 15 minutes in the autoclave or for
2. Pig's stomach, minced 400.0 g. 30 minutes at 100C. on 2 suc-
3. Liver, minced (beef pla- cessive days.
centa or blood clots) 400.0 g. (8) Inoculate (7) with 1.0% of a
4. HCl (Baker Chemical Co.). 40.0 g. 24 hour old broth culture of B.
5. K2HPO4 8.0 g. saccharolyte or B. coli and incu-
6. Agar 80.0 g. bate for 12-18 hours at 37C.
Preparation (9) Steam 20 minutes.
(1) Wash clean and mince fine five or (10) Adjust to desired reaction.
more large pigs' stomachs. Mix an (11) Add 5 and 6 to (10).
equal amount of clean pig or beef (12) Autoclave at 10 pounds pressure
liver, cheap fat-free beef, placenta for 45 minutes or heat in double
or blood clots. boiler to 100C. until the agar
(2) Mix one of 3, and 4 in 1 at 50C.
2, is dissolved.
and keep at 50C. for 18-24 hours. (13) Restore the volume lost by evap-
(3) Make a Biuret and trj-ptophane oration.
test. When both are + the digest (14) Adjust to slightly alkaline to lit-
is yellowish green and contains very mus or pH = 7.3 by using 2N
littleundigested debris. KOH or NaOH.
(4) Transfer to large bottles and steam (15) Cool to 60C. and add white of egg
for 10 minutes at 100C. to stop with crushed shells or for sake of
digestion. economy, ordinary beef or sheep
(5) Strain thru cotton or preferably serum in the quantity of 25-50 cc.
store over night in the ice chest and per liter.
decant after 24 hours. (16) Autoclave for one hour at 115C.

(6) Warm (5) to 70C. and neutralize (17) Filter thru cotton and distribute in
with 2N Na2C03 to litmus. 200.0 to 500.0 cc. quantities.
(7) Add 5 and 6 to (6). (18) Sterilize at 100 for 30 minutes on
(8) Autoclave at 10 pounds pressure for 2 successive days or at 10 pounds
45 minutes or heat in double boiler pressure' for 15 minutes.
to 100C. until the agar is dissolved. (b) Stickel and Meyer prepared a me-
(9) Restore the volume lost by evapo- dium as follows:
ration. (1) Obtain 10 liters fresh beef blood
(10) Adjust to slightly alkaline to litmus from the abattoir.
or pH = 7.3 bv using 2N KOH or (2) Decant and store the serum (which
NaOH. has separated on standing) in a
(11) Cool to 60C. and add white of egg refrigerator.
with crushed shells or for sake of (3) Weigh the blood clots and mix
economy, ordinary beef or sheep 100.0 g. with 1 liter tap water.
serum in the quantity of 25-50 cc. (4) Place mixture in an enameled pot,
per liter. bring slowly to a boil and boil
(12) Autoclave for one hour at 115C. slowly for 5 minutes, stirring con-
(13) Filter thru cotton and distribute in stantly.
200-500 cc. quantities. (5) Wash and mince pig's stomach.
Sterilization: Sterilize at 100 for 30min- (6) Cool (4) to 50 C. and add 100.0 g.
utes on 2 successive days or at 10 pounds minced pig's stomach for each
pressure for 15 minutes. liter of (4).
Use: General inexpensive culture medium. (7) Transfer to glass or porcelain
Variants receptacle and finally add 1.0%
(a) The authors prepared a sugar free HCl.
medium as follows: (8) Digest at 50C. for 18-24 hours.
(9) Make a biuret and tryptophane (8) Filter thru well-wetted thick filter
test, when both are +, the digest paper while hot.
is yellowish green and contains (9) Steam 30 minutes.
very little undigested debris. (10) Filter again thru well-wetted thick
(10) Transfer to large bottles and filter paper.
steam for 10 minutes to stop (11) Mince finely, fat free veal, and add
digestion. 500 g. to 1000 cc. water.
(11) Strain thru cotton or preferably (12) Incubate for 18 hours at 37C.
store over night in ice chest and (13) Pour the mi.xture on a thick clean
decant after 24 hours. cloth.
(12) Warm (11) to 70C. and neutralize (14) Collect the fluid, and that obtained
with 2N Na2C03 to litmus. by squeezing the cloth and its
(13) Filter into a flask. contents.
(14) Add 5 and 6 to (13). (15) Mix (14) with an equal volume of
(15) Adjust to desired reaction using pepsin digest solution (10).
litmus or preferably to a definite (16) Heat to 70C.
H-ion concentration (pH = 7.0 (17) Make the reaction neutral to litmus.
to 7.5). (18) Add 7 cc. of normal NaOH per liter.
(16) Clear (15) by adding 5-10.0% of (19) Filter thru well-wetted thick filter
the decanted beef serum. Steam paper.
45 to 60 minutes. (20) Add sufficient agar to give a solid
(17) Remove from steamer and
(16) medium.
allow the clot to form as a compact (21) Steam 30 minutes.
mass. Decant or better centri- (22) Filter while hot thru well-wetted
fuge the medium to remove it. thick filter paper.
(18) Sterilize at 100 for 30 minutes Sterilization : Method of sterilization of the
on 2 successive days. agar base not given.
Reference: Stickel and Meyer (1918 Added nutrients The author used the agar
:
pp. 79, 81). base in the following manner:

2037a. Harvey's Basal Peptic Digest Agar (A) Defibrinated blood.
(1) Mix one part defibrinated blood
Constituents: with three parts of distilled water.
1. Distilled water.
(2) Add 0.5 cc. of 10% NaOH to 100 cc.
2. Veal, fat free.
of (1).
3. Minced stomach. the autoclave at
(3) Sterilize (2) in
4. HCl.
112C.
5. Agar.
(4) Mix one part (3) with two parts
Preparation
sterile agar, base at 80C. under
(1) Clean and wash a number of pigs'
aseptic conditions.
stomachs.
(5) Slant.
(2) Mince (1) finely.
(B) Formalinized Serum.
(3) Add 10.0 cc. of strong hydrochloric
Add 1 cc. formalin to 500 cc. horse
(1)
acid to 1000 cc. water at 50C.
serum.
(4) Add 200 g. (2) to (3) and keep at
(2) Add 1% ammonia solution to (1)
50C. for 20 hours.
to neutralize to litmus.
(5) Raise the temperature to the boiling
(3) Mix one part (2) with two parts dis-
point.
tilled water.
(6) Pour the mi.xture on a thick clean
(4) Sterilize at 110C. for 15 minutes.
cloth. Collect the fluid and that ob-
tained by squeezing the cloth and (5) Mix one part (4) with three parts
its contents. sterile agar base.
(7) Heat the fluid to 80C. and make Use: Isolation and cultivation meningo-
faintly alkaline to litmus while at coccus.
80C. Reference: Harvey (1921-22, p. 74, 81).
2038. Besredka and Jupille's Egg Stomach (7) Heat to 70C.

Digest Agar (8) Make the reaction neutral to litmus.
Constituents : (9) Add 7.0 cc. N/1 sodium hydroxide
1. Distilled water per liter.
2. Beef or veal 750.0 g. (10) Filter thru well-wetted, thick filter

3. Egg paper.
4. Stomach (hog) (11) Dissolve 2.0% agar in (10).
5. Agar 2% (12) Steam 30 minutes
Preparation (13) Filter while hot thru well-wetted,
(1) Macerate 750.0 g. beef in 1200.0 cc. thickpaper.
filter
water. (14) Distribute the filtrate into flasks.
(2) Heat slowly at first and then boil for (15) Add one of the added nutrients.
30 minutes. Sterilization: Method of sterilization not
(3) Concentrate to one liter. given.
(4) Filter and dissolve 2.0 g. of agar for Use: Cultivation of meningococci.
every 100.0 cc. of filtrate. Added nutrients:
(5) Make slightly alkaline and boil for (a) Defibrinated blood. The blood was
25 minutes. prepared and added as follows:
(6) Filter and distribute in tubes. (1) Add three parts distilled water to
(7) Add 4.0 cc. of egg stomach digest one part defibrinated blood.
medium 1007 for prepara-
solution (see (2) Add 0.5 cc. of a 10.0% NaOH per
tion) each tube of sterile (6).
to 100.0 cc. (1).
(Ordinary peptone agar may be used.) (3) Sterilize in the autoclave at 112C.
Sterilization: Sterilize (6) at 115. (The alkali prevents coagulation.)
Use: Cultivation of gonococcus, whooping (4) Mix one part (3) with two parts
cough bacillus, pnemnococci, tubercle basal agar at 80C. The blood may
bacilli and other forms difficult to cul- be made just slightly alkaline to
tivate. litmus by the addition of HCI
Reference: Besredka and Jupille (1914 before the addition to the agar.
p. 577). (b) Serum.
(1) Add 1.0 cc. formalin to 500.0 cc.
2039. Sellards and Bigelow's Veal Digest horse serum.
Blood Agar Add 1.0% ammonia
(2) to (1) to neu-
Same as medium 1000 but solidified by the tralize to litmus.
addition of 2.0% agar. (3) Sterilize at 110C. for 15 minutes.
(4) Mix one part (3) with 3 parts
2040. Martin's Basal Stomach Digest Veal basal agar.
Infusion Agar (Harvey) (c) Serum (alkaline). Same as for (a)
Constituents above, but use serum instead of
1. Water 1000.0 cc. blood.
2. Veal 500.0 g. Variants: Harvey used the basal medium
3. Stomach digest solution 1000.0 cc. without any additions.
Preparation Reference: Harvey (1921-22 pp. 74, 81, 99).
(1) Mince finely fat-free veal.
(2) Add 500.0 g. to 1000.0 cc. water.
2041. Harvey's Serum Digest Agar
(3) Place 18 hours at 37C. Constituents:
(4) Pour the mixture onto a thick, clean 1. Distilled water.
cloth. 2. Veal, fat free.
(5) Collect the fluid which drains thru 3. Minced stomach.
the cloth together with that obtained 4. HCI.
by squeezing the meat in the cloth. 5. Formalin.
(6) Mix the fluid collected with an equal 6. Ammonia.
volume of stomach digest solution 7. Serum.
(see medium 998) 8. Agar.
Preparation Use: Isolation and cultivation of meningo-

(1) Clean and wash a number of pigs' coccus.
stomachs. Reference: Harvey (1921-22, p. 81).
(2) Mince (1) finely.
2042. Harvey's Defibrinated Blood Digest
(3) Add 10 cc. of strong hydrochloric
acid to 1000 cc. water at 50C. Agar
(4) Add 200 g. (2) to (3) and keep at Constituents:
50C. for 20 hours. 1. Distilled Water.
(5) Raise the temperature to the boiling 2. Veal, fat free.
point. 3. Minced stomach.
(6) Pour the mixture on a thick clean 4. HCl.
cloth. Collect the fluid and that 5. Defibrinated blood.
obtained by squeezing the cloth and 6. NaOH.
its contents. 7. Agar.
(7) Heat the fluid to 80C. and make Preparation
faintly alkaline to litmus while (1) Clean and wash a number of pigs'
at 80 C. stomachs.
(8) Filter thru well-wetted thick filter (2) Mince (1) finely.
paper while hot. (3) Add of strong hydrochloric
10 cc.
(9) Steam 30 minutes. acid to 1000 cc. water at 50C.
(10) Filter again thru well-wetted thick (4) Add 200 g. (2) to (3) and keep at
filter paper. 50 C. for 20 hours.
(11) Mince finely, fat free veal, and add (5) Raise the temperature to the boiling
500 g. to 1000 cc. water. point.
(12) Incubate for 18 hours at 37C. (6) Pour the mixture on a thick clean
(13) Pour the mixture on a thick clean cloth. Collect the fluid and that
cloth. obtained by squeezing the cloth and
(14) Collect the fluid, and that obtained its contents.
by squeezing the cloth, and its (7) Heat the fluid to 80C. and make
contents. faintly alkaline to litmus while at
(15) Mix (14) with an equal volume of 80C.
pepsin digest solution (10). (8) Filter thru well-wetted thick filter
(16) Heat to 70C. paper while hot.
(17) Make the reaction neutral to litmus. (9) Steam 30 minutes.
(18) Add 7 cc. of normal NaOH per liter. (10) Filter again thru well-wetted thick
(19) Filter thru well-wetted thick filter filter paper.
paper. (11) Mince finely, fat free veal, and add
(20) Add sufficient agar to give a solid 500 g. to 1000 cc. water.
medium. (12) Incubate for 18 hours at 37C.
(21) Steam 30 minutes. (13) Pour the mixture on a thick clean
(22) Filter while hot thru well-wetted cloth.
thick filterpaper. (14) Collect the fluid, and that obtained

Add formalin to 500
by squeezing the cloth and its con-
(23) 1 cc. cc. horse
tents.
serum.
(15) Mix
(14) with an equal volume of
(24) Add sufficient 1% ammonia solu-
pepsin digest solution (10).
tion to neutralize (23) to litmus.
(16) Heat
to 70C.
(25) Mix one part (24) with two parts dis-
(17) Make the reaction neutral to litmus.
tilled water.
(18) Add 7 cc. ofnormal NaOH per liter.
(26) Mix one part sterile (25) with three (19) Filter thru well-wetted thick filter
parts sterile (22). paper.
Sterilization: Method of sterilization of (20) Add sufficient agar to give a solid
(22) not specified. Sterilize (24) at medium.
110C. for 15 minutes in the autoclave. (21) Steam 30 minutes.
(22) Filter while hot thru well-wetted Sterilization: Not specified.
thick paper.
filter Use: General culture medium.
(23) Mix one part defibrinated blood Reference: Harvey (1921-22 p. 77).
with 3 parts distilled water.

(24) Add 0.5 cc. of 10% NaOH per 100 cc. 2045. Deycke and Voigtlander's Pepsinized
(23) to (23) . (This permits steriliza- Meat Agar
tion in the autoclave without coagu-
Constituents:
lation.)
(25) Mix one part sterile (24) with two
parts sterile (22) under aseptic
3. HCl (50.0%) 2.0 cc.
conditions.
4. Pepsin (Witte's) 3.0 g.
(26) Slant.
5. NaCl 8.0 g.
(22) not specified. Sterilize (24) at
7. Agar
112C. in the autoclave.
Preparation
Use: Cultivation of meningococcus, etc.
(1) Grind up 125.0 g. of lean horse meat
with 3.0 g. of pepsin and 400.0 g.
2043. Martin's Digest Ascitic Fluid Agar distilled water with 2.0 cc. of HCl.
(2) Place in an Erlenmeyer flask and
Constituents
incubate at 37C. until there is com-
1. Martin's digest veal infusion
plete solution.
agar 200.0 cc.
(3) Dilute to 2 liters, neutralize with
2. Ascitic fluid 100.0 cc.
NaoCOa and add 8.0 g. of NaCl.
Preparation
(4) Add 150.0 g. glycerol and sufficient
(1) Mix 5.0 cc. of 10.0% NaOH and
agar to solidify.
100.0 cc. of ascitic fluid.
(2) Make sterile cool (1) faintly alkaline
1-15
Use: Cultivation of parasitic and sapro-
to litmus by the addition of
phytic forms.
HCl.
Reference: Deycke and Voigtlander (1901
(3) Mix one part with
sterile (2) at 80C.
p. 625).
two parts sterile Martin's Digest Veal
Infusion Agar (Harvey) (see medium
2046. Kamen's Pepsinized Yeast Agar
2040).
(Klimmer)
Sterilization: Sterilize (2) and (3) at 112C.
in the autoclave. Method of sterilization Constituents
Water 1000.0 cc.
of agar not given. 1.
Use: General culture medium. 2. Yeast

Reference: Harvey (1921-22 p. 84). 3. NaCl 3.0 g.
4. Agar 22.0 g.
2044. Harvey's Pepsinized Blood Agar Preparation:
Constituents: (1) Autoclave 10,000.0 g. yeast with
1. Distilled water 1000.0 cc. 20 liters water for 2 hours at 1.5 to
2. Blood, ox 500.0 g. 1 atmosphere pressure.
3. Pepsin powder 15.0 g. (2) Allow to settle and decant the

4. Agar liquid.
Preparation (3) Add pepsin and about 10 liters of

(1) Mix 500.0 cc. of ox blood, 15.0 g. 0.5% HCl solution to the thick
powdered pepsin, 15.0 g. HCl and yeast residue from (2).
1000.0 cc. distilled water. (4) Incubate (3) for 5 days at 37C.
(2) Keep for 12 hours at 40 to 42C. (5) Allow to settle and decant the
(3) Boil 30 minutes. liquid or filter.
(4) Filter. (6) Neutralize the liquid by the addi-

(5) Make faintly alkaline to litmus. tion of NaOH.
(6) Solidify with agar. (7) Add the liquid from (2) to (6).
(8) Heat at 50C. until all the liquid has Preparation

evaporated. (1) Dissolve 27.0 g. of Bacto Peptonized
(9) Heat at 100 to 105C. for one hour. Milk Agar (Dehydrated) in 1000.0 cc.
(10) Pound to a powder. distilled water by boiling or auto-
(11) Dissolve 6.0 g. (10), 3.0 g. of NaCI claving.
and 22.0 g. agar in a liter of water (2) Restore lost weight if necessary.
by heating in the steamer. (3) If sterilized at 15 pounds pressure
(12) Add 10.0% soda solution until the for 20 minutes pH = 6.5.
reaction is 0.07% alkaline.
0.05 to Sterilization: Sterilize in the autoclave at
(13) Boil for another hour in the steamer. 15 pounds pressure for 20 minutes.
(14) Allow to stand over night at room Use: For bacteriological examination of
temperature. dairy products.
(15) Cut away the bottom of the agar Reference: Digestive Ferments Co. (1925
containing the sediment. p. 14).
(16) Melt the clear agar and flask.
Sterilization: Sterilize on each of 3 suc- 2050. Cole and Onslow's Trypsinized Casein
cessive days in the steamer for 1.5 hours. Agar
Variant:Klimmer used 10.0 g. (10), 8.0 g. Solidify medium 1130 with agar. The
NaCl, KNO3, 22.0 g. agar and 0.2 g.
0.1 g.
agar is added at step (18). (See me-
Na2C03 in a liter of water instead of the dium 1130.)
quantities given above.

Reference: Klimmer (1923 p. 171). 2051. Teruuchi and Hida's Trypsinized
Casein Agar
2047. Jensen's Milk Digest Agar
Constituents:
Same as medium 1112, but solidified 1. Water 1000.0 cc.
with agar. 2. Casein 100.0 g.
3. NasCOa (10.0% soln.)
2048. Bacto Nutritive Caseinate Agar
4. Pancreatin
(Dehydrated)
5. NaCl (0.5%)
Constituents 6. Agar (2.0%)
1. Distilled water 1000.0 cc. Preparation
2. Peptonized milk Bacto 7.0 g. (1) Dissolve 100.0 g. of pure casein in
3. Sodium caseinate 3.0 g. 1 liter of 0.8% NajCOa (anhydrous).
4. Agar 12.0 g. (2) Add 5.0 to 10.0 g. of pancreatin
Preparation: (Gehe and Co.) and shake with
(1) Dissolve 22.0 g. of Bacto Nutritive chloroform.
Caseinate Agar (Dehydrated) in (3) Place for 3 to 5 days in the incubator,
1000.0 cc. distilled water by boiling. shaking occasionally.
(2) If sterilized at 15 pounds for 20 min- Each day test the mixture for trj^pto-
(4)
utes pH = 6.5. phane, by adding a few drops of
Sterilization: Sterilize in the autoclave at acetic acid to a sample and then
15 pounds pressure for 20 minutes. adding bromine water.
Use: For differential and comparative milk (5) When the tryptophane content has
counts. reached its maximum and the tyro-
Reference: Digestive Ferments Co. (1925 sine has separated out as white
p. 13). clumps, heat the fluid for a short
time at 80C.
2049. Bacto Peptonized Milk Agar
(6) Filter and neutralize with a few cc.
(Dehydrated)
of HCl.
Constituents: (7) Heat moderately, in a vacuum if
1. Distilled water 1000.0 cc. possible, and evaporate to a syrupy
2. Peptonized milk 15.0 g. thickness. (Possibly may be fil-
3. Agar, Bacto 12.0 g. tered again.)
(8) Place in a mortar and knead it with (11) Add 2.5 g. NaCl, 0.125 g. of CaCls to
alcohol (amount not specified). each liter of the broth.
(9) Dry in a vacuum desiccator. (12) Sterilize at 115C. for one hour.
(10) Prepare a 2.0% agar solution in (13) Filter and place 100.0 cc. together
0.5% HCl. with 4.0 g. desiccated agar (prepared
(11) To 90.0 CO. of (10) add 4 to 5.0 g. of according to Cunningham, Ind.
(9) and exactly 10.0 cc. of a 10.0% J. Med. Res. April, 1919) into each
Na2C03 solution. round whiskey bottle.

(12) Mix thoroly. SteriUzation: Sterilize (13) at 120 for
(13) Boil for several minutes. two hours.

(14) Pour immediately into Petri dishes. Use: Substitute for meat in growth of
(15) Allow the Petri dishes to remain organisms used for vaccines, B. typhosus,
uncovered until the agar has B. para A and B, and B. cholerae. The
solidified. above quantities yield about 450 bottles.
(16) It is recommended to dry the agar Variants: Harvey prepared the medium as
plates in the incubator 1 to 2 hours follows
before use. (1) Add gradually 100.0 g. commercial
Sterilization: Not specified. casein to 500.0 cc. boiling water con-
Use: Isolation of cholera vibrio. taining 0.8% anhydrous sodium
Reference: Teruuchi and Hida (1912 carbonate.
p. 573). Note: Or double the quantity of
washing soda.
2052. Norris' Trypsinized Caseinogen Agar Raise the temperature to boiling
(2)
Constituents point.
1. Water 50,000.0 cc. (3) Add 500.0 cc. cold water.
2. Na2C03 commercial an- (4) Make the reaction faintly alkaline
hydrous 200.0 g. to litmus.
3. Caseinogen 10.0 lbs. (5) Allow to cool to 45C.
4. Pancreatic extract 2,000.0 cc. (6) Add 400.0 cc. pancreatic extract to
5. NaCl 125.0 g. the mixture.
6. CaCU 6.25 g. (7) Keep 4 hours at 37C.
7. Agar 2,000.0 g. (8) Raise the temperature to boiling
Preparation point.
(1) Boil 25 liters of water in a large tub (9) Pour the digest mixture on to a wet,
of about 70 liters capacity. thick clean cloth.
(2) Add 2. (10) Collect the fluid which drains thru
(3) Gradually shake in 3, and bring the the cloth together with that obtained
whole to the boiling point. by squeezing the cloth.
(4) Add 25 liters of cold water. (11) Filter the fluid collected thru well-
(5) Reaction should be alkaline to wetted, thick filter paper.

litmus. (12) Bring the volume up to 1000.0 cc.
(6) If temperature is not more than by the addition of water.
48C. add 2 liters of pancreatic ex- (13) Add 2.5 g. sodium chloride and
tract (prepared according to Cole, 0.125 g. calcium chloride.
Lancet, July 1, 1916). (14) Adjust the reaction.
(7) Estimate amino acids by Sorensen's (15) Steam 30 minutes.
method. while hot, thru well-wetted,
(16) Filter,
(8) Digest (6) for 3.5 to 4 hours at 37C. paper.
thick filter
(9) Then boil and thru muslin.
filter
(17) Sterilize in autoclave.
(10) Determine amount of amino-acids
(18) Add to 30.0 cc. sterilized casein
present again by Sorensen's method.
digest in large test tubes 4.0 g.
This should show an increase equiva-
N/10 NaOH desiccated agar.
lent to about 3.0 cc.
per 10.0 cc. broth. (19) Sterilize in autoclave.
(20) Add the sterilized solution of agar Add chloroform to prevent bacterial
in quantities of 30.0 cc. to casein growth.
digest already sterilized in quanti- (2) Boil (2) and filter thru cotton.
ties of 70.0 cc. in round quart (3) Add various amounts of sterile (2)
bottles. to an agar solution containing 20.0 g.
(21) Sterilize in autoclave. agar and 5.0 g. NaCl per liter.
Reference: Norris (1919-20 p. 541), Harvey Sterilization: Sterilize (2) in the autoclave.
(1921-22 p. 116). Sterilization of agar or final sterilization
not specified.
2053. Norris' Simplified Caseinogen Agar
Use: To study growth requirements of
Constituents Unna-Ducrey bacillus. Authors re-
1. Tap water (or saline) . 1000.0 cc. ported that the medium did not support
2. Caseinogen (10.0%).. 100.0 g. growth.
3. Washing soda (0.8%) . 8.0 g. Reference: Teague and Deibert (1922
4. Agar 20.0 to 40.0 g. p. 70).
5. Pancreatic extract
2055. Sierakowski's Trypsinized Casein
(0.5%) 5.0 cc.
Lactose Agar
Preparation
(1) Digest in an 0.8% aqueous solution Constituents
of washing soda, 10.0% of caseinogen 1. Water 1000.0 cc.
with 0.5% pancreatic extract at 2. Trypsinized casein (10.0%) 100.0 g.
37C. for 24 hours. 3. Lactose (1.0%) 10.0 g.
(2) Concentrate the products of digestion 4. Brom thymol blue 0.3 g.
on the water bath, yielding a paste 5. Agar (2.0%) 20.0 g.
of the consistency and color of Preparation
Liebig's meat extract. This may be (1) Prepare a tryptic digest of casein and
further dried in a desiccator over adjust to pH = 7.0, Method of
H2SO4 yielding a brittle resinous preparation not given.
mass which may be powdered. (2) Dissolve 10.0% (1), 1.0% lactose, 2.0%
(3) To 1000.0 cc. of saline or tap water agar and 0.3 parts per 1000 brom
add 100.0 g. (10.0%) of (2) to 40.0 g. thymol blue in water.
agar. Sterilization: Not specified.
(4) Adjust reaction to +10. Indicator Use: Isolation of colon-typhoid group.
not specified. Author reported that B. coli and lactose
Sterilization: Method not given. fermenters gave yellow colonies. Ty-
Use General culture medium. The author
: phoid and non-lactose fermenters gave
suggested the term "trypsinoids" for the blue colonies. Staphylococci and other
digest (2). B. typhosus grew well on organisms resisting the gram stain were
this medium. Brewer's yeast and fresh inhibited.
yeast were treated in like manner but Reference: Sierakowski (1923 p. 1003).
caseinogen was the more nutritive of
2056. Harvey's Trypsinized Caseinogen
the two.
Blood Agar
Reference: Norris (1919-20 p. 706).
Constituents
2054. Teague and Deibert's Trypsinized
Casein Agar
2. Blood (human or rabbit) 20.0 cc.
Constituents 3. Trypsinized caseinogen agar. 400.0 cc.
2. Casein (10.0%) 10.0 g. (1) Add 20.0 cc. sterile human or rabbit
3. Trypsin blood to 100.0 cc. distilled water in a
4. Agar solution flask furnished with a rubber cork
Preparation and containing glass beads.
(1) Prepare a 10.0% casein solution and (2) Shake vigorously to prevent forma-
digest with trypsin at 37C. for 8 days. tion of large fibrin masses.
Add 5.0 cc. pancreatic extract, 5.0 cc. (11) Mix one part (10) and two parts
(3)
enterokinase solution, and 1.5 cc. water.
chloroform. (12) Make distinctly acid to litmus by
Shake to mix. the cautious addition of strong
(4)
Incubate 8 days with daily shaking hydrochloric acid.
(5)
the first three days. (13) Add agar 2.0%,.
Remove the flask without disturbing (14) Steam 150 minutes.
(6)
the sediment. (15) Filter, while hot, thru well-wetted,
Pipette off for use, with sterile pre- thick, filter paper by placing filter
(7)
cautions, as much clear supernatant funnel, stand, and receptacle for
fluid as possible. filtrate in the steamer and steaming

till filtration is completed.
(8) Filter the remainder thru sterilized
filter paper. (16) Add per liter of melted agar at 65 C.,
Mix one part sterile (9) with four 50.0 cc. defibrinated sheep blood and
(9)
parts sterile trypsinized caseinogen the beaten up whites of 2 eggs.
agar (see Harvey's variant of me- (17) Steam 50 minutes.
dium2052) at 45C. (18) Strain the mixture thru a fine wire
Sterilization: Method of sterilization of sieve and squeeze the fluid out of
agar not given. the clot.

Use: Cultivation of B. influenzae. (19) Filter thru glass wool, taking care
Reference: Harvey (1921-22 p. 117). to keep the mixture hot during the
process.
2057. Harvey's Blood Tryptamine Agar (20) Adjust the reaction.
Constituents (21)^ Distribute in test tubes.
1. Distilled water 3000.0 cc. Sterilization: Sterilize 15 minutes at 100C.
2. Casein 100.0 g. on three successive days.

3. Agar 60.0 g. Use: General culture medium.
4. Blood, defibrinated 150.0 cc. Reference: Harvey (1921-22 p. 74).
Preparation
2058. Teague and Deibert's Serum Tryp-
(1) Prepare a suspension of one part
sinized Casein Agar
casein in 10 parts water in a well
stoppered bottle. Constituents:
(2) Shake well to break up clumps. 1. Water 100.0 cc.

Casein (10.0%) 10.0 g.
(3) Adjust the reaction if necessary 2.
with the help of cresol red. 3. Trypsin

Note: The optimum reaction for 4. Serum (Rabbit or sheep)
the tryptic digestion of casein is 5. Agar solution
about pH = 8.1 at which point Preparation
cresol red indicator solution gives a (1) Prepare a 10.0% casein solution
and
reddish violet color and phenol- digest with trypsin at 37 C. for
phthalein remains colorless. 8 days. Add chloroform to prevent
Add per liter 60.0 cc. pancreatic bacterial growth

(4)
extract and 5.0 cc. toluol. (2) Boiland filter thru cotton.
(5) Shake to mix. (3) Obtain sheep or rabbit serum from
(6) Digest at 39C. 10 days, with daily
freshly drawn defibrinated blood.
shaking and addition of more toluol (4) Heat minutes at 55C.
(3) for 15
if necessary. (5) various amounts of sterile (3), to

Add
(7) Add per liter, 100.0 cc. 7.5% hydro- an agar solution containing 20.0 g.
chloric acid. agar, and 5.0 g. NaCl per liter.
Steam 20 minutes. Sterilization: Sterilize (2) in the autoclave.

(8)
thru well-wetted, thick, filter Sterilization of agar, or final sterilization,
(9) Filter
paper. not specified.
(10) Make the reaction nearly neutral to Use: To study growth requirements of
litmus with 5.0% sodium hydro.xide. Unna-Ducrey Authors reported
bacillus.
that sheep serum medium gave no growth. (10) Distribute in quantities equal to
Remaining media gave good or excellent 10.0 g. of the original casein into
growth. flasks.
Variants: The author added various (11) When ready for use add water to
amounts of the following blood cell infu- make 1000.0 cc. to each sterile flask
sion to the medium prepared as given and dissolve 3.0 g. commercial meat
aVjove extract, agar to give the desired
(1) Add 2.0 cc. of red blood cells (obtained consistency, and 1.0% galactose in
by centrifuging defibrinated blood each flask.
(rabbit) and removing the serum) to Sterilization: Sterilize (10) in the auto-
10.0 cc. of normal NaCl solution. clave. Final sterilization not specified.
(2) Keep the temperature at 100C. for Use: Cultivation of Lactobacillus acid-
3 minutes. ophilus and Lactobacillus bulgaricus.
(3) Shake the tubes and allow to cool. The authors reported that any other
(4) Centrifuge and obtain the super- carbohydrate might be used instead of
natant fluid. galactose, but galactose favored the
Reference; Teague and Deibert (1922 growth of the organisms most. This me-
p. 70). dium is slightly inferior to a medium
prepared from klim powdered skim milk.

2059. Kulp and Rettger's Trypsinized See the following medium.
Casein Agar Variants: Any other carbohydrate may be
used instead of galactose.
Constituents:
Reference: Kulp and Rettger (1924 p. 363).
1. Water 2000.0 cc.
2. NajCOa (1.0%) 20.0 g. 2060. Kulp and Rettger's Trypsinized
3. Casein 200.0 g. Klim Agar
4. Trypsin (Fairchild's) 3.0 g.
Constituents:
5. Galactose (1.0%) 20.0 g.
1. Water 2000.0 cc.
2. NaoCOj (1.0%) 20.0 g.
7. Agar to solidify
3. Klim (powdered skim milk) 200.0 g.
Preparation
(1) Prepare a 1.0% solution of NaaCOs
5. Trypsin (Fairchild's) 3.0 g.
6. Galactose (0.5%) 10.0 g.
(2) Add 20.0 g. of granulated casein to
7. Agar to solidify
2 liters of boiling (1). Add the
Preparation
casein in small quantities at a time,
(1) Prepare a 1.0% solution of Na2C03.
and stir after each addition until
(2) Add 200.0 g. of klim (powdered skim
complete solution has taken place.
milk) to 2000.0 cc. of cold (1). Add
(3) Cool to about 40C.
the klim in small quantities and stir
(4) Add 10 or 12.0 cc. water containing
after each addition, until solution
3.0 g. of fresh Fairchild's trypsin.
is complete.
(5) Mix well and add 25.0 cc. chloroform.
(3) Heat to about 40C.
(6) Flask and incubate at 37C. for
(4) Add 10.0 to 12.0 cc. of water contain-
48 hours.
ing 3.0 g. of fresh Fairchild's trypsin.
(7) Neutralize to litmus by the addition Mix thoroly and add 25.0 cc. chloro-
(5)
of HCl. form.
(8) Heat double boiler to remove
in a
(6) Flask and incubate at 37C. for
the chloroform. 48 hours.
(9) Filter. chemically pure casein is
If (7) Add HCl to neutralize to litmus.
used the filtrate will be clear and (8) Hea: in a double boiler to remove the
have a bright amber color. Com- chloroform.
mercial casein makes a darker (9) Distribute into 100.0 cc. lots.
colored product due to the small (10) When ready for use add water to
lactose content. each sterile flask to make 1000.0 cc,
agar to give the desired consistency (16) Adjust the reaction.

and 0.5% galactose. (17) Steam 30 minutes.
Sterilization: Sterilize (9) in tlie autoclave. (18) Clarify and filter.
Final sterilization not given. (19) Bleed a rabbit directly from the
Use: Cultivation of Lactobacillus acid- carotid into 1.5% sodium citrate
ophilus and Lactobacillus bulgaricus. solution.
The authors reported that any other (20) Dilute with 0.85% sterile salt solu-
carbohydrate might be used instead of tion to give a 5.0% suspension of
galactose, but galactose favored the blood.
growth of the organisms most. This (21) Add 10.0% ether.
medium is slightly superior to preceding (22) Shake to mix.
casein digest medium. (23) Leave the sediment 24 hours.
Variants: Any other carbohydrate may be (24) Draw off the laked blood into a
used instead of galactose. The carbo- sterile bottle.
hydrate may be used in 1.0% instead of (25) Add an excess of ether.

0.5% concentration if desired. Sterilization: Final sterilization not spec-
Reference: Kulp and Rettger (1924 p. 363). ified.
Use: Cultivation of highly pathogenic
2061. Harvey's Trypsinized Ox Heart Agar organisms.
Constituents Variants: The author cultivated meningo-
1. Water 400.0 cc. cocci in a medium prepared as follows
2. Heart, ox (1) Steam 50.0 g. pea flour and 100.0 g.
3. NaCl 1.0 g. NaCl in 1000.0 cc. water for 30 min-
4. CaCl2 0.5 g. utes, with occasional stirring.
5. Blood (citrated laked) 10.0 cc. (2) Filter thru thick filter paper.
Preparation (3) Sterilize (method not given).
(1) Mince finely an averaged sized ox (4) Mix 5.0% of (3) with the agar ob-
heart. tained at step (18) in the medium
(2) Add to 400.0 cc. tap water. given above. 25.0% rabbit or horse
(3) Heat slowly to 75C. serum may be added if desired.
(4) Allow to cool to 45C. Reference: Harvey (1921-22 pp. 75, 77,
(5) Add trypsin solution to 1.0%. 114, 119).

Note: e.g. Liq. trypsin Co. (A
2062. Norris' Trypsinized Mutton Agar
and H).
(6) Place in incubator 150 minutes. Constituents
(7) Test for peptone by the Biuret test. 1. Water 2000.0 cc.
Note: Add 1.0 cc. 5.0% copper 2. Mutton, fat-free 2.0 lbs.
sulphate to 5.0 cc. trj^psin digest, 3. NaCl 5.0 g.
followed by 5.0 cc. N/1 potassium 4. CaCls 0.25 g.

hydroxide. Note the color change. 5. Agar 80.0 g.
If the color is pink, peptonization is Preparation

complete, if bluish purple incom- (1) Mince two pounds fat free mutton.
plete. (2) Place 2 liters of water and (1) in a
(8) Make faintly acid to litmus with 3 liter pot.

4.0% acetic acid. (3) Autoclave at 130C. for one hour.
(9) Boil 15 minutes. (4) Keep over night, but if urgently
(10) Allow the solid matter to settle. wanted, cool to 45C. and continue
Note: Or simply strain thru cloth. as below.
(11) Pour off the supernatant fluid. (5) Test amino acidity by Sorensen's
(12) Add: sodium chloride 1.0 g., calcium method.
chloride 0.5 g. (6) Add 40.0 cc. pancreatic extract per
(13) Make faintly alkaline to litmus. liter.
(14) Steam 45 minutes. (7) Digest four hours at 37C.

(15) Bring up to original volume. (8) Retest amino acid acidity.
(9) Filter thru muslin to remove fat. Keep (11) immersed for 1 to
Sterilization:
(10) Add 1.0 cc. glacial acetic acid per 2 hours in boiling water or in a steam
pot. sterilizer to bring the medium into
(11) Sterilize by heating up to 110C. solution and to sterilize.
and then cool. Use: Desiccated nutrient medium.
(12) Filter to remove mince. Variants: Vardon prepared a similar me-
(13) Make
slightly alkaline to litmus and dium as follows:
then add 2.5 g. NaCl and 0.125 g. (1) Wash a quantity of agar fiber in tap
CaCl2 per liter. water containing 0.25% acetic acid.
(14) Sterilize at 115C. for one hour. (2) Wash in several changes of tap water
(15) Filter thru paper. until the washings are neutral to lit-
(16) Put 100.0 cc. broth into each whiskey mus paper.
bottle. (3) Place the washed fiber on perforated
(17) Sterilize at 120C. for one hour. or wire gauze trays and cover with
(18) Add 4.0 g. desiccated agar to each thin muslin.
bottle. (4) Expose the agar in the trays to the
Sterilization: Sterilize (18) at 120C. for sun to dry.
one hour. (5) Prepare a double strength^ (composi-
Use: Growth of organisms for producing tion not given) tryptic digest of
vaccines. The quantities employed mutton.
yielded about 15 or 16 bottles. Authors (6) Filter thru paper and measure the
reported this medium inferior to medium filtrate.
2052 for vaccine production. (7) Adjust the reaction of the filtrate to
Reference: Norris (1919-20 p. 542). pH = 8.0.
(8) Add2 after this adjustment 25.0%

2063. Harvey and Iyengar's Desiccated more alkali of the strength used.
Tryptonized Mutton Bouillon Agar (9) Add 9.0%3 by weight of washed and
dried agar fiber.
Constituents:
(10) Place the mixture for one hour in
1. Trypsinized mutton 1000.0 cc.
the autoclave, at 120 C. to thoroly
2. Agar
dissolve the agar.
Preparation
(11) Allow the nutrient agar to solidify^
(1) Prepare a tryptic digest of mutton
in a metal funnel.
bouillon (method not given).
(12) Turn out the clear solidified mass
(2) Adjust (1) to pH = 8.0.
and cut into pieces.
(3) Cut up agar fiber into small pieces.
Pass thru a meat mincing machine
(13)
(4) Add (3) to (2). (6.0% by weight.)
with finely perforated outer disc.
(5) Autoclave for one hour at 120 to
(14) Spread the minced agar on metal
melt the agar.
trays.
(6) Filter thru cotton, wool and muslin
(15) Dry in hot air oven at a tempera-
into a tin receptacle.
ture not exceeding* 60C.
(7) Cut the agar out of the receptacle Remove as soon as the agar is thoroly
(16)
and into slices. dried.
(8) Paes slices thru meat mincing ma-
(17) Scrape the dried material off the
chine with a finely perforated outlet
trays and grind to powder.
disc.
(18) Replace the powder in the hot oven
(9) Spread minced nutrient agar on metal and complete the drying process.
or other type of trays.
(19) Record the final^ weight of the
(10) Dry in hot air oven or any other con- powder.
venient way. (20) Store in tight stoppered bottles.
(11) Store powder in sterile glass stop- Notes: Âll the constituents of
pered bottle. the ordinary tryptic digest bouillon
(12) Add 4.0% to 6.0% by weight of (11) (Douglas' medium) are doubled,
to water in test tubes or flasks. i.e.the meat, the liquid pancreatic
extract, the sod. chloride, and the powder to produce a single strength
calcium chloride, ^xhis addition medium. As a percentage this
was found by trial to be necessary would be given as 4.0% of powder
in order to give a final medium of to be added to the water.
pH = 7.0 to 7.2. Variations in the (22) Add the requisite amount of water.
mode of manufacture from that (23) Place in an autoclave for 30 minutes
here given would demand trials to at 120C. to dissolve the powder.
determine the exact amount of addi- (24) Distribute the liquid nutrient agar
tional alkali required. ^Pound to in test tubes in amounts of 7 to
be the requisite amount by trial. 10.0 cc.
^By keeping the agar at a tempera- autoclave 30 minutes

in the steamer for
120C. or
ture at which it remains fluid and at
allowing time for sedimentation, 20 minutes on three successive days.
undissolved matter may be collected (26) Slope the tubes.
at the extremity of the cone. This References: Harvey and Iyengar (1921-22
extremity may be cut off and rejected p. 365), Vardon (1923-24 p. 429).
after the agar allowed to solidify.

is
2064. Harvey and Iyengar's Desiccated Bile
In practice the undissolved material Salt Trypsinized Mutton Agar
may be left in the agar, for it collects
untimately in very small amount in Constituents
1. Tryptic digest of
the butt of the sloped agar medium
mutton 1000.0 cc.
and does not at all inferfere with
its transparency. Ât higher tem- 2. Agar 60.0 g.
peratures the medium is apt to 3. Sodium taurocholate. 5.0 g.

Neutral red (1.0%) ... 2.0 to 5.0 cc.
acquire a permanently brown colora- 4.
tion. 60n this weight taken in 5. Lactose 10.0 g.
conjunction with the volume of Preparation

(1) Prepare a tryptic digest
of mutton.
nutrient bouillon converted into
(2) Adjust (1) to pH
based the calculation of the
= 8.0.
agar is
(3) Cut up agar fiber into
small pieces.
amount of water which it is neces-
(4) Add (3) to (2). (6.0% by weight.)
sary to add to the powder in order
120 for an hour to
to give the medium required for use. (5) Autoclave at
melt the agar.
(21) Weigh out a quantity of dried powder
sufficient for the purpose in hand. (6) Filter thru cotton, wool and muslin
Note: This may be an amount to into a tin receptacle.
make a single test tube of medium (7) Cut the agar out of the receptacle
or several tubes. If the requisite
and into slices.
amount is weighed out with pre- (8) Pass slices thru meat mincing ma-
caution into each test tube sepa- chine with a finely perforated out-
let disc.
rately, or roughly measured out, the
sterilization required, if the test (9) Spread minced nutrient agar on
metal or other type of trays.
tubes are themselves sterile, need
not under ordinary circumstances (10) Dry in hot air oven or any other
convenient way.
be more than is required to dissolve
Store powder in sterile glass stop-
the agar in the water. As an exam- (11)
ple of the amount of powder required

pered bottle.
Dissolve 40.0 g. of (11) and 5.0 g. of
we may take it that, say 1,600.0 cc. (12)
from 3 in 1000.0 cc. distilled water by
of filtrate was obtained
heating in the steamer.
2,000.0 cc. of unfiltered bouillon and
Prepare fresh 1.0% solution of
that in dried powder this repre- (13)
neutral red.
sented 128.0 g. Then, we recollect
Add to (12) 0.2% to 0.5% of (13)
that the original bouillon is double, (14)
strength, it would require an addi-

and 1.0% lactose. Dissolve the lac-
tose by gentle heating. (Lactose
tion of 1,600.0 cc. water to 64.0 g. of
may be added in the form of a strong (16) Add, while liquid, 2.0 cc. 10.0%
sterilized solution and added to the freshly prepared sodium sulphite.
medium after sterilization.) (17) Pour plates or prepare slopes.
Sterilization: Sterilize 20 minutes at 100C. Sterilization: See step (15) above.
on each of 3 successive days. Use: Desiccated culture medium.
Use: Desiccated medium for colon typhoid Reference: Vardon (1923-24 p. 432).
group.
Reference: Harvey and Iyengar (1921-22 2066. Deycke and Voigtlander's Tryptic
p. 366). Meat Agar
Constituents
2065. Vardon's Desiccated Aronson's Agar
1. Water 3000.0 cc.
Constituents 2. Glycerol 190.0 g.
1. Tryptic digest of mutton. . 1000.0 cc. 3. Pancreas
2. Agar (9.0%) 9.0 g. 4. Meat 200.0 g.
3. Na2C03 (10.0% anhydrous 5. NaOH (3.0%) 300.0 cc.
solution) 12.0 cc. 6. Agar to solidify
4. Sucrose (20.0% solution) . . 10.0 cc. Preparation
5. Dextrin (20.0% solution) . . 10.0 cc. (1) Allow the finelj' chopped pancreas
6. Fuchsin, basic (sat. alcoholic of hogs to remain on ice for 24 hours.
solution) 0.8 cc. (2) Mix with 40.0 g. glycerol and
Preparation 160.0 cc. water.
(1) Add 9.0% washed fiber agar to (3) Press out the fluid from this mixture.
1000.0 cc. of double strength tryptic (4) Dissolve 200.0 g. meat in 300.0 cc. of
digest of mutton. (See variant of 3.0% NaOH solution.
medium 1715.) (5) Filter (4) and neutralize.
(2) Heat for 60 minutes at 120C. to (6) Add 0.25% NaaCOs (siccum) to (5).
dissolve the fiber agar. (7) Sterilize (method not given).
(3) Add 12.0 cc. 10.0% anhydrous so- (8) Add 50.0 g. of (3) to (7).
dium carbonate to the nutrient agar. (9) Incubate for 7 to 10 hours at 37C.
(4) Steam 15 minutes at 100C. (10) Neutralize with HCl.
(5) Add while hot 10.0 cc. 20.0%o sucrose, (11) Dilute with water to 3 liters.
10.0 cc. 20.0%, dextrin, and 0.8 cc. (12) Add 150.0 g. glycerol and the usual
saturated alcoholic solution of basic amount of agar to (11).
fuchsin. Sterilization: Not specified.
(6) Allow to solidify. Use: Cultivation of parasitic and sapro-

(7) Cut up the solidified medium and phytic forms. Authors reported that it
pass thru a meat-mincing machine was necessary to add glucose to culti-
with small outlet holes. vate streptococci. Reitz used this me-
(8) Spread the minced medium on metal dium in the bacteriological examination
trays. of butter for the detection of diphtheria
(9) Dry at a temperature not exceeding bacilli.
60C. References: Deycke and Voigtlander (1901

(10) Grind the medium when dried to a p. 626), Reitz (1906 p. 723).
powder.
(11) Weigh the powder obtair.^d. 2067. Murray and Ayrton's Heart Infusion
(12) Complete the drying at a tem- Agar
perature not exceeding 60C. Constituents:
(13) Record the final weight. 1. Distilled water 2000.0 cc.
(14) Add 100.0 cc. water to the requisite 2. Agar 40.0 g.
amount of (13). (See step (21) of 3. Heart, ox 1000.0 g.
variant of medium 2063.) 4. NaCl (0.25%) 2.5 g.
(15) Dissolve the powder by steaming 5. KCl (0.01%) 0.2 g.
15 minutes at 110C. 6. CaCl2 0.1 g.

Preparation (20) Filter while hot through paper.

(1) Mince fresh fat free ox heart muscle (21) Add 0.25% NaCl, 0.02% KCl, 0.01%
and suspend in twice its weight of CaClj and the desired amount of
distilled water in an open can in a (20) to (12),
steamer. (22) Adjust to pH =
7.2.
(2) Raise the temperature gradually to (23) Tube.

70-75C. and hold for three hours. Sterilization: Autoclave at 120C. for
(3) Raise the temperature to 100C. and 20 minutes.
keep at this temperature for Use: Cultivation of meningococci.
15 minutes. Reference: Murray and Ayrton (1924-25
(4) Pour the infusion and meat on a p. 49).
wetted butter muslin and filter until
2068. Hottinger's Agar (Park, Williams and
clear.
Krumwiede)
(5) This constitutes the stock infusion
and may be sterilized or used im- Constituents
mediately to prepare a 4.0% agar 1. Water 1500.0 cc.
solution. 2. Meat 750.0 g.
(6) Prepare an infusion as above, steps 3. NaaCOa 3.0 g.
(1) and (2). 4. Agar 1.5%
(7) Filter until clear thru the meat Preparation
particles suspended on well wetted (1) Free meat from fascia and cut in
butter muslin. finger thick pieces.
(8) Prepare an equal volume of 4.0% (2) Heat 1500.0 cc. of water to boiling.
agar solution in the stock infu- (3) Drop 750.0 g. of (1) into (2), piece
sion (5). by piece, stirring constantly.
(9) Cool (8) to 70C. and mix with (7). (4) Boil strongly and remove from the
(10) Heat in the steamer for one hour. fire.
(11) Allow to set and stand over night. (5) Remove the meat and run thru a
(12) Melt the following morning and chopping machine.
strain through lint. (6) Cool the water to 37C. and add
(13) Suspend the residual meat and fine 1.5 g. NaaCOa per liter.
coagulum from (4) and (7) in a (7) Put the chopped meat in 2 liter
quantity of N/100 HCl equal to the Erlenmeyer flasks, 550.0 g. per flask.
weight of the original raw meat. (8) Add (6) to each flask, filling the
(14) Raise to 100C. and autoclave at flask to the neck.
130C. (25 lbs. pressure) for 30 (9) Add 3.0 g. pancreatin, 10.0 cc. chloro-
minutes. form and 10.0 cc. toluol to each flask.
(15) Allow to cool to 37C., incubate 24 (10) Cork tightly and shake well.
hours, to test sterility. (11) Incubate at 37C. over night.
(16) Add 2.0% of sterile pancreatic ex- (12) Shake the next day and add more
tract and incubate for 5 to 15 hours. pancreatin unless the fluid shows a
(17) Add 0.8% anhydrous NaoCOs in the yellow color and particles of meat
form of a sterile 32.0% solution. look smaller.
(18) Allow digestion to continue until a (13) Continue digestion for four or five
Sorensen figure of not less than 20 days at room temperature, or for
isproduced. (Titrate the digest in two or three days in the incubator.
the presence of neutralized formalin, The meat should be finely divided
using phenolphthalein as an indi- at the end of this time.
cator, and express the result in cc. (14) Decant the liquid thru cheese cloth.
of N/10 NaOH required to neu- (15) Add anequal volume of water to the
tralize the amino acids in 10.0 cc. residue, shake well and again decant.
of the filtered digest.) (16) Place the meat on cheese cloth and
(19) Add 2.0% of N/10 HCl and auto- allow to drain.

clave. (17) Boil the filtrate for a few minutes.
(18) Filter thru absorbent cotton and (13) Mix acid and agar thoroly and leave
paper until clear. for 15 minutes.
(19) Mix the filtrate with an equal (14) Pour off the water and wash thoroly
amount of water. until the agar is free from acetic
(20) Solidify (19) by the addition of 1.5% acid.
agar in the usual manner. (15) Add the agar obtained from (14) to
Sterilization: Sterilize at 15 pounds pres- 1000.0 cc. of (10).
sure for 30 minutes. (16) Add 0.125 g. CaCU.
Use: General culture medium. (17) Autoclave for 45 minutes to dissolve
Reference Park, Williams and Krumwiede
: the agar.
(1924 p. 119). (IS) Neutralize to phenolphthalein by the
addition of N/10 KOH
while hot.
2069. Gordon and Hines' Trypagar (19) Cool to 60C. and add the white of
2 eggs beaten up with the crushed
Constituents shells.
1. Water (20) Autoclave again at 118C. for
2. Pea flour 100.0 g.
75 minutes or heat in the steamer for
3. NaCl 100.0 g.
2 hours.
4. Heart bullock 500.0 g.
(21) Filter.
5. Agar 20.0 g.
(22) Add 2.0% of sterile (3).
6. CaCl2 0.125 g.
Preparation Sterilization: Sterilize (23) in the ordinary
(1) Add a liter of water to a 100.0 g. of
way.
pea flour and NaCl.
100.0 g.
Use: Cultivation of meningococci. Wood
(2) Mix thoroly and steam for 30 min- used a similar medium for the cultivation
utes, stirring occasionally.
of diphtheria bacilli.
(3) Allow to settle and filter the super- Variants: Wood added 0.3 cc. of a sterile
natant liquid.
1.0% telluric acid solution to 10.0 cc. of
(4) Add 1000.0 cc. of water to 500.0 g. of the agar for the cultivation of diphtheria
finely chopped bullock heart and
bacilli.
make slightly alkaline to litmus by Reference: Gordon and Hines (1916
the addition of 20.0% KOH
solution.
p. 682), Wood (1921 p. 562).
(5) Heat (4) slowly at 75 to 80C. for
5 minutes. Duval
2070. and Harris' Tryptic Digest
(6) Cool to 37C. and add 1.0% liquor Agar
trypsinae (Allen and Hanbury's)
and incubate at 37C. for two and Constituents:
one-half to three hours. 1. Water 1000.0 cc.
(7) Test for peptone using the Biuret 2. Agar (3.0%) 30.0 g.
test. 3. Tryptic Digest Solution, . . . 1000.0 cc.
(8) Render slightly acid to litmus by the Preparation
addition of glacial acetic acid and (1) Prepare a 3.0% agar solution in water.
boil for 15 minutes. (2) Tube.
(9) Leave over night in a cool place and (3) Melt sterile tube of (2) and cool to
siphon off the clear liquid in the 45C.
morning. (4) Mix equal parts of the sterile agar
(10) Make faintly alkaline to litmus. with Duval and Harris' Tryptic Di-
(11) Weigh out 20.0 g. agar and cut in gest Solution (See medium 1134).
fine pieces with a scissors. (5) Slant and cool.
(12) Wash with water and drain

(11) Sterilization: Method of sterilization of
thoroly. Add
a sufficient quantity agar solution not given. The Tryptic
of water to cover the agar and add Digest Solution is sterilized by filtering
2.5 g. of glacial acetic acid per liter thru a Berkefeld filter.
of water. Use: Cultivation of leprosy bacillus.
Variants: If desired, glycerol, peptone, (6) Make the thick brownish fluid
salt, etc., may be added in the preparation slightly alkaline to litmus.
of this agar. (7) Add 1.0% pancreatic extract and
Reference: Duval and Harris (1911 p. 169). incubate at 37C. for 5-24 or 48 hours.
(8) When the process is sufficiently ad-
2071. Distaso's Trypsinized Serum Agar
vanced, render slightly acid with
Constituents glacial acetic acid and boil slowly
1. Water 1500.0 cc. 15 minutes.
2. Serum (beef or sheep). 500.0 cc. (9) Either filter or decant the clear
3. Agar 30.0 to 40.0 g. fluid which results on placing the
Preparation digest over night in a cool place.
(1) Mix equal parts (500.0 cc.) of water (10) Adjust the reaction as desired.
and sheep or beef serum. (11) Dissolve 3 and 5 in (10).
(2) Sterilize at 120C. for 15 minutes. (12) Adjust to desired reaction using lit-
(3) Digest for 24 hours at 60C. with a mus or preferably to a definite H-ion
pancreatic extract from a hog in the concentration (pH = 7.0 to 7.5).
presence of chloroform. Activate the (13) Clear (12) by adding 5-10.0% of the
extract with an extract of the upper decanted beef serum. Steam 45 to
portions of the small intestine. 60 minutes.
(4) Filter on paper. (14) Remove (13) from steamer and
(5) Add 30.0 or 40.0 g. of agar to 1000.0 cc. allow the clot to form as a compact
of water. mass. Decant or better centrifuge
(6) Sterilize (5). (Method not given.) the medium to remove it.
(7) Mix equal parts (4) and (6). Sterilization: Sterilize at 100 for 30 min-
(8) Tube. utes on two successive days.
Sterilization: Method of final sterilization Use: General inexpensive culture medium.
not given. Authors reported that this medium was
Use: Culture medium for tubercle bacilli, excellent for primary isolation of highly
strict anaerobes, B. proteus and others. parasitic organisms.
Reference: Distaso (1916 p. 600). Reference: Stickel and Meyer (1918 p. 81).
2072. Stickel and Meyers' Trypsinized

2073. Stickel and Meyer's Tryptic and
Blood Clot Agar
Peptic Digest Agar
Constituents:
Constituents
2. Blood clots 500.0 g. 1. Tap water 4000.0 cc.
K2HPO4 2.0 g. 2. Pig's stomach (minced) .... 400.0 g.

3.
10.0 g. 3. Beef, liver, placenta, blood
4. Pancreatic extract
20.0 g. clots (minced) 400.0 g.
5. Agar
Preparation
4. HCl (Baker Chemical Co.) . 40.0 g.
blood from 5. Pancreatic extract or

(1) Obtain 10 liters fresh beef
the abbatoir.
"Bacto" trypsin 40.0 g.
Decant and store the serum (which 6. K2HPO4 8.0 g.

(2)
has separated on standing) in a 7. Agar 80.0 g.
refrigerator. Preparation
Weigh the blood clots and mix (1) Wash clean and mince fine 5 or more
(3)
500.0 g. with 1 liter tap water. large pigs stomachs. Mince an
Place the mixture in an enameled equal amount of clean pig or beef
(4)
liver, cheap fat-free beef, placenta
pot, bring slowly to a boil and boil
slowly for 5 minutes stirring con- or blood clots.
stantly. (2) Mix 2, one of 3, and 4 at 50C. and
Strain fluid thru cheese cloth and keep at 50C. for 18 to 24 hours.
(5)
pass the residue thru a fruit press, (3) Make a biuret and tryptophane test.
cool to 37C. When both are + the digest is yel-
lowish green and contains very little (8) When the digest is sugar free add
undigested debris. 4.0 g. CaCOs and 80.0 g. agar.
(4) Transfer to large bottles and

steam (9) Autoclave at 10 pounds pressure for
for 10 minutes to stop digestion. 45 minutes or heat in double boiler to
100C. until the agar is dissolved.
(5) Cool to 80C. and make faintly
alka-
line to litmus using 2N KOH
or 2N Remainder of the preparation same
NajCOs. medium given above.
as
(6) Cool to 37C. and add 1.0% pan- Reference: Stickel and Meyer (1918 p. 80).
creatic e.xtract or "Bacto" trypsin.

(7) Keep the mixture at 37C. for 3 to 2074. Deycke and Voigtlander's Tryptic and
10 hours depending on the action of Peptic Meat Digest Agar
the trypsin and the digestion de-
Constituents
Control the process by re-
sired.
1. Distilled water to 1950.0 cc.
peated tests for tryptophane. 125.0 g.
2. Meat, horse
(8) When trypsinizing is sufficiently ad- HCl (5.0%) 2.0 cc.
3.
vanced render slightly acid with
4. NaoCOa (siccum) 3.7 g.
glacial acetic acid, and bring slowly
5. Pancreatin (Merck's gly-
to boiling point for 10 minutes. 15.0 cc.
cerol)
(9) Filter thru paper or keep in cool
6. NaCl 6.0 g.
place over night and
decant the
7. Glycerol
clear liquid in the morning.
8. Agar
(10) Add agar and K2HPO4 and autoclave Preparation:
at 10 pounds pressure for 45 minutes
(1) Grind up 125.0 g. of lean horse meat
or heat in double boiler to lOO^C.
with 3.0 g. pepsin and 400.0 cc.
until the agar is dissolved.
distilled water with 2.0 cc. of HCl.
(11) Restore the volume lost by eva-
(2) Place in an Erlenmeyer flask and
poration.
incubate at 37C. until there is
complete solution.
or pH = 7.3 by using 2N KOH or
Neutralize with NaaCO 3 (anhydrous)
(3)
NaOH. and divide into 3 portions.
(13) Cool to 60C. and add white of egg
(4) Sterilize each portion (exact method
with crushed shells or for sake of
not specified).
economy, ordinary beef or sheep
(5) Add to each portion with a sterile
serum, in the quantity of 25-50.0 cc.
pipette 5.0 cc. of Merck's glycerol
per liter.
pancreatin.
(14) Autoclave for one hour at 115''C.
(6) Keep one portion in the incubator 6
(15) Filter thru cotton and distribute in
hours, another 24 hours and the other
200-500.0 cc. quantities.
48 hours.
Sterilization: Sterilize at 100 for 30 min-
(7) Sterilize in streaming steam.
utes on two successive days or at
(8) Neutralize with HCl.
(9) Dilute each portion to 650.0 cc. and
Use: General inexpensive culture medium. NaCl to each portion.
add 2.0 g.
Variants: The authors prepared a sugar- and agar as usual
(10) Add glycerol
free medium as follows:
(amount not given).
(1) Carry out procedure as above steps
(1) thru (5).
Use: Cultivation of parasitic and sapro-
(6) Cool to 37C. and add 1.0% pan-
phytic Authors reported that
forms.
creatic extract or Bacto trypsin,
8.0 g. K2HPO4 and 1.0% of a culture some organisms developed better on the
of B. saccharolyte.
medium with the longer pancreatin
digestion, others required a shorter
(7) Keep the mixture at 37C. for 3 to
10 hours depending on the action of period.
the trypsin and digestion desired. Variants:

Control the process by repeated tests (a) Bosse cultivated diphtheria bacilli
for tryptophane. on a medium prepared as follows:
(1) Free horse meat from all fat and (2) Digest for two days at 37C.
tendons. (3) Filter, and if positive to the
(2) Chop intosmall pieces with a Biuret test add 3.9 g. dry NajCOs.
knife, a scraper or meat grinding (4) Add 15.0 cc. of a glycerol extract
machine until there are only very of a hog's pancreas to (3).
loose particles. (5) Incubate for 6 hours at 37C.
(3) To 125.0 g. of (2) add 3.0 g. of (6) Sterilize in the steamer.
fresh Witte-Rostock pepsin 400.0 (7) Neutralize with HCl.
cc. distilledwater and 2.0 cc. of a (8) Add 1950.0 cc. water, 6.0 g. NaCl
50.0% HCl solution. and 39.0 g. agar to (7) and boil for
(4) Place in an Erlenmeyer flask and 3 hours in the steamer.
incubate at 37 C. (9) Filter thru cotton.
(5) Incubate for 2 days, shaking oc- (10) Distribute in flasks.
casionally and adjusting the re- (11) Sterilize.
action with the addition of a 50.0% (c) Harvey gave the following method of
HCl solution. preparation:
(6) Filter the grey white precipitate Mix 150.0 g. of finely
(1) minced horse
which has formed at the end of heart, 5.0 g. pepsin, 2.0 cc. of
this time.
50.0% HCl and 400.0 cc. of distilled
(7) Test a portion of the filtrate by water.
the Biuret reaction (violet colora- Leave
(2) to digest 2 days at 37 C
tion with KOH and CUSO4) which (3) Filter.
must be positive. Add 40.0 cc. of glycerol
(4) and 160.0
(8) Add 3.9 g. of NaaCOs (siccum) to cc. distilled water to a finely
the filtrate. chopped pig pancreas.
(9) Sterilize (Method not given). (5) Infuse(4) for 3 days in an ice chest,
(10) Cut a hog's pancreas into small adding a small piece of camphor.
pieces with a knife, and place in Add
(6) 3.9 g. anhydrous NaaCOj and
the ice box for 24 hours. 15.0 cc. of (5) to the filtrate from
(11) Add 40.0 cc, of glycerol and 160.0
(3).
cc. distilled water, and extract for
(7) Leave 6 hours at 37C.
several days in the ice box. (8) Sterilize at 100C.
(12) Press out the juice and add a small (9) Neutralize with HCl.
piece of camphor to the liquid. (10) Add 1950.0 cc. water, 6.0 g. sodium
(13) Add 15.0 cc. of (12) to (9) by means chloride, and 39.0 g. agar.
of a sterile pipette.
(11) Steam 3 hours.
(14) Incubate at 37C. for six hours. (12) Filter.
(15) Sterilize immediately in the auto- (13) Distribute into test tubes and
clave, and neutralize with 50.0% flasks.
HCl. (14) Sterilize.
(16) Add 1950.0 cc. of water, 6.0 g. (d) Deycke (Klimmer) gave the follow-
NaCl, 39.0 agar to (15). ingmethod of preparation
(17) Boil in the autoclave for 3 hours. Mix 125.0
(1) g. of finely chopped
(18) Filter thru cotton and distribute horse meat with 400.0 cc. distilled
into little flasks. water and 3.0 g. of Witte's pepsin.
(19) Sterilize as usual (exact method (2) Add 2.0 cc. of 50.0% concentrated
not given). HCl.
(b) Bosse (Kolle and Wasserman) pre- (3) Incubate at 37C. for 48 hours,
pared the medium as follows: adding more HCl if necessary.
(1) Mix 150.0 g., of finely chopped (4) Filter.
horse heart with 3.0 g. of Witte's Add
(5) 3.9 g. water free soda to the
pepsin (fresh) 2.0 cc. of 50.0% filtrate.
HCl and 400.0 cc. of distilled (6) Sterilize (method not given).
water. Chop the pancreas
(7) of a hog into
small pieces, and store in the ice (6) Make up the volume to 1500.0 cc.
box for 24 hours. (7) Solidify with agar.
(8) Add 40.0 cc. of glycerol and 100.0 References: Kligler (1919 p. 186), Abt and
cc. water to (7) and place in the Blanc (1921 p. 452), Harvey (1921-22 p.
ice box for several days. 120).
(9) Filter the juice from (8).

Add 15.0 cc. of to and 2076. van Steenberge's Yeast Extract Agar
(10) (9) (6)
incubate at 37C. for 6 hours. Constituents
(11) Sterilize (method not given). 1. Yeast water agar 1000.0 cc.
(12) Neutralize with 5.0% HCl. 2. Esculin 1.0 g.
(13) Add 1950.0 cc. water, 6.0 g. NaCl, 3. Iron citrate 1-0 g.
and 39.0 g. agar, and prepare as Preparation
ordinary nutrient agar. (1) Dissolve 2 and 3 in yeast water agar.
References: Deycke and Voigtlander (1901 Sterilization: Not specified.
p. 624), Bosse (1903 p. 472), Kolle and Use: Cultivation of lactic acid bacteria.
Wass&rmann (1912 p. 413), Harvey (1921- Variants The author added 50.0 g. glucose.
:
22, p. 100), Klimmer (1923 p. 221). Reference: van Steenberge (1920 p. 841).
2075. Kligler's Yeast Autolysate Agar

2077. Jotten's Yeast Autolysate Agar
Constituents
Same as medium 983,and variant (b)
1. Yeast autolysate 1000.0 cc.
medium 983, but solidified by the addition
2. Agar 15.0 g.
of 2.2% agar.
Preparation
(1) Prepare yeast autolysate as in
2078. Couret and Walker's Autolyzed
medium 982, but do not filter.
Tissue Agar
(2) Add agar to (1) and stir thoroly to
soften agar thread or powder. Constituents
(3) Heat over a free flame to dissolve 1. Agar solution.
agar. 2. Tissue.
(4) Readjust the reaction to pH = 7.4. Preparation

(5) Heat on a water bath or in an Arnold (1) Collect bits of sterile liver, kidney
for ^ hour and decant the partially and brain of healthy rabbits, guinea
clear fluid to an Erlenmeyer flask or pigs, kittens and human placenta,
other vessel. in sterile flasks.
(6) Cool the agar (exact temperature not (2) Seal and place in the thermostat at
given), and add one whole egg to 40 C. for 10 to 20 days.
clear. (3) Titrate the fluid tissue to a neutral or

Steam in the Arnold for 30 to 45 slightly acid reaction (not exceeding
(7)
minutes. 1.5% acid) by the addition of N/1
(8) Filter and tube. NaOH.
Method not given.
Sterilization: (4) The agar is to be prepared in the
Use: General culture medium. usual manner (exact method not
Variants given) but is to contain no peptone,
(a) Abt and Blanc solidified medium 984 beef extract, etc.
with the addition of agar. (5) Adjust the reaction to neutral or to
(b) Harvey prepared a similar medium an alkalinity not to exceed 0.5%.

as follows: Tube and slant.
(1) Keep 100.0 g. yeast at 50C. for 24 (6) Distribute 0.1 to 0.5 cc. of (3) (auto-
hours or until liquefaction is com- lyzed tissue) over the surface of each
plete by autolysis. agar slant.
(2) Dilute with water to 400.0 cc. (7) Keep several hours in the horizontal
(3) Steam 30 minutes. position to allow the agar to absorb
(4) Adjust reaction. the tissue juice.

(5) Filter. (8) Incubate to determine sterility.
(9) The agar may be melted, and cooled Pinoy's Flax Seed Agar 2101
to 50C. and the tissue juice added. Omeliansky and Ssewerowa's Flax
In this case it is necessary to break Fiber Agar 2102
the surface of the slants before using. Heider's Wheat Agar (Klimmer)... . 2103
Sterilization: Not specified. Klimmer's Oat Agar 2104
Use: Cultivation of amoeba. Bacto Corn Meal Agar (Dehydrated). 2105
References: Couret and Walker (1913 p. C2.* Fruits or their derivatives used.
253), Stitt (1923 p. 51). Milburn's Basal Prune Agar 2106
Reed and Cooley's Prune Agar 2107
Bacto Prune Agar (Dehydrated) . . . 2108
Basal or complete media containing agar Dombrowski's Raisin Must Agar . . . 2109
with constituents of plant origin (exclusive Perold's Grape Juice Agar 2110
of digests) of unknown chemical composi- Richter's Meat Infusion Wine Agar. . 2111
tion. (Animal products may also be Jenkins' Tomato Infusion Agar 2112
present.) C3. Tubers or their derivatives used.
Ai. Containing bacteria, yeast, other fungi Di. Not containing animal products.
or their derivatives. Graham-Smith's Potato Agar 2113
Gassner's Yeast Autolysate Agar. . . 2079 Dawson's Potato Juice Agar 2114
Harvey's Yeast Extract Agar 2080 Gaehtgens' Potato Agar 2115
Beijerinck's Yeast Water Agar 2081 Bacto Potato Dextrose Agar (De-
Duval's Parasite Agar 2082 hydrated) 2116
Gassner's Asparagin Yeast Water Kellerman and McBeth's Potato
Agar 2083 Agar 2117
Emulsion Agar. 2084
v. Eisler's Bacterial Lubinski's Glycerol Potato Agar. 2118 . .
Lanken and Meyers' Fungus Infu- Picker's Glycerol Potato Juice

sion Agar 2085 Agar 2119
Avers and Mudge's Milk Powder Weinzirl's Sucrose Potato Water
Yeast Agar 2086 Agar 2120
A2. Containing flowering plants or their Rochaix's Carrot Agar 2121
derivatives. Schardinger's Hay Infusion Agar 2122
Bi. Non leguminous plants used. D2. Containing animal products.
Ci. Grains or their derivatives used. Bordet and Gengou's Potato Blood
Di. Malts, beerworts or their derivatives Agar (Roos) 2123
added.
C4. Non leguminous plants or their deriva-
Park, Williams and Krumwiede's
tives other than Ci to C3 used.
Basal Beer Wort Agar 2087
Schardinger's Straw Infusion Agar. 2124
Heinemann's Beer Wort Agar 2088
Reed and Cooley's Spinach Agar. . . 2125
Groenewege's Indican Malt Agar. . 2089
Owen's Cane Juice Agar
.
2126
Peklo's Malt Infusion Agar 2090
Owen's Molasses Agar 2127
Fulmer and Grimes' Malt Infusion
Le Fevre and Round's Cucumber
Agar 2091
Juice Agar 2128
Rettger, Reddish and McAlpine's
B2. Leguminous plants or derivatives em-
Malt Extract Agar 2092
ployed.
Tanner and Deck's Acid Near Beer
Agar 2093 Ci. Seeds used.
Janke's Lager Beer Agar 2094 Bacto Lima Bean Agar (De-
hydrated) 2129
Sobel's Lactose Beer Agar 2095
Stutzer's Basal Legume Seed (In-
Difco Wort Agar (Dehydrated) 2096
fusion) Agar 2130
D2. Materials other than Di.
Wyant and Tweed's Pea Agar 2131
Meacham, Hopfield and Acree's Corn
Kaufmann's Jequirity Seed Agar. . . 2132
Meal Agar 2097
Maze's Sucrose Bean Agar 2133
Buchanan's Silage Agar 2098
Reed and Cooley's Lima Bean Agar. . 2134
Reed and Colley's Corn Meal Agar.. 2099 .
Barlow's Sucrose Corn Agar

(Tanner) 2100 See C3 andC4.
Thorn's Bean Agar (Tanner) 2135 (2) Heat the mixture 20 minutes at a
Stutzer's Bean Infusion Agar 2136 temperature not exceeding 50C.
Vogel and Zipfel's Legume Flour (3) Steam 2 hours.
Agar (Klimmer) 2137 (4) Filter and refilter thru well-wetted,
C2. Materials other than seeds used. thick, filter paper.
Bacto Bean Pod Agar (Dehydrated) 2138 . (5) Mix two parts (4), cooled to 15C.
Didlake's Soy Bean Root Agar 2139 with three parts melted 2.5% agar
Zipfel's Legume Leaf Agar 2140 solution of a reaction pH = 7.4.
Simon's Legume Agar (Klimmer and (Agar may have peptone and NaCl
Kriiger) 2141 in it if desired.)
2079. Gassner's Yeast Autolysate Agar Use: Cultivation of meningococci. Harvey
Constituents: reported that with a semi-liquid agar
1. Water. (0.5%) viability can be preserved in stab
2. Yeast, Brewer's. cultures much longer than with a stiffer
3. Agar (3.0%). agar.
4. NaCl. Reference: Harvey (1921-22 p. 120).
Preparation
2081. Beijerinck's Yeast Water Agar
(1) Place about 10 liters of brewer's yeast
and wash with water. Allow
in a flask Constituents
to stand for 30 minutes and pour off 1. Water tap 1000.0 cc.
the liquid. 2. Yeast 80.0 g.
(2) Repeat the washing process until the 3. Agar 0.75 g.

wash water is no longer brown, but 4. Glucose 5.0 to 10.0 g.
slightly turbid. Washing five times 5. Chalk

is usually sufficient. Preparation:
(3) After pouring off the last water add (1) Boil 8.0 yeast in 100.0 cc. tap water.
18 liters of water to the remaining (2) Dissolve 0.75 g. agar and 5.0 to 10.0 g.
washed yeast cells. glucose in (1) by boiling.

(4) Boil in the autoclave or steamer as (3) Filter.
meat bouillon.
in the preparation of (4) Add a layer of pure chalk suspension.
(5) Allow to stand for a suitable length Sterilization: Not specified.
of time and remove the liquid from Use: To study the fermentation of glucose
the sediment or filter thru filter by bacteria.

paper. Reference: Beijerinck (1891 p. 782).
(6) This bouillon may be used exactly

2082. Duval's Parasite Agar
in the same manner as meat bouillon.
(7) To prepare agar dissolve 30.0 g. of Constituents
agar and 5.0 g. NaCl in 1000.0 cc. 1. Water 1000.0 cc.
of (6). 2. Agar
(8) Adjust to slightly alkaline to litmus. 3. Entameba coli
Sterilization: Not specified. 4. B. typhosus

Use: General inexpensive culture medium. Preparation :
Variants: The author added 0.3 or 0.5% (1) Prepare a solution of agar (amount
lactose. not given) in water.

Reference: Gassner (1916-17 p. 311). (2) Adjust to 1.0% alkaline to phenol-
phthalein.
2080. Harvey's Yeast Extract Agar Tube.
(3)
Constituents (4) Incubate the sterile slants of (3) with

1. Water 1000.0 cc. a mixture of Entameba coli and B.
2. Yeast (bakers or brewers) . . 100.0 g. typhosus.
3. Agar (2.5% solution) (5) Incubate at room temperature for 48
Preparation: hours.
(1) Mix one part bakers or brewers yeast Sterilization: Method of sterilization of
with ten parts water. (3) not given.

Use: Cultivation of B. leprae. Inoculate (5) Heat the emulsion for an hour and a
the slant with an emulsion prepared from half at 56C. or boil for one-half hour.
leprous tissue. The author reported that (6) Add from 1.0 to 3.0 cc. of the emulsion
the organism grew without the presence to each sterile tube of agar melted and
of B. typhosus. cooled to 50C.
Reference: Duval (1910 p. 653). Sterilization: Methodof sterilization of
agar not given. See step (5) for steriliza-
2083. Gassner's Asparagin Yeast Water tion of bacterial emulsion.
Agar Use: To study growth of bacteria on killed
Constituents bacteria. Author reported that B. coli
1. Water 900.0 cc.

grew only fairly well. Did not grow at
all on the sediment. Grew well on sta-
3. Agar 30.0 g.
phylococcus organisms, but poorly on the
4. NaCl sediment. Scant growth of B. coli on
5.0 g.
5. Asparagin B. typhosus Staphylococcus aureus
cells.
10.0 g.
6. Water blue (0.5%) 250.0 cc.
grew well on B. coli, typhosus or Staphy-
Preparation lococcusemulsion; B. typhosus grew on

(1) Prepare yeast water. Staphylococcus aureus emulsion, but very
Mix equal parts yeast water and poorly on B. coli or typhosus emulsions.
(2)
water. Variants: The author specified that the
(3) Dissolve 30.0 g. agar, 10.0 g. aspara- emulsion might be centrifuged for quite a
gin, and 5.0 g. NaCl in (2). long time and the supernatant, cell free
To each
80.0 cc. of (3) add 20.0 opalescent fluid be added to the agar, or
(4) cc.
0.5% solution of water blue.
of a the sediment be mi.xed with a volume of
Sterilization: Not specified. NaCl solution, equal to that removed
Use: To study the nitrogen requirements from the sediment, and added to the
for growth of the colon-typhoid and agar.
dysentery group. The author reported Reference: v. Eisler (1918 p. 197).
that asparagin was utilized as a nitrogen

2085. Lanken and Meyers' Fungus Infusion
source by the typhoid and dysentery
Agar
group as well as by Bad. coli.
Variants The author added 0.1, 0.5 or 1.0%
:
Solidify medium 1150 and variant (b)
glucose. medium 1150 by the addition of 2.0% agar.
Reference: Gassner (1917-18 p. 260). 2086. Ayers and Mudge's Milk Powder
Yeast Agar
2084. V. Eisler's Bacterial Emulsion Agar
Constituents:
1. Water 100.0 cc. 2. Skimmed milk powder 5.0 g.
2. NaCl (0.5%) 0.5 g. 3. Na2HP04-2H20 (Sorensen's
3. Agar 3.0 g. phosphate) 1 .0 g.
4. Bacterial emulsion 4. Yeast (dry fresh) 10.0 g.
Preparation 5. Agar (3.0% washed) 500.0 cc.
(1) Dissolve 3.0% agar in a 0.5% NaCl Preparation
solution. (1) Pour 5.0 g. skimmed milk powder
(2) The reaction is usually slightly into 20.0 cc. cold distilled water.
alkaline. Stir until solution is complete.
(3) Distribute in 5.0 cc. lots and sterilize (2) Dissolve 1.0 g. Sorensen's phosphate
(method not given). (Na2HP04.2H20) in 5.0 cc. distilled
(4) Prepare bacterial emulsions by wash- water by heating.
ing bacteria {B. coli, typhosus, and (3) Add (2) to (1), place at 30C. in a
Staphylococcus pyogenes aureus were water bath and heat to eO^C. in 10
used) from large 24 hour agar surfaces minutes. A flocculent grayish pre-
with 25 to 30.0 cc. salt solution. cipitate appears.
Steam until the precipitate turns Added nutrients The authors added 2.0%
:
(4)
white. ofany desired carbohydrate, alcohol, etc.
(5) Dilute about one-third with distilled Reference Park, Williams and Krumwiede
:
water and steam 5 more minutes. (1924 p. 134).
(6) Decant the solution and filter, keep-

ing most of the precipitate in the 2088. Heinemann's Beer Wort Agar
beaker until most of the liquid has Constituents
passed thru the filter.
(7) Wash the precipitate with a little
2. Agar 1.5% 15.0 g.
distilled water. The filtrate is
Preparation
cloudy. (1) Autoclave beer wort at 120 for 5
(8) Make up to 250.0 cc. by the addition minutes.
of distilled water. Cool.
(2)
(9) Steam 10.0 g. yeast in 100.0 cc.
(3) Filter.
distilled water for 45 minutes in an Dissolve 1.5% agar in the filtrate.
(4)
Arnold. Filter until filtrate is clear Clarify with the white of egg.
(5)
and brilliant. Sterilization: Not specified.
(10) Wash with 100.0 cc. distilled water. Use: Cultivation of molds, yeast, etc.
(11) Make up to 250.0 cc. with distilled Variants
water. (a) Dombrowski cultivated milk yeast
(12) Place 30.0 g. agar in 2000.0 cc.
in a medium prepared by dissolving
distilled water and let stand for 24 1.5%agar in wort.
hours. (b) Janke cultivated acetic acid bacteria
(13) Pour off the water and add an equal found in beers on a medium prepared
volume of fresh water. by dissolving 2.0% agar and 2.0%
(14) Allow to stand for 24 hours, pour on gelatin in hopped Lager beer wort of
cotton flannel cloth in a funnel and 12.75 Ballings, clarified with egg
wash with a liter of distilled water. white, filtered thru cotton, distributed
Press out as much water as possible. in 50.0 cc. lots and sterilized by the
(15) Add enough water to have the total discontinuous method in the steamer.
weight 1030.0 g. (c) Harvey cultivated trichophyta,
(16) Dissolve the agar by heating in the molds, yeasts, etc., in a medium
Arnold, and filter thru cotton flannel prepared by dissolving 1.5% agar in
until clear. beer wort diluted to a specific gravity
(17) Mix equal parts of (16) and (8). of 1.1.
(18) The usual pH = 6.7. References: Heinemann (1905 p. 128),
Sterilization: Method not specified. Dombrowski (1910 p. 380), Lohnis (1913 p.
Use: To determine the bacterial count of
88), p. 6), Harvey (1921-22
Janke (1916
milk. p. Klimmer (1923 p. 207), Park,
113),
Variants: Any desired indicator may be Williams and Krumwiede (1924 p. 134)
added.
Reference: Ayers and Mudge (1920 p. 581).
2089. Groenewege's Indican Malt Agar
2087. Park, Williams and Krumwiede's Constituents:
Basic Beer Wort Agar 1. Malt agar 100.0 cc.
Constituents 2. Indican (0.1%) 1.0 g.
1. Beer Wort 1000.0 cc. Preparation

2. Agar (1.5%) 15.0 g. (1) Prepare malt agar.
Preparation: (2) Add 0.1% indican to (1).
(1) Dissolve 2.0% of any of the added Sterilization: Not specified.
nutrients in beer wort (see medium Use: Cultivation of Phytobacter lycopersi-

1218) that has been solidified by the cum n. sp. (causing tomato rot). Author
addition of 1.5% agar. reported that the indican was de-
Sterilization: Not specified. composed.
Use: Cultivation of molds. Reference: Groenewege (1913 p. 24).
2090. Peklo's Malt Infusion Agar Preparation

Same as medium
1214 but solidified by the (1) Add 15.0 g. agar to 400.0 cc. malt
addition of 1.0% agar. drink (near beer) and 600.0 cc. dis-
tilled water.
2091, Fulmer and Grimes' Malt Infusion (2) Boil to dissolve the agar,
Agar (3) Tube.
(4) Just previous to use add 0.4 cc. of
Constituents: 5.0% sterile solution of lactic acid to
1. Distilled water 1150.0 cc. each sterile tube of (3).
2. Malt, distillers 360.0 g. Sterilization: Method of sterilization of
3. Agar (1.5%) 15.0 g. agar or lactic acid not given.
Preparation Use: Cultivation of yeast causing sore
(1) Mash at 55 C. 360.0 g. malt with throat probably Endomyces or Monilia.
1150.0 CO. distilled water for 24 hours. Reference: Tanner and Deck (1923-24
p
(2) Filter thru towelling, then thru filter 285).
paper.
(3) Heat for 30 minutes under 15 pounds 2094. Janke's Lager Beer Agar
pressure.
Constituents
(4) Allow to stand 3 days and filter.
1. Lager beer.
(5) Add agar.
2. Gelatin.
(6) Heat in autoclave 30 minutes at 15
3. Alcohol.
pounds.
4. Agar.
(7) Filter thru absorbent cotton and
Preparation:
tube.
Sterilization: Sterilize in live (1) Evaporate lager beer to one-half its
steam for 30
original volume by steaming.
minutes on 2 successive days.
Use: Growth of yeast Sacch. cerevisiae and (2) Add water to its original volume.
Torula sphaerica. (3) Add 2.0% agar and 2.0% gelatin to
(2).
Reference: Fulmer and Grimes (1923 p
(4) Clarify with white of egg.
586).
2092. Rettger, Reddish and McAlpine's (6) Make up to the original volinne by
Malt Extract Agar
(7) Distribute in 10.0 cc. lots in Freuden-
Constituents: reich flasks.
1- Water 900.0 cc. Sterilization: Sterilize on each of 3 succes-
2. Malt extract powder, Difco . 100.0 g. sive days in the steamer.
Preparation Use: Cultivation of acetic acid bacteria
(1) Dissolve 2 in 1. from beers.
(2) Reaction is about pH = 5.5 to 5.6. Reference: Janke (1916 p. 6).
Use: Isolation of yeast from feces. 2095. Sobel's Lactose Beer Agar
Authors reported that the reaction was
too acid for the development of bacteria.
Constituents:
Reference: Rettger, Reddish and McAlpine 1. Water 500.0 cc.

2. Beer 500.O cc.
(1924 p. 328).
3. Agar 15.0 g.
2093. Tanner and Deck's Acid Near Beer 4. Lactose 5.0 g.
5. Congo red 1.0 g.
Agar
Preparation
Constituents Boil 500.0
(1) cc. of beer, (preferably
1. Distilled water 600.0 cc. stale Pilsner beer) for 5 minutes.
2. Malt drink (near beer) 400.0 cc. Cool and bring to
(2) 1 liter volume by
3. Agar 15.0 g. the addition of water.
4. Lactic acid (5.0% soln.) Add
(3) 15.0 g. agar and allow to soak for
2 hours. Then boil until the agar has

dissolved.
(4) Add 5.0 g. of dry sterilized lactose to
boiling (3) and 1.0 g. of dry sterilized
Congo red.
(5) Boil vigorously for several minutes
and add the amount of water lost by
evaporation.
(6) Pour directly into sterile Petri dishes
if the medium is to be used at once.

Sterilization: If the medium is not to be
used at once sterilize in the steamer on
two successive days.
Use: Diagnosis of cholera, typhoid fever
and dysentery. The author gave the
following reactions:
ORaANISM
2. Corn meal 50.0 g. 2102. Omeliansky and Ssewerowa's Flax

3. Agar (1.5 or 2.0%) .... 7.5 to 10.0 g. Fiber Agar
(1) Allow 50.0 corn meal to simmer
g. of 1- Water iqqo.O cc.
in 500.0 cc. of water for two hours. 2. Flax fiber 50.0 g.
{Phaseolus lunatus, lime bean meal 3. Mannitol 20. g.
may be used instead of corn meal.) 4. Potassium phosphate 0.2 g.
(2) Strain thru a cloth. 5- Agar 15 og.
(3) Add 1.5 to 2.0% agar. Preparation
(4) Melt and filter thru cotton. (1) Heat 50.0 g. of flax at 115C. in the
Sterilization: Sterilize fractionally on three autoclave with a liter of water
successive days. (2) Filter.
Use: Cultivation of Heterosporiutn vari- (3) Neutralize to slightly alkaline re-
ahile. Authors reported this a very good action by adding NaoCOa.
medium for fungi. (4) Dissolve 3, 4 and 5 in (3).
Reference: Reed and Cooley (1911-12 p. Sterilization: Not specified.
49). Use: To study pigment
production of
azotobacter, Azotobacter
chroococcum.
2100. Barlow's Sucrose Corn Agar (Tanner) Authors reported that on the medium
given above brown pigment appeared
Constituents:
after ten days. The substitution of flax
1. Water 4000.0 cc.
extract for water increased the luxuriance
2. Corn 1000.0 g.
of growth of Azotobacter, Clostridium,
3. Sucrose 80.0 g.
Pasteurianum, Bac. cellulose, Nitrobac-
4. Agar 60.0 g.
ter and other forms.
5. Litmus 100.0 g.
Variants: The authors reported that using
Preparation
a medium prepared as above but sub-
(1) Stir 1000.0 g. of corn into boiling
stituting 20.0 to 30.0 g. of dextrin for
water.
mannitol, and adding 20.0 g. of chalk that
(2) Add 3, 4 and 5 to (1).
pigment was produced after 3 to 4 days.
(3) Boil 5 minutes.
The addition of the chalk favored the
(4) Heat in an autoclave and cool slowly.
production of pigment. Pigment more
The heavy matter settles off.
luxuriant in the variant than in the
(5) Draw ofT and tube the supernatant
medium given above.
agar.
Reference: Omeliansky and Ssewerowa
(1911 p. 645).
Reference: Tanner (1919 p. 50). 2103. Heider's Wheat Agar (Klimmer)
Constituents:
2101. Pinoy's Flax Seed Agar 1. Water 1000.0 cc.
Constituents 2. Wheat 500.0 g.
1. Water 1000.0 cc. 3. Agar (1.5%) 15.0 g.
2. Agar 20.0 g. Preparation
3. Flax seed 50.0 g. (1) Soak 500.0 g. of wheat in a liter of
Preparation: water for 24 hours.
(1) Prepare a medium from 1 liter of (2) Filter.
water, 20.0g. of agar and 50.0 g. (3) Dissolve 1.5% agar in the filtrate.
flaxseed. (Method not given.) (4) Neutralize to litmus.
(2) Heat at 117C. Sterilization: Not specified.
(3) Distribute in^culture apparatus. Use: Spore production by Anthrax bacilli.
Sterilization: Sterilize at 115C. for 15 Variants: Klimmer prepared a similar
minutes. medium as follows
Use Cultivation of Bacterium fimhriatum.
: (1) Macerate 500.0 g. of wheat with a
Reference: Pinoy (1907 p. 626). liter of water for 24 hours.
(b) tartaric acid 0.2 or0.5%.

(2) Filter.
(3) Make up the filtrate to a liter. The glucose seemed to suppress the
Dissolve 5.0 g. NaCl and 17.5 g. of conidia development. There was no

(4)
agar in (3). yellow color with either glucose or
Make slightly alkaline to phenol- tartaric acid. Color was dark green
(5)
phthalein. using tartaric acid.
(6) Sterilization not specified. (c) NH4XO3 0.5or 1.0%.

Reference: Klimmer (1923 p. 207). (d) KNOs 0.5 or 1.0%.
(e) K3PO4 0.5 or 1.0%.
2104. Klimmer's Oat Agar (f) K2HPO4 0.5 or 1.0%.

(g) KH2PO4 0.5 or 1.0%.
Same as the variant of medium 2103, but
(h) Dipotassium ammonium phosphate.
using oats instead of wheat.
(i) tyrosine.
(j) leucine.
2105. Bacto Corn Meal Agar (Dehydrated)
(k) oxamide.
Constituents: (1) legrimin.
1. Distilled water (m) casein.
2. Corn Meal Agar (Bacto).... 20.0 g. (n) peptone.
Preparation (p) blood serum.
(1) Dissolve 20.0 g. Bacto corn meal agar (q) meat extract.
(Dehydrated) in 1000.0 cc. distilled (r) asparagin.
water by boiling or autoclaving, (s) acetamide.
preferably the latter. Bacto corn (t) creatin.
meal agar ais synthetic medium (u) albumin.
representing the water soluble in- (v) glucose.
gredients of corn meal solidified by (w) nuclean.
the addition of 1.5% agar. Good growth and conidia were
(2) Distribute as desired. colored dirty green and greyish white
Sterilization: Sterilize for 20 minutes at 15 with tyrosine, asparagin, leucine and
pounds pressure. acetamide. Little growth and no
Use: General culture medium. color with oxamide. Creatin, legu-
Reference: Digestive Ferments Co. (1925 min albumin, casein and blood serum
p. 17). gave good growth a greenish and
yellow color. Remaining materials
2106. Milburn's Basal Prune Agar gave excellent growth of a yellow
Constituents: color.
1. Water 1000.0 cc. (x) glucose 5.0 to 40.0%.

2. Agar High concentrations of glucose
3. Prunes 1 lb. tended to slow up growth and no

Preparation color was formed.
(1) Soak 1.0 pound of dry prunes in 1 liter (y) Knops' nutrient solution 0.25, 0.5,
of water for 12 hours. 1.0, 2.0 or 5.0%.
(2) Filter thru a towel and then cotton. (z) Knops' nutrient solution 0.25, 0.5,
(3) Concentrate to a specific weight of 1.0, 2.0 or 5.0% + glucose (amount

1.06 to 1.08. of glucose not given).
Prepare media using 40.0% of the The presence of Knops' mineral salts
(4)
prune infusion, one of the added seemed to aid the conidia production.
nutrients and solidify with agar Hence more greenish color was pro-
(method not given) duced. More green was produced
Sterilization: Not specified. without dextrose than with it present.
Use: To study color formation. Reference: Milburn (1904 p. 133).
Added nutrients: The author added one Reed and Cooley's Prune Agar
2107.
of the following materials and gave the
following reactions Constituents
or 5.0%. 1. Water 600.0 cc.
(a) glucose 1.0, 2.0
2. Prunes 100.0 g. Preparation

3. Agar (1.5%) 9.0 g. (1) Dissolve 15.0 agar in 1000.0 cc.
g.
Preparation distilled water.
(1) Boil 100.0 g. of dried prunes in 600.0 (2) Add 10.0 g. of gelatin to (1).
cc. water. (3) Filter thru a towel in the steamer
(2) Filter. (requires about an hour).
(3) Dissolve 1.5% agar in (2). (4) To each 100.0 cc. of (3) add 6.0 cc.of
Sterilization: Sterilize by the fractional grape juice.
method in the steamer. Sterilization: Sterilize for one hour in the
Use: Cultivation of Heterosporium vari- steamer.
abile. Author reported that the fungus Use : Cultivation of bacteria from wine.
grew profusely on this medium and Reference: Perold (1909 p. 18).
bacterial contamination was easily
prevented. 2111. Richter's Meat Infusion Wine Agar
Variants Klimmer used 100.0
: g. prunes in Constituents
500.0 cc. of water. 1. Water 250.0 cc.
Reference: Reed and Cooley (1911-12 p. 2. Meat 250.0 g.
50), Klimmer (1923 p. 206). 3. Agar agar lO.O g.
4. Moselle wine 150.0 cc.
2108. Bacto Prune Agar (Dehydrated) 5. Gelatin (2.0%) 7.0 g.
1. Distilled water (1) Pour 250.0 cc. of water over finely
2. Bacto prune agar 24.0 g. chopped meat and allow to infuse
Preparation over night. (Temperature not
(1) Dissolve 24.0 g. of Bacto prune agar specified.)
in 1000.0 cc. distilled water by boiling (2) Add 150.0 cc. of Moselle wine
or autoclaving, preferably the latter. to 10.0 g. of shredded agar.
Bacto prune agar represents
a (3) Allow to stand several hours and
clarified infusion of the best grade of then heat to boiling in a water bath.
prunes, made solid by the addition of Heat until solution is complete.
agar. (4) Allow to stand over night (solidifica-
(2) Distribute as desired. tion takes place).
minutes at 15 (5) On the following morning, liquefy the
pounds pressure. agar by heating.
Use: General culture medium. (6) Neutralize (5) by the addition of
Reference: Digestive Ferments Co. (1925 NaoCOs solution.
p. 17). (7) Dissolve 2.0% gelatin in the infusion
from (1).
2109. Dombrowski's Raisin Must Agar (8) Mix liquid (6) and (7) and boil for
15 minutes.
Constituents
1. Raisin must (9) Filter thru a simple filter using a hot
1000.0 cc
2. Agar (1.5%) 15.0 g.
water funnel. (It may be necessary
to refilter the first 20.0 or 30.0 cc.)
Preparation
Dissolve 1.5% agar in raisin must. (10) Refilter thru the same filter until
(1)
all turbidity has disappeared.
Use: Cultivation of milk yeast. Sterilization: Not specified.
Use Culture medium for anthra.x, cholera,
Reference: Dombrowski (1910 p. 380). :
typhoid and chicken cholera bacilli.

Author reported that medium is slightly
2110. Perold's Grape Juice Agar
yellow but clear and transparent.
Constituents Reference: Richter (1887 p. 600).
2. Agar 15.0 g.
2112. Jenkins' Tomato Infusion Agar
3. Gelatin 10.0 g. Constituents
4. Grape juice 60.0 cc. 1. Water.
2. Tomato. that diphtheria bacilli colonies after 24

3. Agar. hours at 37''C. were either opaque or
Preparation transparent. Pseudo-bacilli formed me-
(1) Place small pieces of tomato

in a glass dium sized, round, whitish, opaque
jar and add sufficient water to just dome shaped colonies. Acid agar clear
cover the topmost layer. and opalescent. Alkaline agar clear
(2) Steam for 2 hours. but of a brownish color.

Variants: Thomas (Tanner) prepared a
(3) Filter.
(4) Add 1.6% agar to the filtrate. similar medium as follows:
(.5) Heat at 120C. for 30 minutes. (1) Wash, pare and slice potatoes.
(6) Add 40.0% NaOH to obtain a reaction (2) Heat one volume of (1) in two
of a minus 5 on the Eyres scale. volumes of water slowly for 2 hours.
and resterilize the medium in (3) Boil, after heating for two hours.
(7) Filter
the autoclave. (4) Filter thru cloth.
Add water to make up the loss due
(8) Readjust the reaction to a minus 5. (5)
Tube while hot. to evaporation.

(9)
Sterilization: Final sterilization not (6) Filter.
specified. (7) Add 1.0% shredded agar to the
Use: Cultivation of streptococci and filtrate.

Heat in the autoclave for 30 minutes
pneumococci. (8)
Variants: Green or canned tomatoes may at 15 pounds pressure.

be used instead of fresh red ones. (9) Filter, if desired, and tube.
Reference: Jenkins (1923 p. 116). (10) Sterilization not specified.

References: Graham-Smith (1904 p. 278),
2113. Graham-Smith's Potato Agar Tanner (1919 p. 61).
Constituents
1. Water 500.0 cc. 2114. Dawson's Potato Juice Agar
2. Potato pulp 500.0 g.
Constituents
3. Agar 30.0 g.
Water 1000.0 cc.
1.
Preparation
2. Agar 20.0 g.
(1) Wash and peel potatoes and crush
3. Potato juice 500.0 g.
in a mincing machine.
Preparation
(2) Add water in the ratio of 1.0 g. of and
(1) Use 500.0 g. unskinned potatoes
potato pulp to 1.0 cc. of water.
1000.0 cc. water and obtain potato
(3) Allow to stand in a flask for 12 hours.
juice.
Filter thru a filter paper.
(4)
(2) Free juice from starch (method not
(5) The medium may be left in the given).
acid condition or to give an alka-
(3) Dissolve agar in (2).
line medium, neutralize the acid
with N/1 caustic soda, using litmus
Use: To show bacterial variation of B. coli.
as the indicator and then adding
Reference: Dawson (1919 p. 142).
3.0 cc. of alkali per liter.
(6) Treat agar as usual (treatment not Agar
2115. Gaehtgens' Potato

specified) and add 3.0% to (4).
Place in steam sterilizer at 100C. Constituents
(7)
1. Water 1000.0 cc
until agar is dissolved.
Potato 500.0 g.
(8) When cool add white of egg and 2.
steam sterilizer.
clarify in the 3. Agar
(9) Filter thru Chardin filter paper.
4. NaCl
(10) Tube. Preparation
Sterilization: Sterilize on 3 successive days (1) Carefully wash and peel 500.0 g.
in steam at 100C. potatoes.

Differentiation of diphtheria and (2) Grind the potatoes fine by means of a
Use:
Author reported porcelain mortar. This is to be
diphtheria-like bacilli.
carried out under the water as far as To

(2) 100.0 g. of (1) add 800.0 cc. of water
possible. and steam for 30 minutes.
(3) Allow to stand for 3 hours at room (3) Filter thru cotton.
temperature. (4) Dissolve 3, 4, 5, 6 and 7 in 1000.0 cc. of
(4) Filter thru a clean towel. water.
(5) Heat at 100C. for one hour. Mix 500.0
(5) cc. of (4) and 500.0 cc. of the
filtrate from (3).
(7) Dissolve NaCl and agar (amounts not Dissolve 10.0
(6) g. of agar in (5).
given) in (6). Sterilization: Not specified.
(8) Adjust to the desired reaction by the Use: Cultivation of Bacillus flavigens,
addition of soda. Bacillus amylolyticus, Bacillus rossica.
Sterilization: Sterilize for one hour in the Variants: Tanner used 15.0 g. agar instead
autoclave (temperature or pressure not of 10.0 g.
given). References: Kellerman and McBeth (1912
Use: Inexpensive culture medium. Tanner (1919
p. 487), p. 60).
Variants: The potato water may be steri-
lized for an hour in the autoclave and 2118. Lubinski's Glycerol Potato Agar
kept until ready for use, and then add Constituents
the NaCl and agar as above. 1- Water 1500.0 cc.
Reference: Gaehtgens (1916 p. 46). 2. Potato 1000.0 g.
2116. Bacto Potato Dextrose
3. Glycerol 60.0 g.
Agar
4. Agar 15.0 to 20.0 g.
(Dehydrated)
Preparation
Constituents: Wash
(1) potatoes clean and cut into
1. Distilled water small pieces.
2. Potato infusion, dry 5.0 g. (2) Boil for 3 or 4 hours under a free
3. Glucose, Bacto 20.0 g. flame or in a steamer.
4. Agar, Bacto 15.0 g. Filter.
(3)
Preparation Add
(4) 1.0 to 1.5% agar.
(1) Dissolve 40.0 g. of Bacto Potato (5) Boil to dissolve agar and filter.
Dextrose Agar (Dehydrated) in (6) Add 4.0% glycerol.
1000.0 cc. of distilled water by boiling (7) Neutralize or may be used without
or autoclaving, preferably the latter. neutralization.
The dry potato infusion is prepared Sterilization: Not specified.
by infusing 50. parts dried potatoes. Use: Cultivation of tubercle bacilli.
(2) Distribute. Reference: Lubinski (1895 p. 126).
Sterilization: Sterilize at 15 pounds pres-
sure for 20 minutes. 2119. Picker's Glycerol Potato Juice Agar
Reference: Digestive Ferments Co. (1925 Constituents
p. 17). 1. Potato juice 1000.0 cc
2117. Kellerman and McBeth's Potato Agar
2. Agar (1.0%) lO.O g.
3. Glycerol (2.0%) 20.0 cc.
1- Water 1000.0 cc. Method of preparation
(1) of potato
2. Potato 100.0 g juice not given.
3. K2HPO4 l.Og. (2) Dissolve 1.0% agar in (1).
4. MgS04 l.Og. (3) Add 2.0% glycerol to (2).
5. NaCl l.Og (4) Reaction may be adjusted neutral,
6. (NH4)2S04 2.0 g. or slightly acid or alkaline.
7. CaC03 2.0 g. Sterilization: Not specified.
S. Agar 10.0 g. Use: Cultivation of tubercle bacilli. The
Preparation author reported that best growth was
(1) Peel, steam and mash a quantity of obtained on a neutral or slightly alkaline
potatoes. medium.
Variants: Klimmer gave the following Use General culture medium. The author
:
method of preparation: reported that the addition of glycerol

Thoroly wash and scrub medium favored the growth of some organisms
(1)
sized, sound unimpaired potatoes (tubercle bacilli).
with a scrubbing brush, soap and Variants

water. (a) The author added 10.0% glycerol to
Remove the peeling and wash in the agar.

(2)
running tap water. (b) Harvey prepared a similar medium as
Grind the potatoes to a pulp and press follows:

(3)
(1) Mince finely well washed carrots.
out the juice.
Dissolve 40.0 parts glycerol and 15 (2) Press out the juice from the minced
(4)
parts agar in 1000 parts (3) by carrots.
heating. (3) Mix one part water with four parts
Do not filter, clarify or neutralize. carrot juice.

(5)
(6) Tube. (4) Add agar to 3.0%.
(method not given). (5) Steam for 45 minutes.

(7) Sterilize
References: Ficker (1900 p. 509), Klimmer (6) Clear with white of egg.
(1923 p. 206). (7) Adjust the reaction.
(8) Filter.
2120. Weinzirl's Sucrose Potato Water Agar (9) Distribute into test tubes.
(10) Sterilize.
Constituents Harvey
References: Rochaix (1913 p. 604),
1. Water 800.0 cc.
(1921-22 p. 119).
2. Potato water 200.0 cc.
3. Sucrose 10.0 g. 2122. Schardinger's Hay Infusion Agar
4. Agar 20.0 g.
Constituents:
Preparation 1000.0 cc.
1. Water
(1) Mix 200.0 cc. of potato water and 800.0
2. Hay 30.0 to 40.0 g.
cc. water. 10.0 to 15.0 g.
3. Agar (1.0 to 1.5%) . . .

Preparation
(1) Boil 30.0 to 40.0 g. of hay with one
Use: Cultivation of molds. liter of water.
Reference: Weinzirl (1917 p. 441).
(2) Filter.
(3) Add 1.0 to 1.5% agar to (2).
2121. Rochaix's Carrot Agar
(4) Boil until agar is dissolved.
Constituents (5) Add NajCOa until the reaction ia
1. Water 800.0 cc. alkaline to litmus.
2. Carrot juice 200.0 cc. Sterilization: Method not given.
3. Agar 30.0 to 35.0 g. Use: Cultivation of protozoa. Wolbach

Preparation and Binger cultivated Spirocheta elusa
Wash carrots well, chop and press out (free living) on a similarly prepared
(1)
the juice. medium.
(2) Dilute 200.0 cc. of the carrot juice Variants Wolbach and Binger mixed equal
:
with 800.0 cc. water. parts of an unheated filtered hay infusion,
(3) Add 30.0 to 35.0 g. of agar to (2). see medium 1179, and a melted and cooled
(4) Heat in the autoclave for 20 to 25 3.0% agar jelly.

minutes. References: Schardinger (1896 p. 541), Wol-
(5) Clarify by the addition of egg white. bach and Binger (1914 p. 10).
Make alkaline (indicator not
(6)
2123. Bordet and Gengou's Potato Blood
specified).
Agar (Roos)
(8) Distribute into tubes. Constituents
or 110. 1. Distilled water 200.0 cc.
Sterilization: Sterilize at 108
2. Glycerol (4.0%) 8.0 g.
(Time not given.)
3. Potato 100.0 g. (4) Add

50.0 cc. of the potato juice to
4. NaCl (0.65) 150.0 cc. 150.0 cc. of (3).
5. Agar 5.0 g. (5) Tube in 2 to 3.0 cc. quantities.
6. Blood, defibrinated horse
Preparation When ready
(7) for use melt the agar
(1) Peel 100.0 g. potatoes, cut in small and add an equal quantity of whole
pieces and wash in running water for or defibrinated human or rabbit
2 hours. blood. (Temperature of the agar
(2) Add (1) to 200.0 cc. of distilled water not specified.)
containing 4.0% double distilled acid (8) Mix well.
free glycerol. (c) Harvey prepared a similar medium as
(3) Autoclave for 40 minutes. follows:
(4) Allow to stand over night and strain Grate finely
(1) washed, peeled
thru cheese cloth. potatoes.
(5) Mix 50.0 cc. (4), 150.0 cc. of a 0.65% (2) Add 100.0 g. to 100.0 cc. water.
NaCl solution and 5.0 g. agar. (3) Heat the mixture 20 minutes to a
(6) Steam in the Arnold sterilizer until temperature not exceeding 50C.
the agar is dissolved (30 to 60 (4) Raise to boiling temperature.
minutes). (5) Boil 10 minutes.
(7) Tube, do not filter. (6) Pour the mixture onto a clean,
(8) When ready for use, melt sterile thick cloth.
tubes of (7), cool to about 45C. and (7) Collect the fluid which drains thru
add the desired amount of sterile the cloth together with that ob-
defibrinated horse blood. tained by squeezing the cloth.
Sterilization: Method of sterilization of (8) Filter the fluid collected thru
agar not given. thick, filter paper.
Use: To maintain stock cultures of menin- Add the filtrate to an
(9) equal
gococci. Author reported that trans- quantity of distilled water.
plants must be made every two weeks. (10) Steam 60 minutes.
Incubate at 37.5C. If tubes are par- (11) Add glycerol to 4.0%.
affined fair growth after 6 weeks. Gono- Mix well.
(12)
cocci grow on this medium when blood (13) Filter.
content high and salt content dimin- Mix
(14) 10 parts (13) with 30 parts
ished. Other authors cultivated the 0.6% NaCl solution and one part
whooping cough bacillus, influenza bacil- agar.
lus, etc., on similar media. (15) Sterilize.
Variants
(a) Bordet and Gengou prepared the (d) Povitzky prepared the medium in the
medium as indicated above. They same manner as Bordet and Gengou
distributed the agar in 2.0 or 3.0 cc. using 2.0% glycerol as a final con-
lots and added an equal volume of centration instead of 1.0% and used
sterile defibrinated rabbit or human defibrinated or citrated horse blood
blood to the sterile agar. instead of defibrinated rabbit or
(b) Bezangon prepared the medium as human blood.
follows: (e) Park, Williams and Krumwiede pre-
(1) Add 100.0 g. of chopped potatoes pared the medium as follows:
to 200.0 cc. of a 4.0% glycerol Add
(1) 500.0 g. sliced potatoes and
solution and boil in the autoclave 80.0 cc. glycerol to 1000.0 cc. water.
(time not given). (2) Heat in the autoclave at 15 pounds
(2) Separate the liquid from the pota- pressure for 30 minutes.
toes (method not given). (3) Pour off the liquid.
(3) Dissolve 6.0 g. NaCl and 50.0 g. C4) To 500.0 cc. of the potato extract,
of agar in a liter of water in the add 1500.0 cc. of a 0.6% salt solu-
autoclave. tion and 60.0 g. agar.
Autoclave to dissolve. (2) After it is well steeped filter and add

(5)
(6) Filter.
2.0% agar.
Tube. (3) Dissolve the agar and filter thru
(7)
in the autoclave for 30 cotton.
(8) Sterilize
minutes at 15 pounds pressure. by the intermittent
When ready for use add an equal

(9)
method using the steamer.
quantity of blood, or more often, Use: Cultivation of Heterosporium vari-
one part blood to four parts agar. abile. The authors reported that this
References: Roos (1916 p. 68), Bordet and was a good medium for the production of
Gengou (1920 p. 734), Bezanôn (1920 p. spores for the hanging drop cultures,
121), Harvey (1921-22 p. 73), Klimmer since the fungus grew very short aerial
(1923 p. 206), Povitzky (1923 p. 8), Park, hyphae.
Williams and Krumwiede (1924 p. 122). Reference: Reed and Cooley (1911-12 p.
50).
2124. Schardinger's Straw Infusion Agar
2126. Owen's Cane Juice Agar
Constituents:
2. Straw 30.0 to 40.0 g. 1. Cane juice (raw) 1000.0 cc.
3. Agar (1.0 to 1.5%)... 10.0 to 15.0 g. 2. Agar (2.0%) 20.0 g.
Preparation: Preparation
(1) Same as medium 2122 but substituting (1) Heat fresh raw cane juice.
straw for hay. (2) Filter thru cotton.
Sterilization: Method not given. (3) Solidify with 2.0%, agar.
Use: Cultivation of protozoa and amoeba. Sterilization: Not specified.
Variants: Use: Bacterial count in cane sugar
(a) Tsujitani prepared a similar medium products.
as follows: Reference: Owen (1914 p. 337).
(1) Cut straw real fine.
(2) Boil a mixture of 30.0 g. (1), 10.0 g. 2127. Owen's Molasses Agar
Gigartina prolifera (Tsunomato)
Constituents:
and 1000.0 cc. water in a Koch
1. Water 1000.0 cc.
steamer for one hour.
2. Molasses (Final 75 Brix) . . . 160.0 g.
(3) Filter thru a fine cloth.
3. Agar 20.0 g.
(4) Add 1.0% to 1.5%, agar and 10.0 cc. Preparation
normal soda solution. final molasses 75
(5) Boil for 30 to 40 minutes.
Brix and 20.0 g. agar in 1000.0 cc. of
water.
sterilize (method not given).
(b) Harvey solidified 1000.0 cc. of a straw
Use: Bacterial count in cane sugar
decoction with 15.0 g. agar. (See
products.
medium 1178 for the preparation of a
straw decoction.)
References: Schardinger (1896 p. 541),
2128. Le Fevre and Round's Cucumber
Tsujitani (1898 p. 667), Harvey (1921-
Juice Agar
22 p. 121).
Constituents
2125. Reed and Cooley's Spinach Agar 1. Cucumber juice 1000.0 cc.
Constituents: 2. Agar (amount not given)

1. Water 500.0 cc. 3. NaCl 100.0 g.
2. Spinach 100.0 g. Preparation

3. Agar (2.0%) 10.0 g. (1) Method of obtaining cucumber juice
Preparation not given.

(1) Steep 100.0 g. of young spinach leaves (2) Dissolve 2 and 3 in (1).
in 500.0 cc. of water. Sterilization: Not specified.

Use: Cultivation and isolation of halo- 2131. Wyant and Tweed's Pea Agar
philic bacteria. Authors reported that
Constituents:
organisms grew just as well on meat
extract with 5.0% salt.
1- Water 4000.O cc.
Reference: Le Fevre and Round (1919 2. Peas (canned) 1000.0 g.
p 3- Agar
178). qqq g
4. Litmus
Preparation
2129. Bacto Lima Bean Agar (Dehydrated)
(1) Boil 3 in 1 until the agar is dissolved.
Constituents (2) Make up to water lost by boiling.
1. Distilled water (3) Mash 1000.0 g. canned peas in their
2. Lime bean infusion (dry)... 8.0 g. juice and add to (2).
3. Agar, Bacto 15.0 g. (4) Autoclave for 30 minutes at 15 pounds
Preparation pressure.
(1) Dissolve 23.0 g. of Bacto Lima Bean (5) Allow the agar to cool slowly. This
Agar (Dehydrated) in 1000.0 cc. of allows the hulls to sink to the bottom.
water, by boiling or autoclaving, (6) Cut away and discard the sediment
preferably the latter. The dry lima after the agar has solidified.
bean infusion is prepared from an (7) Melt the remaining clear agar and
infusion of 50 parts dried lima beans. color with litmus.
(2) Distribute as desired. Sterilization: Sterilize in the autoclave.
Sterilization: Sterilize by autoclaving for Use: Cultivation of organisms causing flat
20 minutes at 15 pounds pressure. sour in peas.
Use: General culture medium. Reference: Wyant and Tweed (1923 p. 12).
p. 17). 2132. Kaufmann's Jequirity Seed Agar
Constituents
2130. Stutzer's Basal Legume Seed 1- Water lOO.O cc.
(Infusion) Agar 2. Jequirity seeds 10.0 g.
Constituents 3. Agar 1.5 to 2.0 g.
1. Distilled water 1000.0 cc. Preparation
2. Legume seed 10.0 g (1) Grind 10.0 g. Jequirity seeds in a
3- Agar 10.0 g. mortar.
Preparation (2) The peeled or shelled seeds now weigh
(1) Same as for medium 1166, but solidified about 8.0 g.
by the addition of 1.0% agar. (3) Add to 100.0 cc. of water.
Sterilization: Not specified. (4) Boil in a steam sterilizer for about 2
Use: Cultivation of nodule bacteria. hours.
Variants Zipfel prepared a similar medium
: (5) Cool and filter.
as follows (6) Dissolve agar in (5).
(1) Mix powdered legume seed
100.0 g. of (7) Reaction is neutral or slightly
with 100.0 cc. N/1 KOH in a mortar. alkaline.
(2) Add 5 liters of water and allow to (8) Distribute into test tubes.
stand for 24 hours. Sterilization: Sterilize in the usual manner
(3) Pour off the clear supernatant fluid. (Method not given).
(4) Neutralize with H3PO4 and make up Use: Cultivation of Bacillus pyocyaneus
to 5 liters volume. and many others. Author reported that
(5) To 1 liter of (4) add 30.0 g. of agar and the medium supported the growth of a
20.0 g. dextrose. great variety of organisms.
(6) Acidify by the addition of 10.0 cc. Reference: Kaufmann (1891 p. 66).
N/1 malic acid.
(7) Sterilization not specified. 2133. Maze's Sucrose Bean Agar
References: Stutzer (1900 p. 898), Zipfel Constituents
(1911-12 p. 10). 1. Water 1000.0 cc.
2. Beans (white) 2137. Vogel and Zipfel's Legume Flour Agar

3. Sucrose (2.0%) 20.0 g. (Klimmer)
4. NaCl (1.0%) 10.0 g.
Constituents:
5. Agar (1.5%) 15.0 g.
1. Water 1000.0 cc.
Preparation
2. Bean flour 50.0 g.
(1) Same as medium 1162 but solidified by
3. Agar 30.0 g.
4. Glucose 20.0 g.
Preparation
Use: To study nitrogen fixation by bacteria
(1) Add water to 50.0 g. of bean flour to
from nodules of leguminous plants.
make a thick paste.
Variants de Rossi Gino prepared a similar
:
(2) Dilute (1) to a liter by the addition
medium as follows:
of water.
(1) Prepare agar using bean extract
(3) Allow to stand for 24 hours, shaking
(method not given).
often.
(2) Add 2.0% saccharose and 0.25% Remove the liquid from the sediment.
(4)
NaCl.
(5) Filter.
(3) Distribute in long necked flasks.
(6) Make up to one liter.
(4) Adjustment of reaction or steriliza-
agar and 20.0
(7) Dissolve 30.0 g. g. glu-
tion not specified.
cose in (6).
References: Maz6 (1897 p. 45), de Rossi
(S) Do not adjust the reaction.
Gino (1909) (1910 p. 271).
Use: Cultivation of nodule bacteria.
2134. Reed and Cooley's Lima Bean Agar
Variants Klimmer used pea flour instead of
:
Same as medium
but Phaseolus
2099, bean flour.
lunatus, lima bean meal was used instead Reference: Klimmer (1923 p. 229).
of corn meal.
2138. Bacto Bean Pod Agar (Dehydrated)
2135. Thorn's Bean Agar (Tanner) Constituents
Constituents 1. Distilled water
1. Water 1000.0 cc. 2. Agar Bacto 15.0 g.
2. Bean, white 200.0 g. 3. Bean Pod Meal Infusion
3. Agar as desired (dry) 7.5 g.
Preparation: Preparation
(1) Heat common white beans with 5 (1) Dissolve 22.5 g. of Bacto Bean Pod
volumes of water. Boiling is stopped Agar (Dehydrated) in 1000.0 cc. of
just before the swelling of the coty- distilled water by boiling or auto-
ledons rupture the seed coats. claving, preferably the latter. Bean
(2) Filter. This solution filters easily. Pod Meal is prepared by infusion of
(3) Add agar as desired. 20 parts bean pod meal.
Sterilization: Not specified. (2) Distribute as desired.
Use: General culture medium. Tanner Sterilization: Heat in the autoclave for 20
reported that this medium contained minutes at 15 pounds pressure.
sufficient nutrients to grow many species Use: General culture medium.
normally. This medium is poor in Reference: Digestive Ferments Co. (1925
available carbon. It may be desirable to p. 17).
add carbohydrates for certain species.
Reference: Tanner (1919 p. 50). 2139. Didlake's Soy Bean Root Agar
Constituents
2136. Stutzer's Bean Infusion Agar Water 1000.0 cc.
1.
Same as variant (c), medium 1166, but 2. Soy bean root

solidified with 1.0% agar. 3. Sucrose (5.0%) 5.0 g.
4. Asparagin (0.25%) 2.5 g. 2141. Simon's Legume Agar (Klimmer and

5. Agar Kriiger)
(1) Prepare a soy bean root infusion agar. 1. Water 1000.0 cc.
(2) Dissolve 0.5% sucrose and 0.25% 2. Agar 15.0 g.
asparagin in (1). 3. Legimae infusion 2.0 cc.
Sterilization: Not specified. 4. Asparagin 10.0 g.
Use: Cultivation of a red pigment produc- 5. K2CO3
ing bacillus isolated from water. Author Preparation
reported that the bacillus produced a (1) Prepare the legume infusion by pour-
red pigment only on this medium. Will ing hot water over finely chopped
grow more or less luxuriantly on soy bean straw of the legumes, mix well,
agar leaving out the asparagin or sucrose, macerate for two days and then boil
on ordinary beef peptone agar with 0.25% an hour. Pour ofl the liquid, filter
asparagin or 0.5% sucrose added or both and evaporate until it contains 10.0%
asparagin and sucrose, or on a plain dry material.
peptone water agar. On these media (2) Dissolve 2 and 4 in 1000.0 cc. of water.
there was no pigment production. (3) Add a knife point of K2CO3 and 2.0
Reference: Didlake (1905 p. 194). cc. of (1) to (2).
(4) Neutralize by the addition of NaOH
using litmus as an indicator.
2140. Zipfel's Legume Leaf Agar
(5) Add malic acid to give a distinct acid
Constituents reaction.
2. Clover plant 1000.0 g. Use: Cultivation of nodule bacteria from
3. Agar 30.0 g. leguminous plants.
4. Glucose 20.0 g. Reference: Klimmer and Kriiger (1914 p.
Preparation 258), Klimmer (1923 p. 228).
(1) Crush 1000.0 g. of closely

picked
clover leaves, (or other legume) to a
pulp. Basal or complete media containing agar,

(2) Extract with hot water until finally with constituents of animal origin (exclu-
10 liters of liquid results. sive of digests) of unknown chemical
(3) Filter the decoction when cold. composition.
(4) Concentrate the filtrate to about Ai. Containing animal products exclusive
900.0 cc. of extracts and infusions.
(5) Neutralize with N/1 NaOH. Bi. Cells, tissues or their derivatives used.
(6) Make up to 1.0 liter. Teague and Deibert's Erythrocyte
(7) Distribute into small flasks. This is Agar 2142.
a concentrated stock solution. Duval's Leprous Tissue Agar 2143
(8) Dissolve 30.0 g. agar in 800.0 cc. Nastiukoff's Egg Yolk Agar (Recht-
water by heating. samer) 2144
(9) Add 20.0 g. glucose and 50.0 cc. of Egg Yolk Agar
Steinschneider's 2145
sterile (7) to (8). Brown's Albumen Agar (Giltner) . . . 2146
(10) Adjust the reaction to neutral. Scales' Ammonium Lactate Egg
(11) Make up to 1 liter volume. Agar 2147
Sterilization: Sterilize (7) in the steamer. Scales'Egg Starch Agar 2148
Sterilization of final medium not given. Despeignes' Egg Yok Milk Agar... 2149
Use: Cultivation of nodule bacteria. Noyes' Gelatin Agar 2150
Standfuss and Kallert's Bone Jelly
Variants The medium may be utilized with
:
Agar 2151
a neutral reaction, or add 10.0 cc. of N/1
B2.* Body fluids used.
malic or citric acid or 10.0 cc. of N/1
Ci. Blood employed.
NaOH to give an alkaline medium.
Reference: Zipfel (1911-12 p. 106). * See B3, page 691.
NicoUe's Blood Agar 2152 Ficker's Spleen Agar 2179

Noguchi's Ringer Solution Plasma Duval's Turtle Agar 2180
Agar (Abbott) 2153 Loeffler's Malachite Green Nutrose
C2. Serous fluids employed. Agar 2181
Tanner's Serous Fluid Agar 2154 D2. Containing additional materials of
Kanthack and Stephens' Serous unknown chemical composition.
Exudate Agar 2155 Meyers' Tissue Agar 2182
Beck's Glycerol Serum Agar (Klim- Williams and Burdick's Egg Veal
mer) 2156 Agar 2183
C3. Bile employed. B2. Extracts specified.
Ecker's Bile Agar 2157 Ci. Additional material, if any, inorganic.
B3. Secretions, excretions or their deriva- (Exclusive of indicators).
tives used. Burgers' Basal Lead Acetate Agar.. 2184
Ci. Milk or its derivatives used. Hiss' Extract Agar 2185
Raskin's Milk Egg Albumin Agar... 2158 C2. Containing additional organic ma-
Reinsch's Alkaline Milk Agar 2159 terials.
Abbott's Milk Agar 2160 Di. All additional organic constituents of

Raskin's Casein Whey Agar 2161 known chemical composition.
Stutzer and Hartleb's Whey Agar. . 2162 Bacto Starch Agar (Dehydrated) . . . 2186
Bronfenbrenner et al. Whey Agar. . . 2163 Hiss' Glucose Extract Agar 2187
Ayres' Casein Agar (Tanner) 2164 Tanner's Malachite Green Extract
Harvey's Nutrose Agar 2165 Agar 2188
C2. Milk or derivatives not used. Noyes' Starch Extract Agar 2189
de Kruyff's Manure Agar 2166 Malenkovic's Xylose Extract Agar. 2190
An. Containing animal extracts or in- Dj. Containing additional organic con-
fusions. stituents of unknown chemical com-
Bi. Infusions specified. position.
Ci. Additional material, if any, inorganic. Hiss' Gelatin Extract Agar 2191
Di. Infusions of animal organs employed. Stapp's Egg White Extract Agar. . . 2192
Henssen's Organ Infusion Agar 2167
2142. Teague and Deibert's Erythrocyte
Mayer's Salivary Gland Agar 2168
Agar
Graham-Smith's Heart Infusion
Agar 2169 Constituents:
D2. Other animal infusions employed. 1. Distilled water.
Kutscher's Meat Infusion Agar 2170 2. NaCl.
Gassner's Basal Fuchsin Infusion 3. Red Blood Cells.
Agar 2171 4. Agar.
Harde and Hauser's Fish Infusion Preparation:
Agar 2172 (1) Add 2.0 cc. of red blood cells (obtained
Pergola's Mussel Infusion Agar 2173 by centrifuging defibrinated blood
C2. Containing additional organic material. and removing the serum) to 10.0 cc.
Di. All additional constituents of known of normal NaCl solution.
chemical composition. (2) Keep the temperature at 100C. for 3
El. Organic nitrogen supplied minutes.
E2. Organic nitrogen not added. (3) Shake the tubes and allow to cool.
Fi. Carbohydrates supplied. (4) Centrifuge and obtain the super-
Harvey's Glucose Starch Infusion natant fluid.
Agar 2174 (5) Add various amounts of (4) to a 2.0%
Vedder's Starch Infusion Agar 2175 water solution containing 0.5% NaCl.
F2. Alcohols added. Sterilization: Method not given.
Kopp's Thyroid Agar 2176 Use: To study growth
requirements of
Wroblewski's Suprarenal Capsule Unna-Ducrey bacillus. The authors re-
Agar 2177 ported that a fair growth occurred when
Ficker's Brain Agar 2178 rabbit serum was added, but no growth
using sheep serum, or without the addi- Sterilization: Sterilize in the steamer.
tion of serum. Use: Cultivation of diphtheria, cholera,
Variants: The authors added various influenza and other pathogenic forms.
amounts of sheep or rabbit serum, heated References: Nastiukoff (1893 ;^33 and 34),
at 55C. for 15 minutes to the medium as Rechtsamer (1895 p. 493).
prepared above.
Reference: Teague and Deibert (1922 p. 2145. Steinschneider's Egg Yolk Agar
70).
Constituents:
2143. Duval's Leprous Tissue Agar 1. Distilled water 800.0 cc.
Constituents: 3. Agar (2.0% soln.)
1. Agar solution. Preparation :
2. Tissue (Leprous). Mix two parts of
sterile distilled water
(1)
Preparation with one part hen or lapwing's egg
(1) Prepare an agar solution. Composi- yolk obtained under sterile con-
tion not given, but the author speci- ditions.
fied that it should contain no peptone.
(2) Prepare a 2.0% agar solution.
(2) Adjust to 1.5% alkaline. Mix one part (1) with two parts
(3) (2).
(3) Place small bits of leprous tissue upon Distribute into tubes.
(4)
the surface of the sterilized and Slant and solidify.
(5)
solidified alkaline agar.
Sterilization: Final sterilization not given.
(4) Moist the surface with a thin suspen- Use: Cultivation of gonococcus. The
sion of an organism capable of oxidiz-
medium was not transparent.
ing nucleo-proteids such as B. typho- Variants: The author prepared a similar
sus, paraiyphosus, prodigiosus, py- medium as follows:
ocyaneus, dysenteriae, etc.
(1) Mix one part yolk of hen or lapwing's
(5) Incubate at 32C. to 35C. for two egg with three parts sterile distilled
weeks. water. Shake thoroly.
Sterilization: Sterilization of agar not To 20.0 g. of (1) add 10.0 cc. of a 20.0%
(2)
given.
Na2HP04 and 3 times, hence 90.0
Use: Indirect culture of B. leprae. cc. ofa 2.5 to 3.0% agar solution.
Reference: Duval (1911 p. 371). Pour into tubes and solidify (method
(3)
2144. Nastiukoflf's Egg Yolk Agar not given).

(Rechtsamer) (4) Final sterilization not specified.
The author reported that this medium
Constituents:
was not yellow and was nearly trans-
parent. Gonococcus growth was not
quite so luxuriant on this medium as on
3. Agar 15.0 to 20.0 g.
serum agar.
Preparation
Reference: Steinschneider (1897 p. 380).
(1) Separate the yolk of egg from the
white by Bunge's method (allow the
2146. Brown's Albumin Agar (Giltner)
whites to roll from a blotting paper
containing the egg). Constituents
(2) Add 0.5 cc. of a 10.0% NaOH solution 1. Distilled water 1000.0 cc.
and 100.0 cc. of egg yolk to 1000.0 cc. 2. K2HPO4 0.5 g.
distilled water. 3. MgS04 0.2 g.
(3) Add 15.0 to 20.0 g. of agar to (2) and 4. Egg albumin (powdered). . 0.1 g.
boil until there is complete solution 5. Glucose 10.0 g.
of the agar. 6. Fe2(S04)3 trace
(4) Allow to stand two hours in the 7. Agar 15.0 g.
steamer. Preparation
(5) Filter the thick liquid thru a hot (1) Dissolve 2, 3, 4, 5, 6, and 7 in 1.
water funnel. (2) Do not filter.
(6) Distribute into sterile test tubes. Sterilization: Not specified.

Use: To determine the bacterial count of Preparation

soil. See medium 1439. (1) Add 100.0 cc. of boiled skim milk,
Reference: Giltner (1921 p. 373). 15.0 g. glycerol and 9.0 g. of agar to
200.0 cc. of Besredka's egg yolk
2147. Scales' Ammonium Lactate Egg Agar solution (see variant of medium 1244)
Constituents: (2) Heat in a salt water bath until the

Distilled water 1000.0 cc. agar is dissolved.
1.
2. Agar 15.0 g. (3) Sterilize in the autoclave at 105C.
3. Na2HP04 2.0 g.
(Time not specified.)
4. Ammonium lactate 10.0 cc. (4) Add 3.0 cc. of 1.0% gentian violet
5. CaCOs 10.0 g.
solution when cool.
6. Egg (5) Distribute in sterile tubes under

aseptic conditions.
Preparation
(6) Slant.
Sterilization: Heat on 3 successive days at
(2) Titrate to +3.
55 to 57C. for 30 minutes.
Add 2.0 cc. of whole egg to 7.0 cc. of
Use: Isolation tubercle bacilli.
(4)
Reference: Despeignes (1922 p. 120).
(3).
(5) Tube.
Sterilization: Sterilize at 12 pounds pres- 2150. Noyes' Gelatin Agar
sure for 15 minutes. Constituents:
Use: To study variation in morphology of 1. Water 1000.0 cc.
^
B. coli. 2. Agar (best) 15.0 g.
Reference: Scales (1921 p. 595). 3. Gelatin 7.5 g.
Preparation
2148. Scales' Egg Starch Agar (1) Dissolve 2 and 3 in 1.
1. Distilled water 1000.0 cc. Use: Cultivation of soil bacteria. Weiss

2. Starch (soluble 0.1%) 1.0 g. used a similar medium to determine the
3. Agar (1.5%) 15.0 g. bacterial count of water.
4. Eggs Variants
Preparation (a) Noyes added 1.0 or 2.0 g. starch.
(1) Strain a fresh whole egg thru four (b) Weiss prepared a similar medium as
thicknesses of cheese cloth four times. follows:
(2) Prepare a soluble solution of agar (1) Dissolve 15.0% gelatin in 500.0 cc.
1.5%, and 0.1% soluble starch in of water from the "Mlihlgraben der
distilled water Schwarzawa."
(3) Add 2.0 cc. of (1) to each 7.0 cc. of (2) Dissolve 1.5% agar in 500.0 cc.
(2).
water.
Sterilization: Sterilize at 12 pounds pres- (3) Mix (1) and (2).
sure for 15 minutes. References: Noyes (1916 p. 93), Weiss (1920

Use: To study variations in morphology p. 25).
of B. coli.
2151. Standfuss and Kallert's Bone Jelly
Reference: Scales (1921 p. 596).
Agar
2149. Despeignes' Egg Yolk Milk Agar Solidify medium 1360 with agar.
Constituents:
2152. Nicolle's Blood Agar
1. Distilled water
2. Milk 100.0 cc. Constituents
3. Glycerol 15.0 g. 1. Water 900.0 cc.
4. Agar 9.0 g. 2. Agar 14.0 g.
5. Gentian violet 3. NaCl 6.0 g.
6. Egg Yolk 4. Blood, rabbit
Preparation (3) Add 25.0% (Use only 5.0% when

(1) Dissolve 2 and 3 in 1. using 0.5% agar) sterile defibri-
(2) Neutralize or make slightly alkaline. nated rabbit blood to sterile melted
(3) Distribute in tubes of the same and cooled (2).
diameter. (e) Stitt gave the following method of
(4) Cool sterile (3) to 48 to 52C. and add preparation of N. N. N. medium for
one third the volume of rabbit blood the cultivation of trypanosomes,
to each tube. Draw the blood di- leishmania and protozoa:
rectly from the heart under aseptic (1) Dissolve 14.0 g. agar and 6.0 g.
conditions. NaCl in 900.0 cc. of water.
(5) Slant the tubes. (2) Tube and sterilize.
Sterilization: Sterilize (3) in the autoclave. (3) To one part of (2) liquified and
Use: Cultivation of kala azar parasite. cooled to 48C. add one third its
Similar media were used for the cultiva- volume of defibrinated rabbit
tion of Prioplasma donovai, Piroplasma blood. (Use human or rat blood for
equi, piroplasma, trypanosomes,
other the cultivation of human trypano-
leishmania, and protozoa. somes.)
Variants (4) Mix thoroly.
(a) Harvey cultivated trypanosomes on a (5) Slant.
medium prepared as follows: (6) Use rubber stoppers or cover cotton
(1) Dissolve 0.6 g. NaCI and 1.5 g. plugs with paraffin.
agar in 100.0 cc. water. References: Nicolle (1909 p. 397), Marzin-
(2) Mix equal parts of (1) and de- owsky (1908-09 p. 419). Bezanôn (1920
fibrinated rabbit blood at 45C. p. 120), Harvey (1921-22 pp. 72, 74),
(b) Harvey prepared a N. N. N. or whole Leiva (1922 p. 179) taken from (1923 p.
blood agar as follows for the cultiva- 342), Stitt (1923 p. 52).
tion of leishmania:
2153. Noguchi's Ringer Solution Plasma
(1) Dissolve 6.0 g. NaCl and 16.0 g. of
Agar (Abbott)
well washed agar in 900.0 cc. of
water. Constituents
(2) Sterilize (1), method not given. 1. Water 1000.0 cc.
(3) Mix one part rabbit blood with 3 2. NaCl 10.0 g.
parts (2) at 50C. 3. KCl 0.2 g.
(4) Slope. 4. CaCl2 0.2 g.
(5) Test sterility. 5. NaHCOa 0.1 g.
(6) Preserve in the dark. 6. Glucose 1.0 g.
(c) Harvey cultivated the trypanosomes 7. Serum, rabbit
of cold blooded animals on the 8. Citrate plasma
following medium 9. Agar (2.0%)
(1) Dissolve 20.0 g. of agar in 1000.0 Preparation:
cc. tap water. (1) Dissolve 2, 3, 4, 5 and 6 in 1. (Ringer
(2) Tube and sterilize. solution.)
(3) Mix equal parts (2) and sterile (2) Add 1.5 parts serum and 1 part citrate
defibrinated rabbit blood. plasma to 4.5 parts (1).
(4) Solidify in the sloped position and 3) Stiffen the medium by the addition of
inoculate in the water of con- "sterile agar (free of peptone and
densation. sugar) in the proportion of 2.0%."
(d) Leiva cultivated Leishmania infan- Sterilization: Not specified.
tum and Leishmania ctenocephali Use: Cultivation of spirochaetes.
on a medium prepared as follows: Variants
(1) Dissolve 20.0 g. agar and 4.0 g. (a) Kaneko cultivated Spirochaeta ic-
NaCl in 1000.0 cc. water. (May ier ohaemorrhagiae and Spirochaeta

use 5.0 g. agar.) hebdomadis on a medium prepared
(2) Tube. as follows:
(1) Dissolve 3.0 g. of agar in 100.0 cc. (b) Teague and Deibert reported that the
Ringer's solution. Unna-Ducrey bacillus would not
(2) Add a drop of dog blood to 2 to 3.0 grow on a medium prepared by mix-
cc. of dog serum diluted with 2 to 5 ing various amounts of sheep or
parts Ringer's solution. (Sera or rabbit serum, heated at 55C. for 15-
ascitic fluids of other animals may minutes with a 2.0% agar solution
be used.) containing 0.5% NaCl.
(3) To 9.0 cc. of (2) add 1.0 cc. of (1). References: Tanner (1919 p. 70), Harvey
(4) Heat at 56 water bath
to 58C. in a (1921-22 p. 79), Teague and Deibert
forabout 30 minutes. (1922 p. 70).
(5) Cover with a layer of liquid sterile
2155. Kanthack and Stephens' Serous
paraffin.
Exudate Agar
(b) Stitt cultivated Leptospira icteroides
(cause of yellow fever) on a medium Constituents:
prepared as follows: 1. Serous exudate (ser-
(1) Mix one part rabbit s erum and 3 um, ascitic fluid, etc.). 100.0 cc.
parts of Ringer's solution solidified 2. KOH 10.0% 2.0 cc.
partially by the addition of 0.3% 3. Agar (1.5 to 2.0%) . . . 15.0 to 20.0 g.
agar. 4. Glycerol (4.0 to
(2) Tube in tall tubes. 5.0%) 4.0 to 5.0 cc.
Introduce 1.0 cc. of yellow fever
(3) Preparation
patients blood into the lower part (1) To 100.0 cc. of a serous exudate add
of each tube. 2.0 cc. of 10% KOH solution. (It is
(4) Pour a thin layer of petroleum over always best to heat a small
first
the top of the medium. portion of the serous exudate in a

References: Abbott (1921 p. 635), Kaneko test tube. If it solidifies or if a large
(1921-22 p. 354), Stitt (1923 p. 54). amount of albumin appears, add at
least a double volume of distilled
2154. Tanner's Serous Fluid Agar water before adding KOH.)
(2) Soak agar in acidified water and wash
Constituents from acid.
free
(3) Add 1.5 to 2.0% agar to (1).
2. Agar 1-5 g.
Boil until the agar is completely
(4)
3. Serum 100.0 cc.
dissolved. (The Mixture appears
Preparation:
clear when agar is dissolved.)
(1) Dissolve 1.5 g. agar in 100.0 cc. dis- thru a coarse paper using
(5) Filter filter
tilled water.
a hot water funnel.
(2) Filter and tube in 5.0 cc. quantities. Add
(6) 4.0 to 5.0% glycerol.
(3) Add an equal volume of sterile serum Distribute into test tubes.
(7)
to each tube of sterile (2) melted and
cooled to 40C. Diagnosis of diphtheria. The authors
Use :
(4) Roll the tube to mix.

reported that staphylococci and B. coli
(5) Cool in a slanting position. communis, etc., development was in-
Sterilization: Method of sterilization of hibited. The sooner the exudate was
(2) not given. used, after it left the human body the
Use: General culture medium. Teague clearer the medium.
and Deibert used a similar medium to Variants
study growth requirements of Unna- (a) The authors reported that the addi-
Ducrey bacillus. tion of 2.0 to 4.0% glucose did not
Variants improve the medium.
(a) Harvey specified that the agar be (b) Harvey prepared a similar medium as
sterilized at 120C., and be mixed follows:
with an equal volume of sterile serum (1) Collect ascitic fluid with sterile
or ascitic fluid. precautions.
(2) Leave overnight to allow for sep- 2157. Ecker's Bile Agar
aration of clot.
Constituents:
(3) Add 50.0 cc. N/1 sodium hydroxide
1. Bile (ox) 1000.0 cc.
per liter.
2. Agar 15.0 g.
Preparation
(5) Add 1.0agar per liter after
g.
(1) Dissolve 2 in 1 as rapidly as possible
making it into a paste or suspen-
by gently boiling.
sion with a little of the alkaline
(2) Filter and allow to cool and again boil
ascitic fluid.
and refilter without titration.
(6) Steam to dissolve the agar.
(3) Tube.
(7) Filter, while hot, thru thick, filter
Sterilization: Sterilize in autoclave for 3
paper by placing filter funnel,
minutes at 15 pounds pressure.
stand and receptacle for filtrate in
Use: To study effect of bile on B. typhosus.
the sterilizer.
The author reported that some strains
(8) Dissolve 10.0 g. glucose per liter
were completely inhibited while others
in the hot, filtered, nutrient agar
were only partially inhibited. Approxi-
(7). (Add 50.0 cc. glycerol per
mately the same results were obtained
liter also if desired.)
using 50.0% or 10.0% bile. (Bile diluted
with distilled water.)
(10) Sterilize.
Reference: Ecker (1918 p. 97).
(c) Harvey prepared a similar medium
from 20.0 g. glycerol, 20.0 cc. of 10.0% 2158. Raskin's Milk Egg Albumin Agar
NaOH, 20.0 g. agar and 1000.0 cc. of a
Constituents
body fluid (ascitic fluid, pleuritic
1. Milk 1000.0 cc.
fluid, hydrocele fluid, ovarian fluid,
2. Egg albumin
milk, urine, etc.).
3. NaCl 5.0 g.
References: Kanthack and Stephens (1896
4. Agar 5.0 to 7.0 g.
p. 609), (1896 p. 835), Harvey (1921-22 p.
Preparation
83).
(1) Stir thoroughly with a glass rod
100.0 g.albumin from fresh eggs in a
2156. Beck's Glycerol Serum Agar
flat bottomed dish and add drop by
(Klimmer)
drop concentrated NaOH until a
Constituents solid opaque gelatinous material is
1. Water 1800.0 cc. obtained.
2. Serum (beef) 200.0 cc. (2) Cut this solid egg albumin in small
3. KHSO4 10.0 g. pieces and place in distilled water.
4. MgS04 5.0 g. Do not allow the alkaline albuminate
5. Asparagin 4.0 g. to stand or the albuminate will
6. Glycerol 40.0 g. become liquid in several hours.
7. Agar (3.0%) 60.0 g. (3) Shake thoroughly and pour off the
Preparation water, continue this washing until
(1) Steam 200.0 cc. beef serum (con- the wash water is only slightly
taining no chloroform) with 1800.0 cc. alkaline.
of water for 1.0 to 1.5 hours. (4) Place the coagulated sodium al-
(2) Filter. buminate in a flask of distilled water,
(3) Add 3, 4, 5 and 6 to the filtrate and plug with cotton and heat in the
steam for 2 to 3 hours. steamer for 15 to 30 minutes. The
(4) Filter while hot. albumin dissolves.
(5) Add 3.0% fiber agar, and heat until (5) Filter.
dissolved. (6) Add 50.0 cc. glycerol and 5.0 to 7.0 g
(6) Tube. of finely divided dried agar to 1000.0
(7) Reaction should be slightly acid. cc. of fresh milk.
Sterilization: Not specified. (7) Allow to stand for 12 to 14 hours (at
Use: Cultivation of tubercle bacilli. room temperature in the winter)
Reference: Klimmer (1923 p. 224). and then boil for 75 to 90 minutes
over a free flame. In order to (12) Distribute into sterile test tubes.
prevent loss of water during boiling (13) Incubate to test sterility.
place a lid over the container or Sterilization: Sterilization of (3) not given.
steam for 3 to 3.5 hours in a steamer. Use: Cultivation of typhoid bacilli and
The
casein coagulates slowly. others. The author reported that the
(8) Press thru four folds of linen

cloth. agar was light yellow, transparent and
slightly slightly opalescent.
(9) The reaction of the fluid is
acid. Variants: The author prepared a slightly
(10) Pour into a tall glass cylinder and darker medium as follows:
place in a warm thermostat. (1) Place 500.0 cc. of fresh cow milk in a
(11) After about 20 or 30 minutes remove separatory funnel and add 1.0 g.
the yellow top layer containing the NaOH (2.5 cc. solution of 400.0 g.
fat by means of a spoon. NaOH in a liter).
(12) Heat the fat-free portion to boiling (2) Shake well and allow to stand at
and add 1.0% of the filtrate from (5). about 18C. for 48 hours.
(13) Add soda to neutralize the reaction. (3) Remove the nearly transparent milk
(14) Add 0.5% NaCl if desired. from the bottom of the funnel and
(15) Filter until clear thru a paper in a add it to a second separatory funnel.
hot water funnel. (4) Add 250.0 cc. ether and shake well.
Sterilization: Not specified. (5) Allow to stand for 48 hours.
Use: Cultivation of pathogenic organisms. (6) Place the opalescent liquid now in a
Reference: Raskin (1887 p. 359). large sterile flask plug with cotton
and heat to 50C.
2159. Reinsch's Alkaline Milk Agar Place under the receiver of a water
(7)
Constituents suction pump for 3 or 4 hours until

1. Water 100.0 cc. all the ether is evaporated.
2. Agar 3.0 to 4.0 g. (8) Add 1.5% pulverized agar.

3. Milk 500.0 cc. (9) Digest for 24 hours at room tem-
4. NaOH 1.0 g. peratures.
Preparation: (10) Heat in steamer for 2-3 hours and
(1) Soak 3.0 to 4.0 g. of powdered agar in filter.
100.0 cc. water for 24 hours. Reference: Reinsch (1892 p. 31).
(2) Add egg white and boil for 3 to 4

2160. Abbott's Milk Agar
hours in a steamer.
(3) Filter.
Constituents:
(4) Place 500.0 cc. of fresh cow milk in a 1. Milk 1000.0 cc.
separatory funnel and add 1.0 g. 2. Agar (1.0 to 1.5%) . . . 10.0 to 15.0 g.
NaOH (2.5 cc. solution of 400.0 g. Preparation:

NaOH in a liter). (1) Solidify cream-free milk by the addi-
Shake well and allow to stand at tion of 1.0 to 1.5% agar.
(5)
about 18C. for 48 hours. Sterilization: Not specified.
(6) Remove the nearly transparent milk Use: General culture medium.
from the bottom of the funnel and Reference: Abbott (1921 p. 139).
add it to a second separatory funnel. Whey Agar
2161. Raskin's Casein
(7) Add 250.0 cc. ether and shake well.
(8) Allow to stand for 48 hours. Constituents
Place the opalescent liquid now in a 1. Milk 1000.0 cc.
(9)
large sterile flask, plug with cotton 2. Agar
and heat to 50C. Preparation
(10) Place under the receiver of a water (1) Allow 1000.0 cc. of skimmed (or
suction pump for 3 or 4 hours until whole) milk to stand 48 hours at
all the ether is evaporated. room temperature.
(11) Mix 1 part of (3) with 3 parts of (10) (2) Remove any cream that settles out
at about 50'"C. and heat the sour milk at 70 to 80C.

(3) Obtain the casein and thoroly press (6) Medium may be employed with a
it dry. neutral, basic, (by adding Na2C03), or
(4) Rub the casein to a powder and wash acid, (by adding lactic acid), reaction.
with 95.0% alcohol, and place in a Sterilization: Not specified.
flask of ether. Use: Stutzer and Hartleb attempted to
(5) Shake for 2 minutes and allow to cultivate the bacterium causing foot and
stand for 18 minutes. mouth disease. Other investigators used
(6) Pour off the ether and add fresh. similar media for the cultivation of a
Repeat this process 3 or 4 times. large number of organisms.
(7) Pour off but half the last portion of Variants
the ether and add alcohol (amount (a) Huss cultivated aroma producing
not specified). bacteria, Bacillus and
esterificans
(8) Shake for 5 minutes. Pseudomonas trifolii on a medium
(9) If drops of fat raise when alcohol is prepared by dissolving 20.0 g. agar
added, repeat the washing of the and 5.0 g. NaCl in 1000.0 cc. of whey.
casein with ether until the casein is (b) Meier made bacterial counts of milk
fat-free. and milk products in a medium pre-
(10) Collect the casein on a filter. pared as follows:
(11) Dry and heat for 5 to 20 minutes at (1) Dissolve 2.5 g. NaCl and 15.0 or
120 to 140C. (10.0 g.) agar in 500.0 cc. of water.
(12) Wash in a moderately concentrated (2) Mix equal parts (1) and whey.
alkali solution. (3) Neutralize by the addition of
(13) This gives a transparent material, KOH. Add KOH
until tumeric
which when dry becomes hard as paper is turned quite weakly brown-
stone. This casein will dissolve by ish-red.
heating in slightly alkaline water. (4) Sterilization not specified.
(14) Prepare 150.0 cc. of an 8.0% solution (c) Fulmer and Grimes used the following
of (13). medium for the cultivation of yeast
(15) Mix 350.0 cc of filtered
. whey contain- found in cream and butter.
ing 1.75% agar with (14). (1) Dissolve 1.5% agar in whey ob-
(16) Heat for 15 to 20 minutes at 60 or tained from skim milk by coagulat-
70C. Do not boil or the casein will ing the casein with rennet.
be coagulated. (2) Filter (1) thru absorbent cotton.
(17) Distribute in sterile test tubes. (3) Tube in 10.0 cc. portions.
Sterilization: Not specified. (4) Sterilize for 20 minutes at 15 pounds
Use : Cultivation or pathogenic organisms. pressure.
References: Raskin (1887 p. 359), Peifer
(5) When using as a plate medium, add
(1888 p. 568). 1.0 cc. of a1.0% tartaric acid solu-
tion to each Petri dish to keep down
2162. Stutzer and Hartleb's Whey Agar
bacterial growth.
Constituents
(d) Klimmer and Sommerfeld (Klimmer)
1. Whey 1000.0 cc.
made bacterial counts of milk on a
2. Agar 20.0 g.
medium prepared as follows:
Preparation
(1) Prepare clear agar solution using
(1) Separate the cream from milk by
20.0 g. agar, 7.0 g. NaCl and 1000.0
centrifugation.
cc. water.
(2) Heat to 40C. and add a little rennet.
(2) Sterilize (1) method not given.
(3) After the casein has coagulated heat
to 75C. and filter. (3) Add a rennet to milk obtained
little
To a liter of (3) add 20.0 g. of finely under the cleanest possible con-
(4)
chopped agar and heat in the steamer ditions and heat at 40C. until the
for 1.5 to 2.0 hours. casein is coagulated.
(5) Neutralize by adding soda (indicator (4) Separate the casein from the whey
not specified). by straining thru a straining cloth.
699
paper Reference: Bronfenbrenner, Davis and

(5) Filter the whey thru filter
and then thru an asbestos filter. Morishima (1918-19 p. 347).
(6) Distribute in sterile flasks.

by the addition of 1.5% 2164. Ayres' Casein Agar (Tanner)
(7) Sterilize
chloroform. Constituents:
(8) Pour in plates.
1. Water 1000.0 cc.
(e) Klimmer whey, prepared
solidified 10.0 g.
2. Casein
from milk by coagulating the casein 3. NaOH (N/1) 7.0 cc.
at 40^C. in the presence of rennet, 4. Agar 10-0 S-
with agar.
Preparation
References: Stutzer and Hartleb (1897 p.
(Eimer and Amend
(1) Add 10.0 g. casein
403), Huss (1907 p. 58), Meier (1918 p.
prepared according to
C.P. casein
435), Fulmer and Grimes (1923 p. 585),
Hammersten) and 7.0 cc. of normal
Klimmer (1923 pp. 172,203).
NaOH to 300.0 cc. of water.
2163. Bronfenbrenner et al. Whey Agar (2) Boil'until solution is complete. It is

desirable to allow the mixture to
Constituents
200.0 cc. stand several hours.
1. Water
100.0 cc. (3) Make up to 500.0 cc. volume.
2. Milk
CR indicator 1.5 cc. (4) Bring the reaction between -|-0.1 and
3.
4. Agar (3.0%) 6.0 g. +0.2 Fuller's scale. Do not allow the

Preparation solution to become alkaline to phenol-
Use fresh milk. Syphon the milk phthalein or over +0.2.

(1)
from under the cream. (5) Filter.
Bring it to boiling and add 2.5 cubic (6) Dissolve 10.0 g. agar in 500.0 cc. of
(2)
centimeters of 10.0% MnCU solution water.
to each 100 cubic centimeters of milk. (7) Filter.
(3) Cool the mixture as soon as clot is (8) Mix (5) and (7).
formed and filter thru a single layer (9) Tube.

of cloth. Sterilization: Sterilize in the autoclave
(4) Titrate an aliquot portion hot and under pressure for 20 minutes and cool the
adjust the bulk of medium to neutral tubes in cold water or ice water.
reaction (1 xlO~^). Use: General culture medium. Harvey
(5) Bring quickly to boiling, cool and cultivated soil bacteria on a medium
filter thru paper. prepared in a similar manner.
(6) At this time prepare an agar jell of
Variants
3.0% concentration in plain water, Harvey prepared the medium as
(a)
sterilize and dilute with an equal
follows:
volume of neutralized milk-whey. Mix 10.0 g. casein with 100.0 cc.
While the mixture is hot add CR
(1)
(7) distilled water.
indicator at the rate of 0.5 cubic
(2) Add N/1 sodium hydroxide 7.0 cc.
centimeters for each 100 cubic centi-
(3) Steam until the casein is dissolved.
meters of medium distributed into
(4) Dissolve 10.0 g. agar in 900.0 cc.
sterile tubes.
distilled water.
(5) Mix the agar solution with the
agar solution (see step (6) above) not solution.
casein
given. Sterilize the final medium for 10
(6) Filter thru thick filter paper.
minutes at 15 pounds pressure.
(7) Adjust the reaction to 1.5% acid to
Use: Cultivation of colon-typhoid group. phenolphthalein.
Authors reported that the medium gave
as good results as lactose peptone agar
(9) Sterilize in autoclave.
as a medium for colon-typhoid bacteria
Reference: Tanner (1919 p. 70), Harvey
and was cheaper and more easily
(1921-22 p. 96).
prepared.
2165. Harvey's Nutrose Agar typhoid and cholera organisms. Livin-

Constituents good used a similar medium using B. coli,
1. Water 1000.0 cc. B. typhosus, B. anthracis, B. diphtheriae,
2. Agar 15.0 g. B. pseudodiphtheria, and reported that
3. Nutrose 15.0 g.
liver gave the best general results.
Preparation Variants: Livingood prepared a similar
(1) Dissolve 15.0 g. of nutrose (or soma- medium as follows:
tose) and 15.0 g. agar in 1000.0 cc. of (1) Pass 1 pound of organs (liver, beef
water. and hog, or spleen, sheep and hog, or

Sterilization: Not specified. or adrenals, beef and sheep) thru a
Use: Cultivation of amoebae. meat chopper and macerate with
Reference: Harvey (1921-22 p. 97). 1000.0 cc. of tap water for 12 hours
on ice.
2166. de Kruyff's Manure Agar (2) Press the juice thru a sterile towel.
Constituents: (3) Force the fluid thru a Chamberland
1. Distilled water 1000.0 cc. filter using carbonic acid pressure and
2. Agar 20.0 g. collect in a sterile flask.

3. K2HPO4 0.1 (4) Prepare a 2.0% agar solution (no
g
4. NH4NO3 0.5 g. NaCl or peptone) sterilize. (Bouil-
5. Stable manure 50.0 g. lon agar, with NaCl and peptone,
Preparation maybe used.)
(1) Dissolve 2, 3, 4 and 5 in 1. Details (5) Mix equal parts of melted (4) and
method
of of preparation not given). (3).
Sterilization: Not specified. (6) Incubate for 24 to 48 hours to deter-

Use: Cultivation of Myxococcus javan- mine sterility.
ensis. References: Henssen (1895 p. 403), Livin-

Reference: de Kruyff (1908 p. 385).
good (1898 p. 981).
2167. Henssen's Organ Infusion Agar 2168. Mayer's Salivary Gland Agar
Constituents
Constituents:
1. Water 200.0 cc.
1- Water 500.O cc.
2. Kidney 100.0 g.
2. Salivary glands 500.0 g.
3. Agar 2.5 g.
3. Agar (1.5%) 15.O g.
Preparation
Preparation
(1) Remove the capsule from the kidney
(1) Chop fresh salivary glands in a meat
(dog, calf and hog were used) and rub
chopping machine.
to a pulp in a mortar with an equal
(2) Mix with an equal weight of water.
amount of water.
(3) Infuse on ice for 24 hours after strong
(2) Allow to extract for 3 hours.
stirring.
(3) Press thru fine pored linen.
(4) Press the mass in a pressing machine.
(4) Prepare a 2^% water solution of agar.
(5) Solidify sterile (6) by the addition of
(5) Heat(4)to40C.
1.5% agar (method not given).
(6) Mix equal parts of sterile (5) and Sterilization: Sterilize(4) in streaming
melted agar cooled to 40C.
sterile
steam for 30 minutes. Final sterilization
(7) Incubate to test sterility.
not specified.
Sterilization: Filter (3) thru a sterile clay
filter using a water suction pump to ported that meat extract media generally
facilitate the filtering. Method of ster-
gave better results.
ilization of agar not given.
Reference: Mayer (1899 p. 747).
Use: To study the effect of kidney infusion
on the growth of some organisms. The
2169. Graham-Smith's Heart Infusion Agar
author reported that the medium checked
the growth of the organisms studied, i.e., Solidify medium 1342 by the addition of
diphtheria bacilli, anthrax bacilli, B. coli, agar.
701
Meat Infusion Agar (6) To each 80.0 cc. of (5) add 5.0 cc. of a
2170. Kutscher's
3.0% acid fuchsin solution and 0.0,
Constituents 0.1, 0.2, 0.5, or 1.0% glucose.

1. Meat water 1000.0 cc. Sterilization: Not specified.
Agar (1.5%) 15-0 g.
2. Use: To study nitrogen source require-
Preparation ments for colon-typhoid and dysentery
(1) Prepare meat water. group. The author reported that as-
(2) Dissolve 15.0 g. agar
in sterile (1) by paragin was utilized as a nitrogen source
boiling over a free flame. by the typhoid and dysentery group as
(3) Make slightly alkaline by the addition well as by Bacterium coli.
of soda solution. Added nutrients: The author added 10.0 g.
(4) Filter. asparagin or 20.0 g. peptone.
Sterilization: Method of sterilization of The author added 0.1, 0.2, 0.5,
Variants:
meat water not given. Method of or 1.0%, glucose. See step (6) above.
sterilization of the final medium not Reference: Gassner (1917-18, p. 263).
given.
2172, Harde and Hauser's Fish Infusion
Use: Isolation and cultivation of Spirillum
Agar
iindula majris. Meier used a similar me-
dium to determine the numbers of bac- Same as medium 1346, but solidified by
teria in milk and milk products. the addition of 15.0 g. agar.
Variants: Meier prepared a medium as
2173. Pergola's Mussel Infusion Agar
follows:
(1) Boil 500.0 g. of fat and tendon free
Solidify medium 1347 by the addition of
beef in 1 liter water. 2.0%, agar.
(2) Filter.
Dissolve 15.0 g. agar and 5.0 g., 20.0 2174. Harvey's Glucose Starch Infusion
(3)
glucose in the Agar
g. or 40.0 g. lactose or
filtrate. Constituents
(4) Neutralize by the addition of KOH. 1 Water 1500.0 cc.
Add KOH until turmeric paper is 2. Beef 500.0 g.
turned quite weakly brownish red. 3. Agar 23.0 g.
(5) Sterilization not
specified. 4. Starch 15-0 g.
References: Kutscher (1895 p. 615), Meier 5. Glucose 15.0 g.
(1918 p. 436). 6. Litmus solution

Preparation:
2171. Gassner's Basal Fuchsin Infusion Macerate 500.0 g. finely minced beef
(1)
Agar in 1500.0 cc. water in the ice chest
over night.
Constituents
750.0 cc. (2) Filter thru doubled gauze.
1. Water
250.0 cc. (3) Bring the filtrate to the boil and add
2. Meat infusion
30.0 g. agar 1.5%.
3. Agar
5.0 (4) Boil 25 minutes.
4. NaCl g.
60.0 cc. Estimate and adjust reaction to 0.5%
5. Fuchsin (acid) (3.0% soln.).. (5)
acid to phenolphthalein.
Preparation
infusion (method not Clarify and filter.
(1) Prepare meat
(6)
Add 1.0%, starch, 1.0%o glucose and
given). (7)
color the medium with litmus solu-
(2) Mix 750.0 cc. of water with 250.0 cc.
tion.
of (1).
Dissolve 30.0 g. agar and 5.0 g. NaCl (8) Steam 45 minutes shaking 3 times
(3)
during this period to distribute the
in (2).
(4) Adjust to a
slight alkalmity usmg starch.
litmus as an indicator.
Sterilization: Sterilize 15 minutes at 10
pounds.
in (4).
Use: Differentiation of meningococci and (9) Distribute into test tubes.

M. catarr halts. Author reported that (10) Sterilize 15 minutes at 10 pounds
meningococci acidified with glucose, M. pressure.
catarrhalis did not. Hereported that there should be an
Reference: Harvey (1921-22 p. 111). abundance of water of condensation
developed, and the agar must not
2175. Vedder's Starch Infusion Agar
exceed 1.5%.
Constituents: (b) Park, Williams and Krumwiede pre-
1. Beef infusion (not pared a medium as follows:
containing peptone (1) Extract 500.0 g. beef in 1000.0 cc.
or salt) 1000.0 cc. of water in an ice box over night.
2. Starch, corn 10.0 g. (2) Boil and strain.
3. Agar 15.0 to 17.5 g. (3) Dissolve 1.5% agar in (2).
Preparation (4) Adjust the reaction to -1-0.2 to
(1) Exact composition of beef infusion -hO.7 to phenolphthalein (pH be-
not given except that it should con- tween 7.0 and 7.6).
tain no peptone or salt. (5) Cool and clarify with eggs.
(2) Dissolve agar in beef infusion, cook, (6) Filter.
clarify and filter according to methods (7) Add 1.0% starch and heat in the
of preparing ordinary agar. Arnold for 45 minutes shaking the
(3) Neutralize so that final reaction will mediimi several times to distribute
be from 0.2 to 0.5% acid to phenol- the starch.
phthalein. (8) Tube.
(4) Grind corn starch in mortar with a (9) Autoclave 15 minutes at 10 pounds
avoid lumps.
little of (3) to pressure.
(5) Boil (4) for a few minutes. References: Vedder (1914 p. 385), Tanner
(6) Tube. (1919 p. 60), Giltner (1921 p. 371), Harvey
Sterilization: Sterilize at not more than 15 (1921-22 HI), Park, Williams
p. and
pounds pressure. Krumwiede (1924 p. 131).
Use: Special culture medium. Author re-
ported that medium will keep gonococci 2176. Kopp's Thyroid Agar
alive for long period of time. be May
used to isolate gonococci and for routine Constituents :
purposes. Park, Williams and Krum- 1. Water 1000.0 cc.

wiede recommended a similar medium 2. Thyroid gland, sheep 500.0 g.
for the cultivation of meningococci. 3. Agar lO.O g.
Variants :
5. NaCl 5.0 g.
follows: Preparation :
Macerate 500.0 (1) Free sheep thyroid glands from fat,

(1) g. finely minced
beef in 1500.0 cc. water in the ice and chop the glands into small pieces.
chest over night. (2) Extract the glands for 3 hours with an
(2) Filter thru doubled gauze. equal weight of sterile water.
(3) Bring the filtrate to the boiling (3) Add (2) to moistened linen and press
point and add 1.5% agar. the fluid thru.
(4) Boil 25 minutes. (4) Dissolve 3, 4 and 5 in 500.0 cc. water.
(5) Estimate and adjust reaction to (5) Mix equal parts of sterile (4) and
0.5% acid to phenolphthalein. sterile (3) melted and cooled to 40C.
(6) Clarify and filter. Sterilization: Filter (3) thru a clay filter to
(7) Add 1.0% starch. sterilize. Method of sterilization of

(8) Steam 45 minutes shaking 3 times agar not given.
during this period to distribute the Use: Cultivation of colon-typhoid group.
starch. Reference: Kopp (1895 p. 81).
703
Sterilization: Sterilize the filtrate from (5)

2177. Wroblewski's Suprarenal Capsule
Agar by steaming for two hours. Sterilization
of the agar not specified. Sterilize (8)
Constituents in steam for 30 minutes.
500.0 cc.
by heating
1. Water of tubercle bacilli.
100.0 g.
Use: Cultivation
2. Suprarenal capsule (ox)
Variants
Agar 7.5 g.
3.
18.0 g.
(a) The author reported that human,
Glycerol
sheep, beef, calf and horse brain were
4.
5. NaCl 3.0 g.
used, but all gave the same results.
Preparation
(b) Abel boiled the brain infusion for 45
(1) Remove the suprarenal capsule from minutes instead of 15 minutes as in
an ox immediately after death. above.
step (4)
(2) Chop to small bits and rub to a pulp. References: Ficker (1900 p. 593), Abel
To 100.0 g. of (2) add 200.0 cc. of
90), Kolle and Wasserman (1912
(3)
(1912 p.
water.
p. 410), Klimmer (1923 p. 224).
(4) Make (3) alkaline with soda and boil
for 2 hours. (Filtration not specified) 2179. Picker's Spleen Agar

(5) Dissolve 7.5 g. agar, 18.0 g. glycerol 2178 but substituting
Same as medium
and 3.0 g. of NaCl in 300.0 cc. of water.
beef spleen for brain.
(6) quantities of (5) and (4).
Mix equal
(7) Sterilize (method not
given). 2180. Duval's Turtle Agar
(8) Filter two or three
times to clarify.
Constituents
Sterilization Sterilize the filtrate (method
:

not given) and store at 36C. for several 20.0 g.
2. Agar
days to test sterility.
3. NaCl 3.0 g.
Use: General culture medium. Author re- Glycerol 30.0 cc.
4.
ported that the medium suported the 500.0 g.
5. Muscle, turtle
growth of some organisms better than
Preparation
others.
(1) Dissolve 2, 3 and 4
in 1.
Reference: Wr6blewski (1896 p. 528).
(2) Clarify and tube
in 10.0 cc. lots.
2178. Picker's Brain Agar (3) Cut 500.0 g. turtle

muscle into 500.0
cc. distilled water.
Constituents
1000.0 cc. (4) Keep in the ice chest 48 hours and
1. Distilled water
500.0 g. filter thru gauze to remove the tissue.
2. Brain, beef
3. Agar (2.5%) 12-5 g. (5) Add 5.0 cc. of sterile (4) to each tube
of sterile (2) that has been melted
4. Glycerol 30.0 g.
and cooled to 42C.
Preparation
fresh brain thru a (6) Agitate thoroly and allow to solidify
(1) Pass 500.0 g.
in the slanted position.
meat chopping machine.
equal weight (500.0 cc.) of Sterilization: Sterilize (2) in the usual
(2) Add an
distilled water. manner, method not given. Sterilize (4)
by thru a Berkefeld filter.

filterting
(3) Stir and heat slowly to a boil.
Boil slowly for 15 minutes. Use: Cultivation of B. leprae. Author
(4)
Filter thru a filtering cloth until the
reported that growth was moist and
(5)
filtrate assumes a pulpy character. orange yellow in color. Growth reached
Press coagulated mass free
the a maximum in 48 to 64 hours.
from any liquid. Reference: Duval (1911 p. 370).
(6) Prepare a 2.5% agar solution in 2181. Loeffler's Malachite Green Nutrose
distilled water and filter. Agar
(7) Mix equal parts of (6) and sterile (5).
Add 3.0% glycerol. Constituents
(8)
1. Water 2000.0 cc.
(9) Tube sterilized (8).
and Beef 1 lb.
(10) Cool to 45C. Mix well solidify 2.
3. Agar 30.0 g.
quickly in a slanted position.
4. Nutrose (10.0% solution) Pour

(5) (3) over tissues, and solidify agar
5. Malachite Green quickly on an iced glass plate.
Preparation (6) Place plates in Novy jar, remove
(1) Prepare a beef infusion of 1 pound in oxygen and fill with hydrogen.
2 liters water. Sterilization: Method of sterilization of
(2) Add 30.0 g. agar. agar not given.
(3) Add 7.5 CO. N/1 HCl. Use: Cultivation of organism causing
(4) Boil 30 minutes. symptomatic anthrax or black-leg in
(5) Add 7.0 cc. N/1 NaOH. swine. The author reported that other
(6) Neutralize to litmus with sodium tissues could not replace that of
the
carbonate. guinea pig.
(7) Add 5.0 cc. N/1 sodium carbonate. Reference: Meyer (1915 p. 467).
(8) Add nutrose to make 1.0% solution
using a 10.0% solution of nutrose for 2183. Williams and Burdick's Egg Veal
this purpose. (0.5% peptone may Agar
be used instead of nutrose.) Constituents
(9) Boil. 1. Distilled water 1000.0 cc.
(10) Distribute in 500.0 cc. flasks. Glycerol (15.0%)
2. 150.0 g.
(11) Let cool slowly in sterilizer following 3. Egg white (10.0% soln.). . . 300.0 cc.
sterilization. 4. Egg yolk (10.0%) 300.0 cc.
(12) Decant clear supernatant liquid. 5- Veal 500.0 g.
(13) To each 100.0 cc. agar add 2.0 to 6. NaCl 5.0g,
2.5 cc. of a 2.0% heated malachite 7. Agar 15.0 g.
green solution, prepared with sterile 8. Gentian violet (1.0% al-
water. coholic solution) 1.0 cc.
(14) Pour 15.0 to 20.0 cc. to each Petri Preparation
dish. Prepare
(1) 300.0 cc. of a 10.0% filtered
(15) Leave dishes open until agar is cool aqueous solution of egg white.
and hard. (2) Prepare 300.0 cc. of a 10.0%, filtered
Sterilization: Sterilize (10) several hours on aqueous solution of egg yolk. To
each of 2 successive days in streaming produce the proper turbidity add
steam. 1.0 cc. of normal NaOH for each
Use Isolation of typhoid bacilli from feces.
:
100.0 cc. of emulsion.
References Loeffler (1906 p. 289), Klimmer Add
:
(3) 500.0 g. chopped lean veal to
(1923 p. 212). 1000.0 cc. of 15.0% glycerol solution
in water.
2182. Meyer's Tissue Agar
(4) Infuse (3) for 24 hours.
Constituents: (5) Filter (4).
1. Brain infusion agar 1000.0 cc. Add NaCl
(6) 5.0 g. to (5) and heat to
2. Glucose 5.0 g. boiling.
3. Tissue (guinea pig)
(7) Filter and render +1.0% alkaline.
Preparation Add
(8) 15.0 g. of powdered agar to
(1) Prepare brain infusion agar (details (7).
of method not given). Add
(9) 1.0 cc. of a 1.0% alcoholic solu-
(2) Dissolve 5.0 g. glucose in (1) and tion of gentian violet to sterile warm
adjust to reaction +0.5. (8).
(3) Boil, cool and inoculate sterile (2) (10) Pour sterile (9) into sterile (1).
with edema, abdominal fluid or heart (11) Add sterile (10) to sterile (2).
blood of infected animal, properly (12) Mix, tube and slant.
diluted. Sterilization: Sterilize (1), (2) and (8)
(4) Cut (method not given) guinea
sterile separately in the autoclave at 15 pounds
pig tissue into small pieces and pressure for 15 minutes.
distribute them over the bottom of a Use: Cultivation of B. tuberculosis. The
sterile Petri dish. authors reported that medium contained
sufficient moisture to prevent its drying Other authors used similar media for the
up under ordinary conditions and was cultivation of amoeba, parasitic flagellata
not liquefied by fast growing types. and ciliata, cladothrix and protozoa.
Reference: Williams and Burdick (1916 p. Variants
413). (a) Hiss omitted the NaCl.
(b) Musgrave and Clegg cultivated
2184. Burger's Basal Lead Acetate Agar
amoeba on a medium containing 20.0
Constituents g. agar, 0.3 toO.5 g. NaCl and 0.3 to 0.5
1. Water 1000.0 cc. g. beef extract per liter. The original
2. Agar 30.0 g. reaction of the medium being 1.5%
3. Lead acetate 1.0 g. alkaline before sterilization, and
4. Meat extract 4.0 g. 1.0% alkaline following sterilization.
Preparation The authors reported that in some
(1) Dissolve 2, 3 and 4 in 1. cases even smaller amounts of salt
(2) Suspend 1.0 g. of one of the added and extract were more desirable
nutrients in physiological salt solu- especially when amoeba were growing
tion and add 3.0% Na2C03. in company with some saprophytic
(3) Add (2) to melted (1), cooled to 56C. bacteria.
Sterilization: Not specified. (c) Linde isolated cladothrix {Clado-
Use : To study the production of hydrogen thrix dichotoma) on a medium con-
sulphide. taining 15.0 g. agar and 5.0 g. meat
Added nutrients: The author suspended extract per liter. Linde reported
1.0 g. of one of the following in physio- that contaminating forms grew but
logical salt solution: cladothrix soon overgrew them. The
cystine best solid medium for cultivation is
taurine 10.0 g. agar with 0.5 g. meat extract
sodium taurocholate in 1000.0 cc. water.
Reference: Burger (1914 p. 202). (d) Zikes cultivated Cladothrix dicho-
toma and Cladothrix natans on
2185. Hiss' Extract Agar
Linde's medium, containing 5.0 g.
Constituents meat extract and 10.0 g. agar per
1. Distilled water 1000.0 cc. liter.
2. Extract, Liebig's 5.0 g. (e) Malm (Besson) prepared the medium
3. NaCl 5.0 g. as follows:
4. Agar 15.0 g. (1) Dissolve 5.0 g. Liebig's meat
Preparation extract (or 20.0 g. Cibils) in 1000.0
(1) Dissolve agar in water by boiling cc. water.
over free flame for 30 to 45 minutes. (2) Soak 20.0 g. chopped thread
of
(2) Dissolve Liebig's extract and sodium agar in water for several
cold
chloride in (1). hours. Squeeze the water thru a
(3) Clarify by the coagulation of two egg cloth.
whites in (2). (3) Heat (2) in (1) at 100C. until the
(4) Filter thru cotton. agar is dissolved.
(5) Reaction is 0.75% acid to phenol- (4) Readjust the reaction if necessary.
phthalein. (5) Allow to cool to 55 or 60C.
(6) Tube in 10.0 cc. lots. (6) Beat the white of an egg in 100.0
Sterilization: Sterilize in the Arnold in the cc. of water and add to (5).
usual manner on three successive days. (7) Mix well.
Use: Differentiation of typhoid, colon (8) Autoclave at 120C. for one hour.
and allied forms. The author reported (9) Filter thru a moistened Chardin
that typhoid colonies were threaded. filter using a hot water funnel.
Colon colonies formed no threads, surface (10) Tube.

colonies dark at center, light at periphery. (11) Sterilize at 115 for 20 minutes.
Little difference, if any, if salt be omitted. (f) Harvey cultivated protozoa on a
medium containing 0.4 g. Lemco medium be clarified with egg whites a

meat extract, 0.4 g. NaCl and 25.0 g. more alkaline medium was obtained which
agar per liter. The reaction of the allowed better differentiation. One cc.
medium was 1.0% alkaline to phen- NaOH added to the medium also allowed
olphthalein. better differentiation.
References: Hiss (1902 p. 158), Musgrave Variants
and Clegg (1905 p. 335), Walker (1908 p. (a) The medium may be clarified with egg
490), Linde (1913 p. 373), Zikes (1915 p. white.
543), Besson (1920 p. 43), Harvey (1921-22 (b) One cubic centimeter of normal
p. 94). NaOH may be added.
(c) The medium may be prepared as
2186. Bacto Starch Agar (Dehydrated)
follows:
Constituents (1) Dissolve 15.0 g. agar in water by
1. Distilled water boiling for 30 to 45 minutes over a
2. Beef extract, Bacto 3.0 g. free flame.
3. Agar, Bacto 12.0 g. (2) Dissolve 5.0 g. Liebig's extract in
4. Starch, Soluble, Difco 10.0 g. (1).
Preparation (3) Coagulate the whites of two eggs in
(1) Dissolve 25.0 g. of Bacto starch agar (2) to clarify.
(dehydrated) in 1000.0 cc. of distilled (4) Filter thru absorbent cotton.
water by boiling or autoclaving, (5) Dissolve 10.0 g. glucose in (4).
preferably the latter. (6) Reaction is 0.75% acid to phenol-
(2) Restore loss if necessary. phthalein.
(3) Distribute as desired. (7) Tube in 10.0 cc. lots.
Sterilization: Sterilize for 20 minutes at 15 (8) Sterilize in the Arnold in the usual
pounds pressure. manner on three successive days.
Use: Culture medium. Reference: Hiss (1902 pp. 156, 158).
p. 14). 2188. Tanner's Malachite Green Extract
2187. Hiss' Glucose Extract Agar Agar
2. Agar 15.0 g. 2. Meat extract, Liebig. 3.0 g.
3. Extract Liebig's 5.0 g. 3. Agar 30.0 g.
4. NaCl 5.0 g. 4. Sucrose 10.0 g.
5. Glucose 10.0 g. 5. Malachite green
Preparation (2.0% solution) 2.0 to 2.9 cc.
(1) Dissolve the agar in water by boiling Preparation

over a free flame for 30 to 45 minutes. (1) Dissolve 2 in 1.
(2) Dissolve Liebig's extract and salt in (2) Acidify with 7.5 cc. normal HCl.
(1). (3) Dissolve 30.0 g. agar in (2) by
(3) Filter thru paper. boiling.
(4) Dissolve the glucose in (3). (4) Neutralize by adding 7.0 cc. normal
(5) The reaction is usually 0.8% acid to NaOH until neutral to litmus.
phenolphthalein. (5) Add 5.0 cc. ofnormal NajCOa.
(6) Tube in 10.0 cc. lots. (6) Heat in an Arnold for several hours.
Sterilization: Sterilize in the usual manner (7) Add 100.0 cc. of 10.0% sucrose
at 100C. on 3 successive days. solution to (6).
Use: Differentiation between typhoid, (8) Store in 100.0 cc. quantities.
colon and other allied bacilli. The (9) Before use redissolve and add 2.0 to
author reported that the typhoid bacilli 2.9 cc. of a 2.0% malachite green
gave threaded irregular colonies. Colon (trade mark Hochst. 120) to each
colonies were large and smooth. If 100.0 cc. of medium. Prepare the
malachite green solution in sterile Use: Differentiation of colon-typhoid

water. Do not boil. group. Author reported that on the
(10) Pour in Petri dishes. medium given above B. typhosus gave a
Sterilization: Not specified. uniform clouding. Colon bacilli gave
Use: General culture medium. only growth where inoculated.
Reference: Tanner (1919 p. 52). Variants
(a) Hiss prepared a similar medium using
2189. Noyes' Starch Extract Agar
10.0 g. agar instead of 5.0 g., and
Constituents: 25.0 g. of gelatin instead of 80.0 g.
1. Water 1000.0 cc. The reaction is adjusted to 2.0% acid
2. Agar (best) 15.0 g. instead of 1.5%. This medium is
3. Extract (Liebig) 5.0 g. poured in plates when ready for use.
4. Starch 4.0 g. On this medium the author reported
Preparation: (1) Dissolve 2, 3 and 4 in 1. that deep B. typhosus colonies under
Sterilization: Not specified. low power were small and generally
Use: General culture medium. Primarily spherical with rough irregular out-
used to study soil organisms. line. By transmitted light they were
Reference: Noyes (1916 p. 93). of a vitreous greenish or yellowish-
green color. Showed threaded
2190. Malenkovid's Xylose Extract Agar
growth. Deep colon colonies were
Constituents usually much larger and were spheri-
1. Water 1000.0 cc. cal or of whetstone form. By trans-
2. Xylan 20.0 g. mitted light they were darker more
3. Meat extract, Liebig's 5.0 g. opaque and less refractive than
Preparation: (1) Dissolve 2 and 3 in 1. typhoid colonies. By reflected light
Sterilization: Not specified. they were pale yellow. Neither of
Use: Cultivation of Merulius lacrymans. the organisms spread thru the me-
Reference: Malenkovid (1906 p. 407). dium. The typhoid colonies ap-
peared to the naked eye as mere
2191. Hiss' Gelatin Extract Agar among
greyish points the much
Constituents: larger and yellowish colon colonies.
1. Water 1000.0 cc. (b) Hiss gave the following method of
2. Beef extract, Liebig's 5.0 g. preparation of a similar medium.
3. NaCl 5.0 g. He reported that typhoid colonies
4. Agar 5.0 g. were small greenish, irregular and
5. Gelatin 80.0 g. fringed with threads. Colon colonies
6. Glucose 10.0 g. were larger and did not form threads.
Preparation (1) Boil 15.0 g. agar in 1000.0 cc.
(1) Dissolve agar in 1000.0 cc. of water distilled water over a free flame for
to which beef extract and NaCl have 30 to 45 minutes to dissolve.
been added. (2) Dissolve 15.0 g. gelatin, 5.0 g.
(2) When agar is completely melted Liebig's extract and 5.0 g. NaCl in
dissolve the gelatin by a few minutes (1).
_
boiling. (3) Clarify by the addition and coagu-

(3) Titrate and adjust to 1.5% normal lation of the whites of two eggs.
acid to phenolphthalein. (4) Filter thru absorbent cotton.
(4) Add one or two eggs beaten in 25.0 cc. (5) Dissolve 10.0 g. glucose in (4).
of water and boil for 45 minutes. (6) The reaction is usually about 1.2%
(5) Filter thru thin filter paper or ab- acid to phenolphthalein and no
sorbent cotton. acid or alkali is added.
(6) Add the glucose after clearing. (7) Tube in about 10.0 cc. lots.
(7) Tube. (8) Sterilize the usual manner at
in
Sterilization: Method of sterilization not 100C. on 3 successive days.
given. References: Hiss (1897 pp. 681, 694),
(1901 p. 728), (1902 pp. 151, 152, 156), 2. Ashes, wood 5.0 g.
Frost (1903 p. 340), Ball (1919 p. 80), 3. Agar 2.0 g.
Tanner (1919 pp. 51, 56) Heinemann 4. Maltose 2.0 g.
(1922 p. 34). Preparation
(1) Dissolve 2.0 g. agar and 2.0 g. maltose
2192. Stapp's Egg White Extract Agar in 100.0 cc. water by heat.
Constituents: (2) Add 5.0 g. wood ashes to 100.0 cc.
1. Water 500.0 cc. water.
2. Egg white (3) Boil and filter.
3. Meat extract 1.33 g. (4) Mix 100.0 cc. of (1), 40.0 cc. of (2),
4. NaCl 0.33 g. and 60.0 cc. of water or mix 100.0
5. Glucose 1.66 g. with 60.0 cc. (2) and add 40.0
cc. (1)
6. Agar 10.0 g. water or mix 100.0 cc. (1) with

cc. of
Preparation 100.0 cc. of (2) and add no water.

(1) Beat fresh egg white until it is a Sterilization: Sterilize in streaming steam
completely uniform liquid. or in the autoclave at 10 pounds for 10 to
(2) Mix with water in the ratio of 1:100 20 minutes.
by thoro shaking. Use: Cultivation of Pseudornonas radici-
(3) Close the flask and heat in the cola and other bacteria from the nodules
steamer for 20 minutes. of leguminous plants.
(4) When all the air is removed from the Variants

flask place the flask in the autoclave (a) Harrison and Barlow gave a number
and heat at 138 for 20 minutes. of different media, using varying
(5) Remove after cooling and filter. amounts of constituents, as in
(6) Mix 200.0 g. of the filtrate, 0.33 g. medium 1377, and then adding 1.2,
NaCl, 1.66 glucose, 10.0 g. agar, 1.33 1.5 or 2.0% agar to solidify.
g. meat extract and 300.0 cc. water. (b) Lohnis prepared the medium as
(The meat extract may be omitted). follows:
(7) Dissolve by heating in the steamer. (1) Infuse 4.0 g. of wood ashes with
(8) Filter while hot. 1000.0 cc. of water.
Sterilization: Not specified. (2) Add 4.0% maltose and 2.0%
Use: Cultivation of Bac. cobayae, Bac. K2HPO4 to (1).
capri, Bac. guano, Bac. musculi, Bac. (3) Solidify with agar.
hollandicus. (4) Sterilization not specified.
Reference: Stapp (1920 p. 5). (c) Harvey dissolved 4.0 g. maltose, 5.0
g. wood ashes and 10.0 g. agar in
Basal or complete media containing agar (d) Percival prepared the medium as
with derivatives of soil, ashes, etc., but not follows:
containing digests. (1) Add 8.0 g. of well burnt wood ashes
Ai. Ashes or derivatives employed. to 500.0 cc. distilled water and boil
Harrison and Barlow's Wood Ash for one minute.
Agar 2193 (2) Filter thru two sheets of paper.
A2. Soil infusions or extracts employed. (3) Add 4.0 g. maltose and 4.0 g. of
Fremlin's Soil Infusion Agar 2194 agar to (2).
Gowda's Soil Infusion Agar 2195 (4) Heat until dissolved.
Conn's Soil Infusion Agar 2196 (5) Filter.
Lohnis' Mannitol Soil Infusion (6) Tube.
Agar 2197 (7) Sterilize in the usual way, method
Perotti's Dicyandiamide Soil Infu- not given.
sion Agar 2198 (8) Slant.
(e) Giltner gave the following method of
2193. Harrison and Barlow's Wood Ash Agar preparation:
Constituents: (1) Stir 5.0 g. of wood ashes (elm,
1. Water 200.0 cc. beech, maple) into 1000.0 cc. of
distilled water for two or three 2195. Gowda's Soil Infusion Agar
minutes only. Constituents:
(2) Filter. 1. Water 2000.0 cc.
(3) Add 1.0% washed agar.
2. Soil 1000.0 g.
(4) Heat in steam for 30 minutes. 3. K0HPO4 1.5 g.
(5) Add 1.0% commercial sucrose. 4. (XH4)2S04 1.5 g.
(6) Boil for 5 minutes over a free flame. 5. MgS04 0.75 g.
(7) Strain while hot thru several thick- 6. reoS04 0.02 g.
nesses of clean cheese cloth. This 7. NaCl 3.0 g.
may be filtered if desired.
8. Na.COs 1.0 g.
(8) Sterilize by the Tyndall method in
9. Agar
the steamer.
Preparation
References Harrison and Barlow (1907 p.
:
(1) Digest 1000.0 g. soil in the autoclave

269), Lohnis (1913 p. 113), Harvey (1921-
for 2 hours with 2 liters of water.
22 p. Ill), Percival (1920 p. 204), Giltner
(2) Soak agar in water for 8 to 10 days.
(1921 p. 377).
(3) Dissolve 3 in 100.0 g. conductivity
2194. Fremlin's Soil Infusion Agar water.

(4) Dissolve 4, 5 and 6 in 100.0 g. con-
Constituents:
ductivity water.
1. Soil infusion 1000.0 cc.
(5) Dissolve 7 and 8 in 100.0 g. con-
2. Agar
ductivity water.
Preparation
(6) Add 1.0 cc. each of sterile (3), (4) and
(1) Solidify soil infusion with agar.
(5) to each agar plate. The details
of preparation of the plates are not
Use: Cultivation of nitroso bacteria.
Francois-Perey cultivated soil protozoa
given, or the amount of soil extract
that is to be added to the plate, or if
on a similarly prepared medium
Variants the washed agar is to be dissolved in
prepared medium the soil extract is not stated.
(a) Lohnis the as
follows: Sterilization: Pass the liquid from (1) thru
(1) Heat 1000.0 cc. of good rich garden a Chamberland filter, then sterilize on 3
soilwith a liter of tap water for 30 successive days in the autoclave under 15
minutes in the autoclave under pounds pressure for four hours. Sterilize
pressure of 1 atmosphere or boil (3), (4) and (5) separately,
method not
with 2 liters of water over a free given.
flame. Use: To study nitrification. Gowda re-
(2) Pour off the turbid liquid. ported that the ammonia was oxidized to
(3) Mix talc with the liquid. nitrites.
(4) Filter thru a double filter paper. Reference: Gowda (1924 p. 255).
necessary. 2196. Conn's Soil Infusion Agar

with agar.
(6) Solidify
Constituents:
1. Water 900.0 cc.
(b) Francois-Perey prepared the medium
as follows:
3. Glucose 1-0 g.
(1) Boil 1000.0 g. soil with 2000.0 cc. of
4. Agar 15.0 g.
water.
(2) Filter and dissolve 2.0% agar in Preparation
the filtrate. (1) Preparation of soil infusion not given.
References: Fremlin (1903 p. 373), Lohnis (2) Dissolve 3 and 4 in 1.
(1913 p. 101), Giltner (1921 p. 370), (3) Mix (1) and (2).
Francois-Perey (1925 p. 315), taken from Sterilization: Not specified.
(1925 p. 93). Use: Cultivation of soil bacteria.

Variants: Harvey prepared a similar me- 4. K2HPO4 (0.05%) 0.4 g.

dium as follows: 5. Agar (1.5%) 12.0 g.
(1) Dissolve 15.0 g. agar in 900.0 cc. of Preparation :
water. (1) Heat 1000.0 g. of good rich garden

(2) Boil 2000.0 cc. water and 1000.0 g. soil with a liter of tap water for 30
garden soil for 2 hours. minutes in the autoclave under pres-
(3) Pour off the turbid fluid. sure of 1 atmosphere or boil with 2
(4) Mix talc with (3). liters of water over a free flame.
(5) Filter until clear thru thick filter (2) Pour off the turbid liquid.
paper. (3) Mix talc with the liquid.
(6) Mix 100.0 cc. of (5) and 900.0 cc. of (4) Filter thru a double filter paper.
(1). (5) Make up the volume to 800.0 cc. if
(7) The reaction should be 0.5% acid to necessary.

phenolphthalein. (6) Dissolve 3, 4 and 5 in (5).
(8) Add 1.0 g. glucose to (7). (7) Tube.
(9) Tube. Sterilization: Sterilize in the autoclave or
(10) Sterilization not specified. stQamer.
References: Tanner (1919 p. 49), Harvey Use: Isolation of nodule bacteria.
(1921-22 p. 105). Reference: Lohnis (1913 p. 112).
2197. Lohnis' Mannitol Soil Infusion Agar

2198. Perotti's Dicyandiamide Soil Infusion
Constituents :
Agar
1. Water 1000.0 cc.
2. Soil 1000.0 g. Same as medium 1392 but solidified by the
3. Mannitol (1.0%) 10.0 g. addition of 1.5% agar. ^
GROUP III. MEDIA SOLIDIFIED BY ADDITION OF GELATIN
SUBGROUPS OF LIQUEFIABLE sporogenes, Bacillus hotulinus. Other
GELATIN MEDIA investigators used similar media for a
-, , iu , .
1 variety of purposes.
Ai. Xot containme
^
organic materials other
,
, .
<=
Variants:
than gelatin
Kufferath used a 10.0, 20.0, 30.0,
Subgroup III-A (Med. 2199-2204) .n -rr, r> ^ I- i ^- ^
jj- 40.0 or 70.0 gelatin solution.
, r^
^
,
As. Containing organic material
.
X 1
m

addi- /, ,,
s j mnrv x-
(b) Meyer used a 10.0% gelatin solution,
x- i i
.
, ^
tion to gelatin j- x j n ôr / xx xxto culti"
ix-
none ooTiN adjustcd to 0.4% to attcmpt
i.
Subgroup TTT Ti /AT J

, '
III-B (Med. 2205-2371) [ . '." ^
vate the causative agent oi symptom-
*^ STTRPROUP TTT-A atica anthra.x, or blackleg in swine.
(c) Conn (1916) cultivated spore forming
Gelatin Media, with Additional bacteria on a 12.0% gelatin in tap

water. The medium was clarified
Constituents Inorganic
with white of egg.
SUBGROUP III-A (d) Conn (1917) cultivated soil micro-
r^ ,
J. ,
. . 1 organisms in a medium prepared by
Basal or complete media containing
,
r V. u
gela- , or>r> n ,
t u
dissolving 200.0 g. of Gold Label
i
.j,.^. .,
tin.
,.,
Additional constituents, if any, in-
, ,
.
^. , ,^
gelatin in 1000.0 cc. tap water, adjust-
.
^ , x ^
.\..
Ai. Containing gelatin
,,.
and water only. , , , ing to 0.5% normal acid to phenol-

n x on n
'
. u-x /-I x- oirn . 1
phthalein /or> (20.0 to 30.0 cc. of normal
i e i
Natsuschita s Gelatin 2199 t'^ ^,

thatxionn
Jv ixu
_, o ix T-i
, r^ , x- ooriA 1
NaOH). He reported x
120.0 g. of
c
Taylor's Salt Free Gelatin 2200 ^ ^

-x j ox x
Bacto or tt Glue r^
^ r^^
--' United States Co.
-x Ti r-i X- , oori
Matzuschita s Basal Gelatin
,, , ,, .
2201
.
. 1
,
1
.
.,^, .
gelatin might be used instead or Gold

,. ^ irrû
A2. Containing added salts. ^ ,. _
Label. ^"i
, ,
. ,
^ ^ J XT xi u> Tiu u X Only 10.0 cc. of normal

Stutzer and Hartleb's Phosphate .^ ___
NaOH Were required xto give xu
-^
j ,
^. , r,r,nr> the de-

(jrelatin zzuz
x- i tx
, , , , ,,T^ ,, ,, nnrvo Sired Teaction. it was necessary ^
to
Stoklasa s "Kollagen' 2203 ..
, ^, ,. ., ,
.., -- . , , , clarify the medium, prepared with

Sohngen's Magnesium
,
Phosphate ....^, ^^ . , J^ ,
x- u
'
r^ ? J.- /Tr- 1- oonA United States Glue Co. gelatin, by ,
Gelatin (Vierling)N 2204 ^, ,,.. . -^ . \ , ,
the addition oi white ot an egg, but

2199. Matzuschita's Gelatin clarification was not necessary when
Constituents: using Bacto gelatin.

1. Water . 1000.0 cc. (^) Committee S. A. B. gave the following
2 Gelatin '
. 100.0 g. method of preparation
Preparation: (1) Dissolve 120.0 g. Gold Label,
Thoroly shake 100.0 g. gelatin in one 100-0 B^^to, or 100.0 g. U. S. Glue
(1)
water.
liter of
^^- gelatin in 1000.0 cc. distilled
water.
(2) Heat until the gelatin is melted.
(4) Boil in the steamer. (3) Adjust the pH between 6.6 and 7.4.
(4) Sterilize.
(5) Filter.
Sterilization: Sterilize in the steamer on (0 Waksman studied the metabolism of
from 2 to 5 successive days for 15 to actinomycetes in a 15.0% gelatin

solution,
30 minutes. Incubate for 2 days at 37C.
to test sterility. (g) Weiss dissolved 15.0% gelatin in
Use: Used by Matzuschita for the cultiva- water and used the medium to de-
tion of spore forming bacilli, Clostridium termine the bacterial count of water.
hutyricum, Bacillus oedematis maligni, (h) Harvey cultivated bacteria found in
Bacillus anthracis symptomatici, Bacillus soil on a medium prepared from one
711
712 CULTURE MEDIA FOR CULTIVATIOX OF MICRO ORGANISMS
part gelatin to five parts of tap water. 2202. Stutzer and Hartleb's Phosphate
The reaction was adjusted to 0.5% Gelatin
acid to phenolphthalein.
Constituents:
Kufferath (1914 p. 559), Meyer 1- Water looo.O cc.
(1915
2. Gelatin lOO.O g.
p. 467), Conn (1916 p. 188), (1917 p. 42),
Committee 3. Potassium phosphate 1.0 g.
S. A. B. (1918 p. 116), Waks-
Preparation
man (1920 p. 22), Weiss (1920 p. 25),
Harvey (1) Dissolve 100.0
gelatin and 1.0 g.
g. of
(1921-22 p. 105), Committee
of potassium phosphate in water.
S. A. B. (1923 p. 10).
(2) Make up the volume to 1 liter.
2200. Taylor's Salt Free Gelatin

(3) Add soda to obtain a neutral reaction.
Constituents: Use: Cultivation of Zoogloea ramigera, a
1. Distilled water 1000.0 cc. nitrate fungus.
2. Gelatin 100.0 g. Reference: Stutzer and Hartleb (1897
Preparation p. 236).
(1) Soak gelatin in weak alkali and after
it has become swollen, wash re- 2203. Stoklasa's "Kollagen"
peatedly by decantation. Constituents
(2) Dissolve gelatin in water by heat. Water
1- 1000.0 cc.
(3) Precipitate by the least effective "Kollagen"
2. with
(c.p.
quantity of alcohol. 17.02% N "Ossein")
(4) Wash with alcohol and ether and dry. 3. K.SO4 1.0 g.
(5) Dissolve 100.0 g. of (4) in 1000.0 cc. 4. MgCl2 0.5 g.
of twice distilled water (conductivity 5. Iron sulphate 0.1 g.
atl6C. tobel.l X lO-^). 6. CaHP04 0.0 or 1.0 g.
(6) Submit the solution to electrolysis for Preparation: Dissolve
(1) 2, 3, 4, 5 and 6
five days, the conductivity reaching a in 1.
constant (8 X 10-). During electro- Sterilization: Sterilize in the autoclave.
lysis,dialysis is taking place at all Use: To study XH3 formation by Bacillus
times by means of a special apparatus. mycoides, Bacillus suhtilis, Bacillus
A
110 volt circuit was used. mesentericus vulgatus.
Sterilization: Not specified. Reference: Stoklasa (1911 p. 476).
Use: Salt free medium. The author re-
ported that the medium
did not support 2204. Sohngen's Magnesium Phosphate
the growth of ordinary saprophytic or Gelatin (Vierling)
pathogenic bacteria.
Constituents
Reference: Taylor (1905 p. 114).
1. Water 1000.0 cc.
2201. Matzuschita's Basal Gelatin 2. Gelatin lOO.O g.
Constituents
3. MgSO^. 1.0 g.
1. Gelatin
4. K2HPO4 1.0 g.
10.0%
Preparation: (1) Solidify one of the added
5. CaCU trace
nutrients by the addition of 10.0% Preparation
gelatin.
(2) Pour sterile (1) into plates.
Sterilization : Method not specified.
Use: Cultivation of intestinal bacteria.
Use: Cultivation of Mycobacteria. The
Added nutrients: The author used one of
author reported that the plates were
the following:
slightly turbid due to a precipitate of
Beer wort
Mg3(P04)2. After 8 days mycobacteria
Urine
caused a clearing of the medium around
Straw Infusion
the colony.
Reference: Matzuschita (1901-02 p. 214). Reference: Vierling (1920 p. 202).
SUBGROUP III-B Munter's Basal Glucose Salt Gela-

tin 2223
Gelatin Media, with Additional Mortensen's Basal Nitrate Gelatin 2224 . .
Constituents Organic A2. Additional organic nitrogen employed.

B:. Containing carbohydrates.
Key to the Sections of Subgroup III-B Ci. Carbohydrates, only, added.
Chemical composition of all constit- Beijerinck's Starch Asparagin Gela-
Ai.
uents known Section 1 (Med. 2205-2234)
.
tin 2225
A2. Chemical composition of one or more

C2. Carbohydrates and other organic car-
constituents not definitely known. bon added.
Grimbert's Starch Asparagin Gela-
Bi. Containing digests
Section 2 (Med. 2235-2323) tin (Charrin and Dissard) 2226
B2. Not containing digests Kappen's Cyanamide Asparagin

Section 3 (Med. 2324-2371) Gelatin 2227
Maassen's Glucose Asparagin Gela-
SUBGROUP III-B. SECTION 1 tin (Klimmer) 2228
Basal or complete media
containing B2. Not containing carbohydrates.
gelatin with additional organic materials Ci. Containing organic acids.
of known chemical composition. van Delden's Lactate Asparagin

Gelatin 2229
Ai. No additional organic nitrogen used.
Frankel's Lactate Asparagin Gela-
Bi. Inorganic nitrogen salts not added.
tin (Klimmer) 2231
Ci. Containing carbohydrates.
Beijerinck's Starch Gelatin 2205 Uschinsky's Glycerol Asparaginate
Gelatin (Klimmer) 2232
Waksman's Starch Gelatin 2206
Dombrowski's Glucose Gelatin 2207 C2. Not containing organic acids.
Fraenkel's Glucose Glycerol Gelatin. 2208 Goslings' Asparagin Gelatin 2233
Beijerinck's Acid Starch Gelatin.. 2209 Sullivan's Nitrate Asparagin Gela-
BobiliofT-Preisser's Basal Glucose tin 2234
Gelatin 2210
2205. Beijerinck's Starch Gelatin
C2. Not containing carbohydrates; other
organic carbon used. Constituents
Sohngen's Petroleum Gelatin 2211
1. Water 1000.0 cc.
Weisser's Glycerol Gelatin 2212
2. Gelatin 100.0 g.
Proskauer and Beck's Glycerol Gela- 3. KH2PO4 0.5 g.
(Klimmer)
tin 2213
4. Starch (soluble) 1-0 g.
Fermi's Phenol Gelatin 2214
Preparation
B2. Inorganic nitrogen salts added.
Sohngen's Ammonium Chloride
Malate Gelatin 2215
Woltje's Basal Sucrose Gelatin
Use: Detection of quinone production.
(Zikes) 2216
The author seeded the medium by pouring
Mortensen's Sucrose Ammonium a culture of S. chromogena in medium 250
Sulphate Gelatin 2217
over the plate. After a few days the
Higgins' Lactose Ammonium Suc- gelatin was melted and the quinone was
cinate Gelatin 2218
extracted with benzol.
V. Tubeuf's Sucrose Ammonium Ci- Reference: Beijerinck (1900 p. 10).
trate Gelatin (Malenkovic) 2219
Cohn's Ammonium Tartrate Gelatin 2206. Waksman's Starch Gelatin
(Klimmer) 2220
Kossowicy's Ammonium Phosphate Constituents
Sucrose Gelatin (Will) 2221 1. Distilled water 1000.0 cc.
V. Tubeuf's Citric Acid Ammonium 2. Gelatin 150.0 g.
Nitrate Gelatin 2222 3. Starch 10.0 g.
Preparation dissolved in dilute HCl, was added the

(1) Dissolve 2 and 3 in 1. gelatin was colored highly blue as far
(2) Tube. as the quinone had diffused from the
Sterilization: Method not given. colony.
Use: To study metabolism of actinomy- Reference: Beijerinck (1900 p. 9).
cetes.
Reference: Waksman (1920 p. 22). 2210. Bobilioflf-Preisser's Basal Glucose
Gelatin
2207. Dombrowski's Glucose Gelatin
Constituents
Constituents
1- Water 1000.0 cc.
2. Hayduck's Mineral Solution 20.0 cc.
2. MgSO, (0.1%) 1.0 g.
Glucose
3. 50.0 g.
3. K2HPO4 (0.2%) 2.0 g.
4. Gelatin (10.0%) IQO.O g.
4. Glucose (5.0%) 50.0 g.
Preparation
5. Gelatin (10.0%) 100.0 g.
(1) Composition of Hayduck's Mineral
Solution not given.
(2) Add 20.0 cc. of (1) to 1000.0 cc. of
Use: Cultivation of milk yeasts.
distilled water.
Reference: Dombrowski (1910 p. 380).
nutrients in (2).
2208. Fraenkel's Glucose Glycerol Gelatin
(4) Distribute in 10.0 cc. lots in small
Constituents Erlenmeyer flasks.
1. Water 1000.0 cc. Sterilization: Sterilize in the steamer.
2.Gelatin 150.0 g. Use: Cultivation of fungi imperfecti,
3.Glycerol 2.5 g. oospora. The author reported that the
4. Dextrose 2.5 g. growth was inhibited using inorganic
Preparation: (1) Dissolve 2, 3 and 4 in 1. nitrogen source. The gelatin was lique-
Sterilization: Sterilize in the usual manner fied.
(method not specified). Added nutrients : The author added one of
Use: Cultivation of nitrate fungi. the following materials:
Reference: Fraenkel (1898 p. 10). Asparagin 2.5 g.
Peptone 21g
2209. Beijerinck's Starch Gelatin (NH4)2S04 2.5 g.'
Constituents
Reference: Bobilioff-Preisser (1916 p. 395).
1- Water 1000.0 cc.

2211. Sohngen's Petroleum Gelatin
2. Gelatin 100.0 g.
3. Lactic acid Same as medium
1460 but solidified by
4. Starch the addition of 10.0% gelatin instead of
Preparation 2.0% agar.
(1) Dissolve 10.0% commercial gelatin
in water. 2212. Weisser's Glycerol Gelatin
(2) Acidify slightly with lactic acid (a few Constituents
drops). 1. Water 1000.0 cc.
(3) Add a little starch (amount not 2. Gelatin (10.0%) 100.0 g.
given). 3. Glycerol (2.0%') 20.0 g.
(4) Four into Petri dishes. Preparation
Sterilization: Not specified. (1) Dissolve 2 and 3 in 1.
Use: To quinone production by
test for (2) Adjust the reaction to a slight alka-
Streptothrix chromogena. The author linity.
reported that the gelatin turned brown Sterilization: Not specified.
due to the oxidation of the formed Use: Cultivation of Neapler cholera bac-
quinone. The acid hindered this oxida- teria (Emmerich).
tion and stored the quinone. When KI Reference: Weisser (1886 p. 326).
715
and Beck's Glycerol 2216. Woltje's Basal Sucrose Gelatin

2213. Proskauer
(Zikes)
Gelatin (Klimmer)
Constituents
Constituents: 1000.0 cc.
1. Water
1. Water 1000.0 cc.
2. K2HPO4 5.0 g.
2. Glycerol 15.0 g.
3. MgS04 2.5 g.
3. (NH4)2C03 3.5 g.
75.0 g.
4. Sucrose
4. MgS04 (Crystalline) 2.5 g.
5. Gelatin 140.0 g.
5. KH2PO4 1-3 g.
Preparation
6. Gelatin 100.0 to 150.0 g.
and 6 (1) Dissolve 2, 3, 4 and 5 in 1.
Preparation: (1) Dissolve 2, 3, 4, 5
(2) Dissolve one of the added
nutrients
in 1.
in (1).
Use: Cultivation of tubercle bacilli. Use: To study perithezien formation by
Reference: Klimmer (1923 p. 172). Aspergillus oryzae.
2214. Fermi's Phenol Gelatin of peptone, 10.0 g. asparagin, 10.0 g.
(NH 4)2804 to the basal medium.
Constituents Variants Zikes used 5.0 g. K2SO4 instead of
:

1. K2HPO4, used 140.0 g. gelatin and used
Gelatin (10.0%) 100.0 g.
2. 10.0 g. asparagin as an added nutrient.
Phenol (0.5%) 5.0 g.
3. References: Zikes (1922 p. 342).
4. Na.COa (1.0 or 2.0%)
Preparation 2217. Mortensen's Sucrose Ammonium
at 80 to 90C. Sulphate Gelatin
(1) Dissolve the gelatin in 1
(2) Add 3 and 4 to (1).

(3) Adjust the reaction as desired, by the addition of gelatin.
addition of soda, mineral or organic
acids. 2218. Higgins' Lactose Ammonium
(4) Distribute in tubes. Succinate Gelatin
Constituents
Use: Prove proteolytic enzyme. The 1. Water 1000.0 cc.
author reported that if a proteolytic en- 2. Glucose 2.5 g.
zyme was present the gelatin was Lactose 2.5 g.
3.
liquefied. 4. Ammonium succinate. 2.5 g.
Variants: The author used 1.0, 2.0 or 5.0% 5. (NH4)2S04 1.0 g.
gelatin instead of 10.0%. 6. KCl 1.5 g.
Reference: Fermi (1906 p. 177). 7. KNO3 2.5 g.
8. Ammonium phosphate 2.5 g.
Gelatin (7.0%) 70.0 g.

2215. Sohngen's Ammonium Chloride 9.
Malate Gelatin Preparation

(1) Dissolve 2, 3, 4, 5, 6, 7, 8
and 9 in 1.
Constituents (2) Neutralize to phenolphthalein.
1. Water 1000.0 cc. Sterilization: Method not given.
2. K.,HP04 (0.05%) 0.5 g. Use: Cultivation of organisms causing
3. NH4CI (0.05%) 0.5 g. cholera, Pictoy cattle disease and others.
4. Calcium malate (0.5%) 5.0 g. Reference: Higgins (1898 p. 668).
5. Gelatin (10.0%) 100.0 g.
Dissolve 4 and 5 in 1. 2219. v. Tubeuf's Sucrose Ammonium

Preparation: (1) 2, 3,
Citrate Gelatin (Malenkovic)
Use: Cultivation of urea splitting Constituents:

organisms. 1. Water 1000.0 cc.
Reference: Sohngen (1909 p. 94). 2. K2HPO4 5.0 g.

3. MgS04 1.0 g. Preparation: Dissolve

(1) 2, 3, 4, 5, 6 and 7
4. Ammonium citrate. . 2.0 g. in 1.
5. Gelatin 50.0 g. Sterilization Method not
: given.
6. Cane sugar 50.0 to 100.0 g. Use: Cultivation of Merulius lacrymans
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 causing dry rot. The author reported
in 1.
that a luxuriant white flaky growth was
Sterilization: Not specified. obtained.
Use: Cultivation of Merulius lacrymans. Variants: The author used 50.0 or 100.0
g.
Reference: Malenkovic (1906 p. 407). glucose or 50.0 or 100.0 g. sucrose instead
of 10.0 g. glucose.
2220. Cohn's Ammonium Tartrate Gelatin Reference: v. Tubeuf (1902 p. 130).
(Klimmer)
2223. Hunter's Basal Glucose Salt
Constituents:
1- Water Gelatin
1000.0 cc.
2. K2HPO4 5.0 g. Same as medium
1442 but solidified by
3. MgS04 (crystalline) 5.0 g. 15.0% gelatin instead of agar.
5. Gelatin 100.0 g. 2224. Mortensen's Basal Nitrate Gelatin
Preparation
addition of gelatin.
(2) Solidify by dissolving 100.0 to 150.0 g.
gelatin in (1). 2225. Beijerinck's Starch Asparagin Gelatin
Use: General synthetic culture medium. Constituents
Reference: Klimmer (1923 p. 172). 1- Water iqqo.O cc.
2. Starch (soluble) 5.0 g.
2221. Kossowicy's Ammonium Phosphate 3. Asparagin 2.5 g.
Sucrose Gelatin (Will)
5. Gelatin 100. g.
Constituents Preparation: Dissolve 2, 3, 4 and 5 in 1.
1- Water 1000.0 cc. Sterilization: Method not given.
2. Sucrose (5.0%) 50.0 g. Use: Cultivation of Saccharomyces ellip-
3. KCl (0.4%) 4.0 g. soideus.
4. (NH4)oHP04 (0.4%) 4.0 g. Reference: Beijerinck (1895 p. 336).
5. MgSO4(0.4%) 4.0 g.
6. CaHP04
(0.04%) 0.4 g. 2226. Grimbert's Starch Asparagin Gelatin
7. Gelatin (10.0%) 10.0 g. (Charrin and Dissard)
Constituents
in 1.
2. Maltose 1.0 g.
Use: Cultivation of non-spore forming 3. Starch (soluble) 2.0 g.
saccharomj^ces.
4. Asparagin 2.0 g.
Reference: Will (1908 p. 387).
2222. V. Tubeuf's Citric Acid (neutral) 2.0 g.
Ammonium g'
Nitrate Gelatin
6. K2SO4 2.0
7. MgS04 2.0 g.
Constituents 8. Ammonium bimalate 2.0 g
1- Water 100.0 cc. 9. MgCOs g."
1.0
2. NH4NO3 10.0 g. 10. Gelatin 150 g
3. Potassium phosphate 5 g 11. KI
4. MgS04 1.0 g. Preparation
5. Citric acid 2.0 g. (1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1.
6. Gelatin 5O.0 g. Melt 150.0 g. gelatin in
(2) (1) by heating
7. Glucose 10.0 g. on a salt water bath.
CULTURE MEDIA FOR CULTIVATION 6F MICROORGANISMS 717
(3) Carefully add KOH

until 10.0 cc. of 7. Mohr's salt trace
the medium will be neutralized to 8. (FeS04) (NH4)2S04
phenolphthalein by 5.0 cc. of lime Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
solution. (This is an equivalent of in 1.
1.0 g. H2SO4 per liter.) Sterilization: Not specified.
(4) Heat in the autoclave for 15 minutes Use: Isolation of Microspira desulfuricans
at 110. and Microspira aestuarii.
(5) Filter.
Variants: The author added 30.0 g. NaCl
(6) Tube in 9.0 cc. lots. for the isolation of Microspira aestuarii.
(7) Just before use add 1.0 cc. of a 10.0% Reference: van Delden (1903-04 p. 88).
KI solution to each tube (liquefied). 2231. Frankel's Lactate Asparagin Gelatin
Sterilization: Final sterilization not spe-
(Klimmer)
cified.
Use: Isolation of typhoid bacilli. Char- Constituents:
rin and Dissard cultivated Bacillus 1. Water 1000.0 cc.
pyocyaneus on this medium. 2. K2HPO4 2.0 g.
Variants: Remy omitted the KI. 3. NaCl 5.0 g.
References: Charrin and Dissard (1896 4. Ammonium lactate 6.0 g.
p. 816), Remy (1900 p. 559), Thoinot and 5. Asparagin 4.0 g.
Masselin (1902 p. 336). 6. Gelatin 100.0 g.

Preparation
2227. Kappen's Cyanamide Asparagin (1) Dissolve 2, 3, 4 and 5 in 1.
Gelatin (2) Make distinctly alkaline (indicator

not specified).
addition of 10.0% gelatin.
2228. Maassen's Glucose Asparagin Gelatin
(Klimmer) 2232. Uschinsky's Glycerol Asparaginate
Gelatin (Klimmer)
Constituents
2. Mgb04 (crystalline). 0.4 g. 1. Water 1000.0 cc.
3. Asparagin 10.1 g. 2. K0HPO4 2.0 to 2.5 g.
4. CaCl2 0.01 g. 3. MgS04 (crystalline).. 0.2 to 0.4 g.
5. Na2HP04 2.0 g. 4. NaCl 5.0 to 7.0 g.
6. Na.COs (crystalline) 2.5 g. 5. Ammonium lactate... 6.0 to 7.0 g.
7. Glucose 5.0 to 10.0 g. 6. CaCU 0.1
8. Malic acid 7.0 g. 7. Sodium asparaginate. 3.5 g.
9. Gelatin 100.0 g. 8. Glycerol 30.0 to 40.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 9. Gelatin 100.0 g.
7 and 8 in 1. Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7,
Sterilization: Not specified. 8 and 9 in 1.
Use: General synthetic culture medium. Sterilization: Not specified.

Reference: Klimmer (1923 p. 172). Use: General synthetic culture medium.
2229. van Delden's Lactate Asparagin
Gelatin 2233. Goslings' Asparagin Gelatin
1. Water ~.
1000.0 cc. 1. Water 100.0 cc.
2. Gelatin 100.0 g. 2. Gelatin 10.0 g.
3. Sodium lactate 5.0 g. 3. Asparagin 10 g.
4. Asparagin 1-0 g. 4. Na2S04 10 g-
5. MgS04 10 g. 5. NaCl 0.5 g.
6. K2HPO4 0.5 g. 6. PbCOs

Preparation: (1) Dissolve 2, 3, 4, 5 and 6 B2. One or more of the additional constit-
in 1.
uents organic.
Sterilization: Not specified. Ci. All additional organic constituents of
Use Cultivation of organism causing water
:
known chemical composition.
rust in flax (Plectridium pectinovorum). Di. No additional nitrogen supplied.
Author reported that if HaS be produced El. Only one type of organic carbon added.
the medium was turned brown. Fi. Carbohydrates employed.
Reference: Goslings (1904 p. 392). C 1. Monosaccharides used.
Utz's Glucose Peptone Gelatin 2246
2234. Sullivan's Nitrate Asparagin Gelatin
Beijerinck's Glucose Peptone Gela-
Constituents: tin 2247
1. Water 1000.0 cc. Pfeilerand Lentz's Ringer Solution
2. Asparagin 10.0 g. Peptone Gelatin 2248
3. MgSOi 0.2 g. Vierling's Glucose Peptone Gelatin. 2249
4. CaCl2 trace Sear's Glucose Peptone Gelatin 2250
5. K0HPO4 1.0 g. Go. Disaccharides used.
6. NaCl 1.0 g Utz's Lactose Peptone Gelatin 2251
7. KNO3 0.2 g. Molisch's Sucrose Peptone Gelatin
8. Gelatin 100.0 g. (Smith) 2252
Preparation: (1) Dissolve 2, 3, 4, 5, Trommsdorff's
6, Sucrose Peptone
7 and 8 in 1.
Gelatin 2253
Sterilization: Method not given. G3. Polysaccharides used.
Use: Used to determine liquefaction of Molisch's Dextrin Peptone Gelatin 2254 . .
gelatin by B. prodigiosus and B. pyo- F2. Alcohols or organic acids employed.

cyaneus, and other organisms. Matzuschita's Glycerol Peptone
Reference: Sullivan (1905-06 p. 119). Gelatin 2255
Molisch's Glycerol Peptone Gelatin. 2256
SUBGROUP III-B. SECTION 2 Freudenreich and Jensen's Lactate
Peptone Gelatin (Boekhout and
Basal or complete media containing Ott de Vries) 2257
gelatin and a digest. E2. More than one type of organic carbon
Ai.* Peptone (or other commercial digest) added.
employed. Matzuschita's Glucose Glycerol Pep-
Bi. Additional constituents, if any, in- tone Gelatin 2258
organic. Dombrowski's Glucose Peptone
Ci. Basal media employed with the addi- Gelatin 2259
tion of other materials. D2. Additional nitrogen supplied.
Will's Basal Salt Peptone Gelatin. 2235 .
Beijerinck's Asparagin Peptone
Matzuschita's Basal Peptone Gela- Gelatin 2260
tin 2236 Remy's Phenolated Asparagin Pep-
Banning's Basal Peptone Gelatin.. 2237 tone Gelatin 2261
Harvey's Basal Neutral Red Pep- Scholz and Krause's Urea Peptone
tone Gelatin 2238 Gelatin 2262
C2. Complete media. C2. One or more of the additional organic
Weiss' N.ahrstoff Heyden Gelatin. 2239 . .
constituents of unknown chemical com-
Jacobi's Peptone Gelatin position.
2240
Metchnikoff's Peptone Gelatin Di. Containing unknown constituents of
(Thoinot and Masselin) plant origin only.
2241
Mprtensen's Peptone Gelatin Beijerinck's Peptone Malt Gelatin. 2263
2242 .
Rossi's Salt Peptone Gelatin d'Herelle's Peptone Potato Gelatin. 2264

2243
Metchnikoff's Nitrate Peptone Gela- Eisner's Peptone Potato Gelatin
tin (Bran) 2244 (Heinemann) 2265
Lichtenstein's Cenovis Gelatin de Rossi's v. Faba Peptone Gelatin. 2266
2245
Molisch's Pea Gelatin (Lohnis) 2267
* See page 720 for A2. Molisch's Peat Gelatin 2268
unknown constituents of Gi. Additional constituents, if any, in-

D2. Containing
animal origin. organic.
El. Animal derivatives (exclusive of ex- Hi. Basal media employed with the addi-
tracts and infusions) employed. tion of other materials.
em- Rosenberg's Basal Extract Gelatin. 2293
Fi. Cells, tissues or their derivatives
Biirger's Basal Fuchsin Sulphite
ployed.
Extract Gelatin 2294
Deycke's Peptone Alkali Albumin
2269 Tausz and Peter's Basal Ragit
Gelatin
2270 Gelatin 2295
Kotlar's Pancreas Gelatin
2271 Buchan's Basal Litmus Extract
Harde's Tissue Gelatin
Gelatin 2296
F2. Body fluids employed.
H2. Complete media.
Pergola's Nitrate Alkaline Blood
Bacto Nutrient Gelatin (Dehy-
Gelatin 2272
drated) 2297
Fs. Secretions or excretions employed.
Heinemann's Peptone Extract Gela-
Gi. Milk or its derivatives added. tin 2298
Matzuschita's Peptone Milk Gelatin 2273
Frost's Peptone Extract Gelatin 2299
Raskin's Whey Peptone Gelatin. . . . 2274
Kohn's Peptone Extract Gelatin .... 2300
Appel's Peptone Whey Gelatin 2275
Buchan's Neutral Red Peptone Ex-
Meier's Whey Peptone Gelatin 2276
tract Gelatin 2301
Wigger's Whey Gelatin 2277
von Varon's Nitrate Extract Gela-
G2. Milk or its derivatives not employed. tin 2302
Piorkoweki's Peptone Urine Gela- G2. Containing additional organic constit-
tine 2278 uents.
Heller's Urine Peptone Gelatin 2279 Henneberg's Glucose Extract Gela-
E2. Animal extracts or infusions employed. tin 2303
Fi. Infusions specified. Gottheil's Glucose Extract Gelatin. 2304

constituents, any, in- Buchner's Sucrose Extract Gelatin 2305 .
Gi. Additional
.
if
organic. Percival's Urea Extract Gelatin... 2306

Schultz's Peptone Infusion Gelatin. 2280 F3. Whether extracts or infusions employed
Smith's Peptone Infusion Gelatin.. 2281 not definitely stated.
Loeffler's Malachite Green Infusion Gi. Additional constituents, if any, in-
Gelatin 2282 organic. (Exclusive of indicators.)
Zipfel's Basal Nutrient Gelatin. 2307
Roux and Rochaix's Peptone Infu- . . .
2283 Wurtz's Nutrient Gelatin 2308

sion Gelatin
Heller's Indicator Nutrient Gelatin 2309
Miiller's Indicator Infusion Gelatin. 2284
.
G2. Containing additional organic ma-

G2. Containing additional organic constit-
terials.
uents.
Hi. Chemical composition of additional
Jackson and Muer's Liver Infusion
2285 constituents known.
Gelatin
Matzuschita's Glucose Gelatin 2310
Fuhrmann's Glucose Infusion Gela-
2286 Ramond's Rubine Acid Lactose
tin
Gelatin 2311
Choquet's Glycerol Phosphate Infu-
Wurtz's Litmus Lactose Gelatin 2312
sion Gelatin (Besson) 2287
Wurtz's Glycerol Gelatin 2313
Teague and Clurman's Congo-Red
Holz's Phenol Gelatin 2314
Brilliant Green Glucose Gelatin. 2288 .
Stutzer and Hartleb's Urea Gelatin. 2315

Kowalki's Glycerol Lung Infusion
2289 H2. Containing additional constituents of
Gelatin
Uffelmann's Methyl Violet Infusion
unknown chemical composition.
2290 Ii. Cells, tissues or derivatives added.
Gelatin
2291 Park, Williams and Krumwiede's
Vincent's Glucose Glycerol Gelatin.
Meat Gelatin 2316
Huntoon's Hormone Gelatin 2292
Worth's Glucose Liver Gelatin 2317
Fj.* Extracts specified.
Nastinkoff's Egg Yolk Nutrient
* See Fs next column. Gelatin 2318
12. Body fluids added. 2.Gelatin (15.0%) 150.0 g.

Pergola's Alkaline Blood Gelatin. 2319
. . 3.Nahrstoff Heyden (0.8%) ... 8.0 g.
Muller's Blood Gelatin 2320 Preparation: (1) Dissolve 2 and 3 in 1.
Mailer's Serum Gelatin 2321 Sterilization: Not specified.
13. Other animal derivatives added. Use: Bacterial count of water. The author
A2. No commercial digest employed. Non- reported that generally an alkaline me-
commercial digest used. dium gave a slightly higher bacterial
Beijerinck's Trypsmized Gelatin... 2322 count.
Jensen's Pepsinized Milk Gelatin. 2323 . .
Reference: Weiss (1920 p. 25).
2235. Will's Basal Salt Peptone Gelatin
2240. Jacobi's Peptone Gelatin
addition of 10.0% gelatin. The author also
Constituents:
solidified the basal solution, see medium 1- Water 1500.O cc.
555, by the addition of 10.0% gelatin. 2. Meat peptone (Kem-
merich's) 22.5 g.
2236. Matzuschita's Basal Peptone 3. Peptone (siccum) 45.0 g.
Gelatin 4. Gelatin 225.0 g.
Same as variant (b) medium 568, but Preparation
solidified by the addition of 10.0% gelatin. (1) Add 2 and 3 in 1.
References: Kita (1913 p. 446), Mat- (2) Boil over a free flame for a minute.
zuschita (1902 p. 286, 288). (3) Cool to about 50 to 60C.
(4) Dissolve the gelatin in (3) without
2237. Banning's Basal Peptone Gelatin further heating.
Same as medium 1535, but solidified by (5) Correct the acid reaction by the
the addition of 70.0 g. gelatin instead of addition of concentrated Na2C03.
10.0 g. agar. (6) Transfer to a large flask.
(7) Add the white of an egg, mi.x well
2238. Harvey's Basal Neutral Red Peptone and steam for | hour.
Gelatin (8) Filter, using compressed air to
Constituents hasten filtration.
1- Water 1000.0 cc. (9) Distribute in 50 to 100.0 cc. lots in
2. Peptone 20 g flasks.
3. KOH(5.0%) 10.0 cc. Sterilization: Place in flowing steam for
4. Gelatin 75.O g. 10 minutes on 3 successive days.
5. Neutral red (1.0%) (0.5%) . 5.0 cc. Use: General culture medium. Other in-
Preparation vestigators used similar media for spe-
(1) Dissolve2, 3 and 4 in 1. cific purposes as indicated below.
(2) Steam 45 minutes. Variants
(3) Filter. (a) Jacobi stated that the medium might
(4) Add 5.0% of a 20.0% solution of one be diluted with distilled water when
of the added nutrients, and 0.5% of a ready for use.
1.0% neutral red solution. (b) Matzuschita dissolved 10.0 g. of
Sterilization: Sterilize for 10 minutes on Koch's peptone in 1000.0 cc. water
each of 3 successive days at 100C. and solidified with gelatin. He culti-
Use: Cultivation and differentiation of vated spore forming bacilli on this
colon group. medium.
Added nutrients: The author added 5.0% (c) Lieske isolated iron bacteria, Lepto-
of a 20.0% solution of any desired sugar. thrix ochracea on a medium made
Reference: Harvey (1921-22 p. 109). slightly alkaline by the addition of
2239. Weiss' Nahrstoff Heyden Gelatin

KOH containing 100.0 g. of gelatin
and 0.25 g. manganese peptone per
Constituents 1000.0 cc. of peat water.
1- Water 1000.0 cc. (d) Weiss used 1.0% peptone instead of
0.8% Xahrstoff-Heyden in the prepa- (8) Final reaction of NaOH portion

ration of medium 2239. -M.7.
(e) Levine and Carpenter studied the Final reaction of Na2C03 portion
liquefaction of gelatin on a medium -M.S.
prepared as follows: (b) Sergent cultivated Bact. zopfi on a
(1) Add 1.0 g. Difco peptone, 35.0 g. medium prepared by dissolving 5.0 g.
Difco gelatin to 1000.0 cc. distilled NaCl, 10.0 g. peptone and 80.0 g.
water and heat, at 60-65C. until all gelatin in 1000.0 cc. distilled water.
material is in solution. (c) Roux and Rochaix dissolved 10.0 g.
(2) Heat in double boiler for 15.0 peptone, 0.5 g. NaCl and 20.0 g.
minutes. gelatin in 1000.0 cc. water and made
(3) Adjust to pH = 8.0. the reaction slightly alkaline (indi-
(4) Make up volume. cator not specified).
(5) Heat 15.0 more minutes. (d) Besson prepared the medium as
(6) Filter thru cotton flannel. follows:
(7) Distribute in 25.0 cc. quantities. (1) Dissolve 10.0 g. Chapoteaut's pep-
(8) Sterilize at 15.0 pounds for 15.0 tone, 5.0 g. NaCl, and 20.0 g.
minutes. gelatin (extra white) in 1000.0 cc.
References: Jacobi (1888 p. 539), Matzu- water.
schita (1902 p. 286), Lieske (1919 p. 417), (2) Make slightly alkaline by the addi-
Weiss (1920 p. 24), Levine and Carpenter tion of soda solution.
(1923 p. 298). (3) Heat 5 minutes at 115C.
thru paper.
(4) Filter
2241. Metchnikoff's Peptone Gelatin Distribute as desired.
(5)
(Thoinot and Masselin) (6) Sterilize at 110 to 115C.
References: Thoinot and Masselin (1902
Constituents:
p. 392), Wherry (1905 p. 320), Sergent
1. Water 1000.0 cc.
(1906 p. 1015), Roux and Rochaix (1911
2. Peptone 10.0 g.
p. 109), Besson (1920 p. 30).
3. NaCl 10.0 g.
4. Gelatin 20.0 g. 2242. Mortensen's Peptone Gelatin
addition of gelatin.
Use: Enrichment of Bacillus of Asiatic
cholera. Sergent cultivated B. zopfi on 2243. Rossi's Salt Peptone Gelatin
a similar medium. Constituents
Variants 1. Distilled water 1000.0 cc.
(a) Wherry gave the following method of 2. Peptone 20.0 g.
preparation: 3. NaCl 5.0 g.
(1) Dissolve 200.0 g.Gold Label gela- 4. Gelatin 100.0 g.
tin, 10.0 g. Witte's peptone and 5. Na.COs 0.6 g.
5.0 g. NaCl in 1000.0 cc. of water 6. K2CO3 0.9 g.
by boiling. 7. NaOH or KOH 2.0 to 4.0 g.
(2) Divide in two 500.0 cc. portions. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
(3) Adjust one portion to +1 reaction in 1.
to phenolphthalein with normal Sterilization: Not specified.

NaOH. To the other portion add Use: To be used in water examination.
an equal volume of normal Na2C03. Reference: Rossi (1893 p. 69).
(4) Cool to 40C., add whites of three
2244. Metchnikoff's Nitrate Peptone
eggs to each.
Gelatin (Brau)
(6) Filter thru cotton and distribute. Constituents
(7) Sterilize in Arnold for 20 minutes 1. Water
on each of 3 successive days. 2. Gelatin. 2.0 g.
3. Peptone (dry) 1.0 g. the medium, it stuck to the plants very

4. NaCl 1.0 g. well.
5. KNO, 0.1 g. References: Utz (1903-04 p. 610), d'Herelle
Preparation: (1) Dissolve 2, 3, 4 and 5 in (1914 p. 310).
sufficient quantity of water.
2247. Beijerinck's Glucose Peptone
Gelatin
Use: Isolation of cholera vibrio. The
author added 200.0 cc. of the water under Constituents
investigation to each flask. 1. Water 100.0 cc.
Variants: Pergola solidified medium 549 2. Glucose 5.0 g.
by the addition of 10.0 to 15.0% gelatin. 3. Peptone 5.0 g.
References: Brau (1905 p. 813), Pergola 4. Gelatin 10.0 g.
(1911 p. 85). 5. Potassium biphosphate 0.02 g.
Preparation: Dissolve 2, 3, 4 and 5 in 1.
2245. Lichtenstein's Cenovis Gelatin
Same as medium 518 but solidified by the Use: Cultivation of vinegar bacteria pro-
addition of gelatin. ducing pigment. The author reported
that the gelatin was first liquefied and
2246. Utz's Glucose Peptone Gelatin
then coagulated.
Constituents Reference: Beijerinck (1911 p. 174).
1. Water 1000.0 cc.
2. Gelatin 100.0 g.
2248, Pfeiler and Lentz's Ringer Solution
Peptone Gelatin
3. Peptone 10.0 g.
4. NaCl 5.0 g. Same as medium 1551 but solidified by
5. Glucose 20.0 g. the addition of 15.0% gelatin instead
Preparation of agar.
2249. Vierling's Glucose Peptone Gelatin
(2) Neutralize.
(3) Mix with egg white. Constituents:
(4) Shake. 1. Water 1000.0 cc.
(5) Boil. 2. K2HPO4 1.0 g.
(6) Filter. 3. CaClz 0.1 g.
(7) Add 20.0 g. glucose. 4. MgS04 0.1 g.
Sterilization: Not specified. 5. FeClj trace
Use: Cultivation organisms found in
of 6. NaCl trace
milk. d'Herelle cultivated the Cocco- 7. Gelatin 100.0 g.
bacillus of the grasshopper (Coccobadllus 8. Peptone 10.0 g.
acridiorium). 9. Dextrose 10.0 g.
Variants: d'Herelle prepared a similar me- Preparation
dium as follows: (1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1.
(1) Dissolve 40.0 g. Chapoteaut's pep- (2) Adjust to a slightly alkaline reaction
tone, 5.0 g. NaCl, 30.0 g. gelatin and by the addition of soda.
5.0 g. glucose in 1000.0 water by Sterilization: Not specified.
boiling. Use: To study proteolytic action, lique-
(2) Make slightly alkaline. faction by mycobacteria. The author
(3) Filter. reported that growth was good, but lique-
(4) Distribute in bottles. faction did not take place. Same results
(5) Plug with cotton and cover the cotton if glucose be omitted.
with a layer of parchment paper. Reference: Vierling (1920 p. 203).

2250. Sear's Glucose Peptone Gelatin
He reported that the virulence of the
culture was maintained for about 15 days Constituents
in this medium. When a culture was 1. Water 1000.0 cc.
powdered due to the gelatin content of 2. Peptone 10.0 g.
3. NaCl 5.0 g. Preparation: (1) Dissolve 2, 3 and 4 in 1.

4. Gelatin 50.0 g. Sterilization: Not specified.
5. Glucose (1.0%) 10.0 g. Use: Cultivation of Leptomitus.
6. CaCO, Reference: Trommsdorff (1918 p. 65).
Preparation
2254. Molisch's Dextrin Peptone Gelatin
(1) D,issolve 2, 3 and 4 in 1.
(2) Clear with white of egg. Constituents

(3) Neutralize to phenolphthalein. 1. Moldau water 1000.0 cc.
(4) Filter till clear. 2. Gelatin lOO.O g.
(5) Add 1.0% glucose (glucose may be 3. Peptone 5.0 g.
omitted). 4. Dextrin 5.0 g.
(6) Distribute both portions into 300.0 cc. Preparation: (1) Dissolve 2, 3 and 4 in 1.
flasks, 200.0 cc. per flask. Sterilization: Not specified.
Sterilization: Sterilize at 15 pounds for Use: Cultivation of purple bacteria.
10 minutes. Reference: Molisch (1907 p. 11).
Use: To study nitrogen metabolism. Am-
monia determined by Folin's method, 2255. Matzuschita's Glycerol Peptone
amino acid by Van Slyke's micro method Gelatin

and total nitrogen by Kj,eldahl-Gunning- Constituents
Arnold method. 1. Water 1000.0 cc.
Reference: Sears (1916 p. 126). 2. Peptone 10.0 g.
3. NaCl 5.0 g.
2251. Utz's Lactose Peptone Gelatin
4. Gelatin 80.0 to 100.0 g.
Same as medium 2246 but used 20.0 g. 5. Glycerol 60.0 g.
lactose instead of glucose. Preparation
(1) Prepare ordinary nutrient gelatin
2252. Molisch's Sucrose Peptone Gelatin
using water instead of bouillon.
(Smith)
(Peptone and NaCl, in the amounts
Constituents: indicated, were considered to be the
1. Water 1000.0 cc. constituents of nutrient gelatin.
2. Gelatin 100.0 g Exact amount of gelatin not specified
3. Sucrose 20.0 g in the article either.)
4. Peptone 10.0 g, (2) Add 6.0% glycerol to (1).
5. K2HPO4 0.25 g Sterilization: Not specified.
6. MgS04 0.25 g, Use: Cultivation of mammalian and
Preparation chicken tubercle bacilli.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. Reference: Matzuschita (1899 p. 128).
(2) Make feebly alkaline by the addition
of NaOH. 2256. Molisch's Glycerol Peptone Gelatin

Use: Cultivation of luminous bacteria. 1. Moldau water 1000.0 cc.
Smith reported that on this medium the 2. Crelatin 100.0 g.
bacteria grew feebly and were non- 3. Peptone 5.0 g.
luminous until NaCl or some equivalent 4. Glycerol 5.0 g.
material was added (3.0%). Preparation: (1) Dissolve 2, 3 and 4 in 1.
Reference: Smith (1905 p. 198). Sterilization: Not specified.
Use: Cultivation of purple bacteria.
2253. Trommsdorff's Sucrose Peptone
Gelatin
Constituents: 2257. Freudenreich and Jensen's Lactate

1. Water 1000.0 cc.
Peptone Gelatin (Boekhout and Ott de
2. Peptone (Witte) (0.5%) ....
Vries)
5.0 g.
3. Sucrose (0.5%) 5.0 g. Same as medium
644 but solidified by the
4. Gelatin (8.0%) 80.0 g. addition of 10.0% gelatin.
Reference: Boekhout and Ott de Vries 2261. Remy's Phenolated Lactose Peptone
(1918 p. 130). Gelatin
Constituents
2258. Matzuschita's Glucose Glycerol 1. Distilled water. .. 1000.0 cc.
Peptone Gelatin 2. Asparagin 6.0 g.
3. Oxalic acid 0.5 g.

Constituents
4. Lactic acid 0.15 g.
1. Water 1000.0 cc.
2. Gelatin (10.0%) 100.0 g
6. Bisodium phos-
3. NaCl (0.5%) 5.0 g
phate 5.0 g.
4. Peptone (1.0%) 10.0 g
7. MgS04 2.5 g.
5. Glycerol (6.0%) 60.0 g
8. K0SO4 1.25 g.
6. Glucose (2.0%) 20.0 g
9. NaCl 2.0 g.
Preparation
10. Gelatin 120.0 to 150.0 g.
(1) Dissolve gelatin in water.
11. Peptone (Witte's). 30.0 g.
12. Lactose
(3) Add 0.5% NaCl, 10.0% peptone, 6.0%
13. Phenol
glycerol and 2.0% glucose.
Preparation
(4) Filter.
(1) Grind 6, 8 and 9 in a mortar.
(1), 2, 3,
(3) Add 30.0 g. peptone (Witte or Griib-
chicken tubercle bacilli. The author
ler's) to (2) and autoclave under
reported that mammalian type gave gray
white flakes which sank to the bottom
(4) Pour the hot solution into a flask
of the liquid at 37C. In gelatin a gray
containing 120.0 to 150.0 g. of gela-
white membrane formed on top. Chicken
tin. Shake thoroly until solution is
type gave large flakes and a gray white
complete.
precipitate at the bottom. A membrane
also formed on the surface.
(5) Make slightly alkaline by the addi-
tion of soda.
Reference: Matzuschita (1899 p. 128).
(6) Heat under pressure
in the autoclave
for 15minutes at 110C.
2259. Dombrowski's Glucose Peptone
(7) Acidify by the addition of 0.5N
Gelatin
H2SO4 so that the acidity of 10.0 cc.
Same as medium 631 but solidified by the of gelatin will be neutralized by
addition of 10.0% gelatin. 0.2 cc. of a 0.5 N soda solution.
(8) Steam again for 8 or 10 minutes.
(9) Filter.
2260. Beijerinck's Asparagin Peptone
(10) Retest the reaction by adding 10.0 cc.
Gelatin
ofgelatin to 100.0 cc. water and
Constituents several drops of phenolphthalein.
1. Water 900.0 cc. Add 0.5 N soda solution drop by
2. Sucrose 10.0 g. drop until a red coloration appears.
3. Asparagin 2.0 g. 0.2 cc. of the soda solution should
4. Peptone 8.0 g. be required.
5. Gelatin 80.0 g. (11) Add 2.5 g. MgS04.
(1) Dissolve 2, 3, 4 and 5 in 1. (13) Just before use, add 1.0 cc. of a
(2) Adjust (1) to neutral or slightly acid. 35.0% lactose solution and 0.1 cc. of
Indicator not specified. a 2.5% phenol solution to each tube
Sterilization: Method not specified. of sterile (12).
Use: To study liquefaction of gelatin and Sterilization: Sterilize by heating on 3
pigment production by Chlorococcum. successive days.
Chlorococcum did not liquefy the gelatin. Use: Isolation of typhoid bacillus.
Reference: Beijerinck (1890 p. 461). Reference: Remy (1900 p. 561).
2262. Scholz and Krause's Urea Peptone (3) Filter.

Gelatin (4) Distribute in bottles.
Constituents (5) Plug with cotton and cover the cot-
Water ton with a layer of parchment paper.
1. 1000.0 cc.
2. Gelatin Sterilization: Sterilize at 120 for 30
30.0 g.
3. Peptone minutes.
5.0 g.
4. Urea Use: Cultivation of Coccobacillus of the
20.0 g.
Sodium or ammonium grasshopper, Coccobacillus acridiorium
5. urate. 3.0 g.
6. (NH4)2C03 This medium was used in field work.
20.0 g.
Preparation See also variant of medium 2246.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. Reference: d'Herelle (1914 p. 310).
(2) Adjust the reaction to slight alka- 2265. Eisner's Peptone Potato Gelatin
linity.
(Heinemann)
Use: Cultivation of colon-typhoid group. Constituents:
The author reported that colon and 1. Water 1000.0 cc.
typhoid colonies could not be distin- 2. Potato 1.0 lb.
guished on this medium. 3. Peptone (1.0%) 10.0 g.

Reference: Scholz and Krause (1900 4. KI
(1.0%) 10.0 g.
5. Gelatin (10.0%) 100.0 g.
p. 431).
Preparation
2263. Beijerinck's Peptone Malt Gelatin (1) Grate 1 pound of potatoes with 1 liter
of water.
Constituents
(2) E.xpress the juice.
1. Malt infusion 890.0 cc.
(3) Filter for 24 hours (method not
2. Glucose 29.0 g. given).
3. Peptone 0.5 g.
(4) Dissolve 1.0% peptone, 1.0% KI and
4. Asparagin 0.5 g.
10.0% gelatin in (3).
Preparation
(1) Method of preparation of malt infu- Reference: Heinemann (1905 p. 129).
sion not given.
(2) Dissolve 2, 3, 4 and 5 in (1). 2266. de Rossi's v. Faba Peptone Gelatin
(3) Adjust (1) to neutral or slightly acid.
Same as medium
1623 but containing
Indicator not specified.
10.0% gelatin instead of 1.5% agar.
Use: To study liquefaction of gelatin and 2267. Molisch's Pea Gelatin (Lohnis)
pigment production by chlorococcum.
Constituents:
The author reported that chlorococcum 1. Water 1000.0 cc.
did not liquefy the gelatin.
2. Peas
3. Peptone, manganese (0.25%) 2.5 g.
4. Gelatin
2264. d'Herelle's Peptone Potato Gelatin
Preparation
Constituents (1) Prepare a pea infusion with boiling
1. Water 1000.0 cc. water (method not given).
2. Potatoes, grated 10.0 g (2) Add 0.25% manganese peptone to (1).
3. Peptone 5.0 g (3) Solidify with agar.
4. NaCl 5.0 g Sterilization: Not specified.
5. Gelatin 20.0 g, Use: Cultivation of iron bacteria.
6. Glucose 5.0 g, Reference: Lohnis (1913 p. 116).
Preparation
Heat 5 and 6 to boiling 2268. Molisch's Peat Gelatin
(1) 2, 3, 4, in 1 and
boil (time not specified). Constituents:
(2) Make slightly alkaline. 1. Water 1000.0 cc.
2. Peat eliminated practically all other intestinal

3. Peptone, manganese 0.25 g. organisms.
4. Gelatin 100.0 g. References: Deycke (1893 p. 888), (1895
Preparation p. 243), Klimmer (1923 p. 220).
(1) Boil a piece of peat brick the size of a
2270. Kotlar's Pancreas Gelatin
fist in a liter of water to prepare the
peat infusion. Constituents:

(2) Dissolve 2 and 3 in (1). 1. Water 400.0 cc.
(3) Make slightly alkaline by the addi- 2. Pancreas (calf) 200.0 g.
tion of NaOH. 3. Peptone (2.0%) 2.0 g.
Sterilization: Not specified. 4. NaCl (1.0%) 1.0 g.
Use: Cultivation of iron bacteria, lepto- 5. Gelatin (20.0%) 20.0 g.

thrix. Preparation
Reference: Molisch (1910 p. 39). (1) Add a little water to 200.0 g. of small
fat and connective tissue free pieces
2269. Deycke's Peptone Alkali Albumin
of calf pancreas and mince to a pulp.
Gelatin
(2) Add 200.0 cc. of water to (1).
Constituents (3) Filter thru a clay filter.
1. Water 1200.0 cc. (4) Prepare a watery solution of 20.0%
2. KOH (3.0%) 36.0 g. gelatin containing 2.0% peptone and
3. Peptone 1.0% 1.0% NaCl.
4. Veal 1000.0 g. (5) Mi.x 100.0 cc. of sterile (4) and 100.0 cc.
5. Gelatin 10.0% of sterile (3).
6. NaCl 1.0% Sterilization: Sterilize (2) by filtering thru
Preparation a clay filter (see step (3) above). Method
(1) Digest 1000.0 g. finely ground and of sterilization of (4) not given.
fat-free veal in 1200.0 cc. of 3.0% Use: Show effect of pancreas extract on
KOH in flask for 2 days at 37C. growth of Bad. coli, Bad. typhosus,
(2) Heat one hour on water bath 60- cholera bacillus, anthrax bacillus and
70C. until all protein dissolves. Staphylococcus pyogenes aureus. Author
(3) Add HCl carefully to precipitate reported that the organisms were in-
albuminate. hibited by the extract.
(4) Collect on filter. Reference: Kotlar (1895 p. 147).
(5) Suspend in distilled water and add

2271. Harde's Tissue Gelatin
concentrated soda solution until
alkaline reaction brings part into Constituents:
solution. 1. Nutrient gelatin.
(6) Dissolve by prolonged heating in 2. Beef or veal.
steam. Preparation: (1) Add a 2 cc. cube of beef or
(7) Correct reaction to weak alkalinity. veal to 20.0% gelatin just before steri-
(8) Determine %
dry weight by drying lization.
100.0 cc. sample at 100C. Sterilization: Method not given.
(9) Dilute (8) so that there is about Use: To obtain anaerobes and aerobea
3.0% dry material present. from exudates.
(10) Add 1.0% peptone, 1.0% NaCl and Reference: Harde (1917 p. 661).
10.0% gelatin.
2272, Pergola's Nitrate Alkaline Blood
(11) Dissolve and neutralize by Dah-
Gelatin
men's method using 0.33% soda
solution. Constituents
Sterilization: Sterilize on 3 successive days 1. Distilled water 1000.0 cc.
for 10 minutes each day in streaming 2. Peptone 10.0 g.
steam. 3. NaCl 10.0 g.
Use: Isolation of cholera bacilli from 4. NasCOs 0.2 g.
stools. The author reported the medium 5. KNO3 0.1 g.
6. Gelatin 100.0 to 150.0 g. (2) Add 60.0 to 100.0 g.

(6.0 to 10.0%
7. Blood gelatinand heat until the gelatin
8. KOH
(normal) has become melted.
Preparation : (3) Boil until the casein is completely
(1) Dissolve 2, 3, 4, 5 and 6 in 1. coagulated.
(2) Filter. (4) Pass thru four folds of linen cloth
(3) Mix blood with equal amounts of separating the casein and liquid.
N/1 KOH. (5) The reaction of the fluid is slightly
(4) Steam (3) for 30 minutes to sterilize. acid.
(5) Mix 30.0 parts sterile (4) with 70.0 (6) Pour into a tall glass cylinder and
parts sterile (2). place in a warm thermostat.
(6) Pour into sterile Petri dishes. (7) After about 20 or 30 minutes two
Sterilization : Method of sterilization of (2) layers are formed, the lower layer
not given. See step (4) for sterilization being transparent and fat free, the
of alkaline blood mixture. upper layer yellowish white, con-
Use: Enrichment medium for cholera taining the fat.
vibrio. (8) Allow to cool and remove the fat
Reference: Pergola (1911 p. 85). by means of a spoon.
(9) Heat the fat free portion to boiling
2273. Matzuschita's Peptone Milk
and add 1.0% peptone.
Gelatin
(10) Add soda to neutralize the reaction.
Constituents (11) Filter until clear thru a paper in a
1. Milk 1000.0 cc. hot water funnel.
3. Peptone 10.0 g. Use: CultivMion of pathogenic organisms.
4. NaCl 5.0 g. Variants
Preparation: (1) Dissolve 2, 3 and 4 in 1. (a) Raskin added 0.5% NaCl.
Method not specified.
Sterilization: (b) Klimmer prepared the medium as
Use: Cultivation of mammalian and follows:
chicken tubercle bacilli. The author (1) Coagulate the casein from milk
reported that chicken type grew slightly by heating at 40 C. in the presence
more luxuriantly than did the mam- of rennet.
malian types. Utz cultivated organisms (2) Separate the whey from the casein.
found in milk on a similar medium. (3) Dissolve 1.0% peptone, 0.5% NaCl
Variants: Utz prepared a similar medium and gelatin whey.
to solidify in the
as follows: not specified.
(4) Sterilization
(1) Dissolve 100.0 g. gelatin and 10.0 g. (c) Cunningham gave the following
peptone in 1000.0 cc. milk. method of preparation:
(2) Neutralize. (1) Warm 2 liters of clean fresh skim
(3) Mix with egg white. milk in a large pot at 37C.
(4) Boil one hour. (2) Add sufficient rennet to curdle.
(5) Filter. (3) Allow to settle for 10 minutes.
(6) Sterilization not specified. (4) Bteak the curd into large pieces
References: Matzuschita (1899 p. 128), by means of a stirring rod.
Utz (1903-04 p. 610). (5) Heat to 80C. to contract the clot
and to express the whey.
2274. Raskin's Whey Peptone Gelatin
(6) Strain thru a cheese cloth.
Constituents (7) Add 1.0% peptone and 0.5% NaCl.
1. Whey 1000.0 cc. (8) Steam
for 30 minutes.
2. Gelatin (6.0 to Neutralize to turmeric paper.
(9)
10.0%) 60.0 to 100.0 g. (10) Steam for one hour.
3. Peptone (1.0%) 10.0 g. (11) Dissolve 10.0% gelatin in (10).
Preparation (12) Filter thru paper until clear.
(1) Heat 1000.0 cc. of milk in a porce- (13) Add 1.0% Andrades indicator.
lain dish at 60 to 70 C. (14) Sterilize intermittently in steam.
References: Raskin (1887 p. 358), Klimmer Use: To determine bacterial counts of milk
(1923 p. 172), Cunningham (1924 p. 102). and milk products.
Reference: Meier (1918 p. 436).
2275. Appel's Peptone Whey Gelatin
Constituents
2277. Wigger's Whey Gelatin
1. Water to 1000.0 cc. Constituents:

2. Whey 1. Whey, goat.
3. Gelatin 100.0 g. 2. Gelatin.
4. Peptone 10.0 g. 3. Peptone.
Preparation :
(1) Add 10 to 12 drops of rennet extract
(1) Heat 1 liter of centrifuged milk to of a strength of 1:10,000 to 1 liter of
40 C. fresh skim milk heated to 30 or 35C.
(2) Add a little rennet and allow to stand This coagulates the casein in 30 or
until coagulation takes place. 45 minutes to a gelatinous mass.
(3) Heat for about 15 minutes on a boil- (2) Heat at 55 to 60C. and the casein
ing water bath. separates out in large lumps leaving
(4) Pour thru a straining cloth, stirring a clear whey.
with a porcelain spatula to cause a (3) Mix sterile (2) with an equal amount
separation of the whey and coagulum. of water.
(5) The yellow colored whey is brought (4) For the preparation of gelatin use
to 1000.0 cc. by the addition of water. (3) instead of meat water, omit the
(6) Add 100.0 g. gelatin, 10.0 g. peptone NaCl and proceed in the usual man-
and 5.0 g. NaCl. If one works fast ner. (It was assumed that peptone
the solution is warm enough to dis- was added.)
solve the gelatin. Sterilization: Sterilize the whey in the
(7) Mix well and autoclave at 105 for autoclave at one-half atmosphere addi-
30 minutes. tional pressure for 45 minutes. Final
(8) Immediately after removing filter sterilization not specified.
thru paper and without allowing the Use: Cultivation of milk bacteria.
gelatin to solidify, distribute into Variants The author added lactic acid to
:
sterile tubes. the undiluted whey and heated to 80 or

Sterilization: Sterilize once more at 105 90C. to precipitate the albumin.
for 30 minutes. Reference: Wigger (1914 p. 3).
Use: Cultivation of organisms found in
2278. Piorkowski's Peptone Urine Gelatin
milk. The author reported that the
gelatin had a high melting point. Constituents
Reference: Appel (1899 p. 763). 1. Urine 1000.0 cc.
2. Peptone 5.0 g.
2276. Meier's Whey Peptone Gelatin
3. Gelatin 100.0 to 120.0 g.
1. Water 500.0 cc. (1) Add and 0.5 g. peptone
100.0 cc. urine
2. Whey (goat milk) 500.0 cc. to a flaskand plug with cotton.
3. Gelatin 110.0 g. (2) Steam for 15 minutes in a steamer.
4. Peptone (Witte) 10.0 g. (3) Dissolve 10.0 to 12.0% gelatin in (2).
5. NaCl 5.0 g. (4) Filter using a hot water funnel or in a
Preparation steamer.
(1) Dissolve 3, 4 and 5 in 1. (5) Distribute into 10.0 cc. lots.
(2) Mix (1) with 500.0 cc. of whey from Sterilization: Sterilize on each of two suc-
goat milk. cessive days for 10 to 15 minutes in the
(3) Neutralize by the addition of KOH. steamer.
Add KOH until turmeric paper is Use: Differentiation of Bad. coli, commune
turned quite weakly brownish red. and Bacillus typhi abdominalis. Urine
Sterilization: Not specified. should be fresh, clear, bright yellow and
of acid reaction. A urine of specific Preparation

gravity of 1.012 gives the best medium. (1) Place 500.0 g. of the best quality
Variants: Piorkowski prepared similar meat, without fat or tendons, in a
media as follows: glass container fitted with a lid.
(a) He reported that on the following me- (2) Pour 1300.0 cc. distilled water over
dium Bad. coli commune colonies the meat.
after 20 hours, under the microscope, (3) Store in a cool place until the next
were round, yellow fine grained and day.
edges were well defined. Bad. typhi (4) Filter thru four thicknesses of
ahd. gave thread like colonies. Do cloth and press the remaining meat
not incubate at a temperature lower to obtain as much fluid as possible.
than 22C. (5) Pour the filtrate into a kettle, add
(1) Allow normal urine to stand for 10.0 g.peptone (siccum) 5.0 g. NaCl
2 days. Specific weight of the urine and the whites of two eggs, beaten
to be 1.020. During this time the up in two or three volumes of water.
reaction has become alkaline. (6) Boil over a gas flame for 15 minutes.
(2) Add 0.5% peptone and 3.3% gelatin (7) Adjust to faint alkalinity, using
to (1). phenolphthalein as an indicator, and
(3) Boil for an hour in a water bath. the end point being a faint red color.
(4) Filter at once without the use of (8) Pour into an iron kettle, add 100.0 cc.
heat. distilled water, boil strongly for
(5) Distribute into test tubes and plug 5 minutes and filter.
with cotton. (9) Dissolve 50.0 to 100.0 g. of gelatin
(6) Sterilize in the steamer for 15 min- in (8).
utes at 100C. and on the following (10) Add
200.0 cc. distilled water to (9).
day sterilize at 100C. for 10 (11) Cool to 40C. and add the whites of
minutes. two eggs beaten up in 2 or 3 volumes
(7) Incubate at 22C. of water.
(b) On the following medium typhoid (12) Boil strongly for 10 to 15 minutes.
colonies were small and colorless, (13) Filter thru four layers of cloth and
with 4 to 6 turft like runners. Bad. then thru a hot water funnel.
coli colonies were large and yellow Sterilization: Sterilizefor 30 minutes on
without the runners. each of 3 successive days in a steamer.
(1) Dissolve 0.5% peptone and 3.3%
gelatin in alkaline urine having a
Variants
specific gravity of 1.020.
(a) Acosta and Grande (1892) (Sentinon
(2) Allow to stand at 22 for 16 to
1893).
20 hours.
(1) Add to 1000.0 of tendon free lean
not specified.
(3) Sterilization
meat a double weight of water.
References: Piorkowski (1896 p. 687), (1899
(2) Boil (time not specified).
p. 145), (1916-17 p. 259).
(3) Skim and strain.
(4) Place once more on the fire and
2279. Heller's Urine Peptone Gelatin
and 0.5% peptone and 0.25% NaCl
Same as medium 745 but solidified by the and add water to bring up to the
addition of 10.0% gelatin. original volume.
(5) Add 16.0 to 18.0% gelatin.

2280. Schultz's Peptone Infusion Gelatin (6) Pour the solution in a clay or glass
Constituents: container that is twice as high as
1. Water 1300.0 cc. wide.
2. Meat 500.0 g. (7) Autoclave at 105 at 0.5 atmos-
3. Peptone (siccum). . 10.0 g. phere pressure for 15 minutes.
4. NaCl 5.0 g. (8) Release the pressure and allow the
5. Gelatin 50.0 to 100.0 g. mi.xture to stand for 24 hours.
(9) Remove from the autoclave, and (d) Jensen (1898) cultivated denitrifying
loosen the solidified gelatin from bacteria on the following medium:
the walls of the container. (1) Prepare an infusion from 500.0 g.
(10) Upset the gelatin on a filter paper. meat and 1000.0 cc. of water.
(11) Cut off the turbid upper layer of (2) Dissolve 5.0 g. NaCl, 10.0 g. pep-
the gelatin cylinder with a thread tone and 200.0 g. of gelatin in (1).
or wire. (3) Adjust the reaction to a slight
(12) Cut the clear gelatin into pieces, alkalinity by adding soda.
place into a flask and melt. (4) Method of sterilization not speci-
(13) Distribute into test tubes. fied.
(14) Sterilize by the discontinuous (e) Committee A. P. H. A. (1899).

method, (details not given). (1) Macerate one part finely chopped
(b) Frothingham (1895). lean meat with 2 parts distilled
(1)Add one pound of finely chopped water in an ice box for 18 to 24
lean meat to 1000.0 cc. water and hours, stirring constantly.
allow to stand from 12 to 24 hours (2) Strain while cold thru a fine cloth.
in a cool place. (3) Add 1.0% peptone and 0.5% NaCl
(2) Strain thru a cheese cloth or coarse to the filtrate. Heat until solu-
towel and squeeze in a meat press tion is complete.
or by twisting the ends of the (4) Add NaOH until the reaction is
cloth until 1000.0 cc. of the meat slightly alkaline (practically neu-
juice is obtained. Make up to tral) to phenolphthalein.
1000.0 cc. by the addition of water (5) Heat on a water bath for 30 min-
ifnecessary. utes and boil 5 minutes over a
(3) Dissolve 5.0 g. NaCl and 10.0 g. free flame.
dried peptone in (2). (6) Filter while hot thru paper or
(4) Add 100.0 g. gelatin to (3). cotton and cloth, and dissolve
(5) Heat in a water bath to
50 until 10.0% gelatin in the filtrate.
the gelatin is dissolved. (7) Add normal HCl to the filtrate to
(6) Make slightly alkaline. obtain the desired reaction. If
(7) Boil for 45 minutes to 60 minutes. the medium is clear distribute in

(8) Filter. tubes or flasks. If not clear,
(c) Forster (1897). clarify by adding the whites of
(1) Prepare Loeffler's bouillon. one egg to the medium cooled to
(2) Sterilize (1) method not given. 50 or 60 and boil vigorously.
(3) Heat to 60C. and dissolve the Filter.
necessary or required amount (8) Sterilize either by the fractional or
(exact amount not given) of continuous method.
gelatin in (1). (f) Migula (1901).
(4) Make slightly alkaline and cool (1) Mix 500.0 g. of finely chopped lean
a little. beef with one liter of water, and
(5) Add the white of an egg. allow to stand in the ice box for
(6) Place the kettle containing (5) in 12 to 24 hours.
boiling water, and stir with a (2) Press the liquid thru a towel and
spoon to insure equal heating. make up the volume to one liter.
(7) Adjust the reaction once more and (3) Boil in the steamer cooker for
heat to 100C. for 15 minutes. 30 minutes.
Leave the lid loosely on the kettle. (4) The infusion may be boiled for an
(8) Heat a water funnel to 60C. (not hour before removing the meat
higher) and filter quickly. and then filtered thru paper. If
(9) Distribute the filtrate into sterile the liquid is still red, boil again
culture tubes. for 15 minutes.
(10) Heat in boiling water or in flow- (5) Filter when cold to remove any fat.
ing steam at 100C. for 17 to 20 (6) Heat to boiling and add 0.5%
minutes. NaCl, 1.0% Witte's peptone and
10.0% gelatin (1.0 to 2.0% glucose (6) Heat over a small flame or on a
may be added). water bath until the gelatin is com-
(7) Neutralize carefully by the addi- pletely dissolved.
tion of concentrated Na2C03 until (7) Make slightly alkaline.
litmus paper is colored violet. (8) Heat minutes at 113-114C.
for 5
(8) Add soda solution as desired. in the autoclave and filter while
Generally 10.0 cc. of 15.0% soda hot thru Chardin paper.
solution is added per liter. (9) Distribute as desired.
(9) Boil for 30 minutes in the steamer. (10) Sterilize at 112C. for 25 minutes.
(10) Filter thru paper. (i) Frost (1903).
(11) The gelatin should be perfectly (1) Remove the fat and connective
all
clear. not cool to 40C., and
If tissue from
500.0 g. beef and
mix thoroly with the white of an mince or use hamburger steak.
egg. (2) Add 1 liter of distilled water to (1),
(12) Boil again for a short time. shake thoroly and set in the ice
(13) Filter. chest for 12 to 24 hours.
(14) Distribute in tubes or flasks. (3) Squeeze thru a cloth and add
(15) Sterilize on 2 successive days in enough distilled water to make one
flowing steam for 20 minutes each liter.
day, and for 10 minutes on the (4) Add 1.0% peptone (Witte) 0.5%
third day, or for 15 minutes on NaCl and from 10.0 to 15.0% of the
each of 3 successive days. Cool best gold label sheet gelatin.
quickly after each heating. (Add 10.0% gelatin in winter and
(g) Roux (Thoinot and Masselin 1902). 15.0% gelatin in summer.)
(1) Macerate 500.0 g. of finely chopped (5) Weigh the solution and the vessel.
beef in a liter of water for several (6) Heat until solution is complete.
hours. (7) Neutralize to phenolphthalein.
(2) Pass thru a linen cloth and express (8) Boil 5 minutes and restore the
the juice from the meat. weight lost by the addition of dis-
(3) Add 10.0 g. peptone, 5.0 g. NaCl tilled water.
and 100.0 g. of light colored gelatin (9) Test the reaction, and readjust if
to (2). necessary.
(4) Heat in a water bath not above (10) Boil until the albumin coagulates
60C. to dissolve the gelatin. and floats in the clear liquid.
(5) Add soda to make slightly alkaline. (11) Filter thru cotton supported on a
(6) Heat in steam at 100 for an hour. wire using a suction pump
coil of
(7) Filter. to hasten filtration.

(8) If not clear, cool to 50C. and add (12) Add 5.0 cc. (0.5%) of a normal
the white of an egg and heat again HCl solution.
to 100C. (13) Tube.
(9) Filter while hot thru wet Chardin (14) Sterilize in the steamer for 30
paper. minutes on 3 consecutive days or
(10) Tube in sterile tubes. in the autoclave at 110 for 15
minutes on each of
(11) Sterilize for 15 minutes.
3 successive days at 100C. (j) Frost (1903).
(h) Thoinot and Masselin (1902). (1) Remove all the fat and connective
(1) Macerate 500.0 g. of lean beef with tissue from 500.0 g. of beef and
a liter of water. mince, or use hamburger steak.
(2) Express the juice and pass thru a (2) Add 1 liter of distilled water.
linen cloth. (3) Place in a vessel for cooking and

(3) Boil several minutes. cook for 30 minutes at about 70C.
(4) Filter thru moistened filter paper. (4) Filter thru paper and make up to
(5) Add 10.0 g. peptone, 5.0 g. NaCl 1 liter.
and 100.0 g. gelatin. (5) Add 1.0% peptone (Witte) 0.5%>

NaCl and from 10.0 to 15.0% of (6) Add 10.0 g. peptone and 5.0 g.
the best gold label sheet gelatin. NaCl and stir with a small wooden
(Add 10.0% gelatin in winter and spoon.
15.0% gelatin in summer.) (7) Add the gelatin and stir until
(6) Weigh the solution and the vessel. dissolved.
(7) Heat until solution is complete. (8) Make up to 1 liter by the addition
(8) Neutralize to phenolphthalein. of (4).
(9) Boil 5 minutes and restore the (9) Add KOH until the reaction is
weight lost by the addition of dis- slightly alkaline to litmus.

tilled water. (10) Add the beaten white of one egg.
(10) Test the reaction and readjust if (11) Steam for 30 minutes.
necessary. (12) Filter thru two thicknesses of
(11) Boil until the albumin coagulates Swedish or Rhenish filter paper
and floats in the clear fluid. moistened with distilled water.
(12) Filter thru cotton supported on a (13) Sterilize in the steamer for 30 min-
wire using a suction pump
coil of utes on each of 2 successive days.
to hasten filtration. (m) Forster (Abel 1912).
(13) Add 5.0 cc. (0.5%) of a normal (1) Chop 500.0 g. of fat free meat and
HCl solution. add to a liter of water at 50C.
(14) Tube. (2) Keep at 50C. for 30 minutes and
(15) Sterilize in the steamer for 30 then boil for 30 to 45 minutes.
minutes on 3 consecutive days or (3) Filter or strain the fluid from the
in the autoclave at 110 for meat.
15 minutes. (4) Make up the fluid to one liter.
(k) Abel (1912). (5) Dissolve 10.0 g. Peptone (Witte or
(1) Chop 500.0 g. of fat free meat and Chapoteaut) and 5.0 g. NaCl in
add to a liter of water at 50C. (4) by heating in the steamer.
(2) Keep at 50C. for 30 minutes and (6) Neutralize to litmus.
then boil for 30 to 45 minutes. (7) Steam for 15 to 30 minutes.
(3) Filter or strain the fluid from the (8) Filter and readjust the reaction.
meat. Filter until clear.
(4) Make up the fluid to one liter. (9) Tube or flask.
(5) Dissolve 1.0% Witte's peptone, (10) Sterilize on each of 3 successive
0.5%, NaCl and 1.0 to 1.5% gelatin days steamer or autoclave
in the
in (4) by heating in the steamer. for 15 minutes at 120C.
(6) Neutralize to litmus. (11) Dissolve 100 to 150.0 g. gelatin in
(7) Steam for 15 to 30 minutes. (10) by heating at 60C.
(8) Filter and readjust the reaction. (12) Add KOH
until the acidity is
If not clear, clarify by the addi- faint and then add NasCOa to
tion of an egg white or meat juice. make slightly alkaline.
(9) Tube or flask. (13) Add the white of an egg.
(10) Sterilize on each of 3 successive (14) Place the vessel in a large pot
days in the steamer or autoclave filled with boiling water.
for 15 minutes at 120C. (15) Boil the gelatin for 15 minutes.
(1) Abel (1912). (16) Filter thru a sterile hot water
(1) Chop 500.0 g. fat free meat and funnel at 60C.
add a water at 50C.
liter of
(17) Tube under aseptic conditions.
(2) Keep at 50C. for 30 minutes and
(18) Heat for 20 minutes in the steamer.
then boil for 30 to 45 minutes.
(19) Immerse in cold water,
meat. (n) Lohnis (1913).
(4) Make up the fluid to a liter. (1) Add 1000.0 cc. distilled water to
Place 500.0 cc. of (4) in a white 500.0 g. of finely minced lean

(5)
enamelled sauce pan and heat to beef.
about 70C. (2) Infuse for 24 hours in the ice chest

or heat for one hour in the water (q) Besson (1920).
bath at 55C. (1) Remove all fat and tendons from

Boil for 60 minutes in the steamer beef and chop into small pieces.
(3)
or in a covered dish. (2) Allow 500.0 g. of (1) to macerate
Filter thru a clean cloth rising with 1000.0 cc. of cold water for
(4)
pressure (meat press). 6 hours, or if one wishes to remove
(5) Cool and remove the fat by filtering the sugar, 12 hours at 37 C.
thru S. and S. filter paper. (3) Place in an enamelled pot and
(6) Make up the volume to 1000.0 cc. bring slowly to a boil.
by the addition of water. (4) Boil for 10 minutes.
(7) Add 1.0% Witte's peptone and (5) Throw on a thick cloth and press
0.5% NaCl. the meat free from juice.
Steam for one-half hour. (6) Filter the juice thru moistened
(8)
paper.
(9) Filter.
(10) Cool. (7) Make up to 1000.0 cc.
Adjust the reaction. (8) Add 10.0 g. Chapoteaut's peptone,
(11)
(12) Steam again for 30 minutes. 5.0 g. NaCl, and 80.0 to 130.0 g.
(13) Filter.
gelatin (extra) (80.0 g. in winter
(14) Add 100.0 g. of the best quality of and 130.0 in summer) to (7).
gelatin and soak 2 hours at room (9) Heat slowly in an enamelled
temperature. kettle.
(10) After solution is complete, boil
(16) Cool. for 2 or 3 minutes.
Make slightly alkaline by the
(17) Titrate and adjust the reaction. (11)

addition of soda.
Heat at 115 for 5 minutes.
(19) Filter thru S. and S. filter paper (12)
washed with sterile boiling water. (13) Filter thru paper in the autoclave
(20) Tube. or use a hot water funnel.
(21) Heat on 3 successive days for (14) Tube.

15 minutes, 10 minutes and 5 (15) Sterilize at 110 for 20 minutes,
minutes, respectively, at 100C. (r) Abbott (1921).
(o) Meier (1918). (1) Add chopped lean beef
500.0 g. of
(1) Boil 500.0 g. of fat and tendon free and soak for
to 1 liter of water
beef in 1 liter of water. 24 hours, kept at ice box tem-
(2) Filter. perature.
(3) Dissolve 110.0 g. gelatin, 10.0 g. (2) Strain thru a coarse towel and
peptone (Witte) and 5.0 g. NaCl press until a liter of fluid is ob-
in the filtrate. tained.
(4) Neutralize by the addition of
Dissolve 10.0 g. (1.0%) peptone,
(3)
KOH. Add KOH until turmeric
5.0 g. (0.5%) NaCl and 10.0 to
paper is turned quite weakly 12.0% gelatin in (2).
brownish red.
(4) Make slightly alkaline or neutral
(indicator not specified).
(p) Worth (1919).
(5) Place in a porcelain lined sauce-
(1)Heat 500.0 g. of chopped beef in
pan, and boil over a flame until
1000.0 cc. water in a water bath
all the albumin is coagulated and
at 50 to 55C. for one hour.
the fluid portion is of a clear pale
(2) Strain thru cloth and restore
straw color.
volume.
NaCl (6) Filter thru a folded paper.
(3) Add 10.0 g. peptone, 5.0 g.
and 100.0 g. gelatin to (2). (7) Sterilize by steam (method not
adjust to 1.0% given).
(4) Dissolve, filter,
acid. (s) Dopter and Sacquepee (1921).
(5) Sterilize for 20 minutes at llO'C. (1) Add 1000.0 cc. of water to 500.0 g.
finely chopped fat and tendon cloth and make up the volume to
free beef. 1 liter.
(2) Allow to stand in the ice box for (4) Inoculate with a culture of the
12 hours, or heat at 50 to 55 for colon bacillus.
30 minutes. (5) Allow to stand at room tempera-
(3) Heat slowly to boiling". ture over night.
(4) Boil slowly for 10 minutes stirring (6) Boil and add 10.0 g. Witte's pep-
constantly. tone and 5.0 g. NaCl.
(5) Strain thru a cloth. (7) Weigh the saucepan and contents
(6) Heat slowly (not above 80C.) and heat to 60C.
and add 20.0 g. peptone, 5.0 g. (8) Make up the loss in weight by the
NaCl and 120.0 g. of gelatin per addition of water.
liter of filtrate. (9) Neutralize to litmus,
(7) Shake until solution is complete. (v) Klimmer (1923).
(8) Add soda solution to neutralize (1) Prepare meat infusion.
to litmus. (2) Dissolve 10.0 g. peptone, 5.0 g.
(9) Heat in the autoclave at 112 for NaCl and 100.0 g.
gelatin in (1)
10 minutes. by heating between 50 and 60C.
(10) Filter thru paper. Do not heat above 60C.
(11) Distribute. (3) Neutralize to litmus.
(12) Sterilize at 110 for 10 minutes. (4) Filter.
(Do not heat over 110C.) (5) Add 5.0 cc. of normal soda solu-
(13) Store in a cool place. tion.
^t) Harvey (1921-22). by heating for 40 minutes
(6) Sterilize
(1) Proceed as in the preparation of on each of 3 successive days for
infusion broth, see variant (bb) 15 minutes in streaming steam.
of medium 779, step (1) thru (9). (w) Park, Williams and Krumwiede
(2) Add 10.0 g. peptone, 5.0 g. sodium (1924).
chloride and 120.0 g. gelatin. (1) Dissolve 100.0 g. gelatin, 10.0 g.
(3) Heat gently with constant stirring. peptone and 5.0 g. NaCl in
(4) Steam or boil 45 minutes to obtain 1000.0 cc. meat juice.
complete solution. (2) Coagulate the albumin present in
(5) Bring the volume up to 1000.0 cc. the meat juice to clarify.
by the addition of hot water. (3) The coagulation of the albumin
(6) Adjust reaction to a definite pH present in the meat juice clears
value, or faintly alkaline to litmus the medium.
or 1.0% acid to phenolphthalein. (4) Filter thru cotton.
(7) Steam 30 minutes. (5) Tube and sterilize in the Arnold
(8) Filter, while hot, thru well-wetted, sterilizer 20 to 30 minutes on 3
thick filter paper by placing filter successive days.
funnel, stand and receptacle for (x) Park, Williams and Krumwiede
filtrate in the steam sterilizer, and (1924).
steaming till filtration is complete. (1) Add "10.0 g. (1.0%) peptone and
(9) Distribute into flasks or test tubes. sodium chloride 0.5 g.," to
(10) Sterilize. 1000.0 cc. of meat infusion.
(11) Cool the flasks or test tubes on (2) Heat nearly to boiling and add
finalremoval from the sterilizer. 100.0 g. gelatin. Dissolve with
(12) Store in a cool place, as little heat as possible.
(u) Pitfield (1922). (3) Adjust the reaction neutral to
(1) Cover 500.0 g. of finely cut fat litmus (pH = 6.8 to 7.0).
free beef with 1000.0 cc. water. (4) Cool to 50C. and clarify by heat-
(2) Shake well and place on ice over ing for 45 minutes in an Arnold
night. sterilizer.
(3) Squeeze out the fluid by means of a (5) Filter.
Tube. References: Koch (1881 p. 169), Hueppe

(6)
the Arnold 20 to 30 min-
(7) Sterilize in
(1885 p. 101-104), Koch (1887 p. 161).
utes on 3 successive days.

2281. Smith's Peptone Infusion Gelatin
References: Schultz (1891 p. 62), Acosta
and Grande (1892 fÛ), Frothingham Constituents
(1895 p. 55), Forster (1897 p. 342), Jensen 1. Distilled water 1000.0 cc.
(1898 p. 406), Committee A. P. H. A. (1899 2. Lean beef 500.0 g.
p. 77), Migula (1901 p. 13), Thoinot and 3. Peptone (Witte) 10.0 g.
Masselin (1902 pp. 30, 32), Frost (1903 4. Gelatin (best white French) 10.0 g.
p. 6), Abel (1912 p. 15, 17), Lohnis (1913 Preparation

p. 14),Meier (1918 p. 435), Worth (1919 (1) Prepare a medium from the constit-
p. 608), Besson (1920 p. 39), Abbott

uents, method not given.
(1921 p. 125), Dopter and Sacquepee (2) Add enough KOH to neutralize to
(1921 p. 124), Harvey (1921-22 p. 70), litmus.

Pitfield (1922 p. 116), Klimmer (1923 Sterilization: Sterilize(method not given).
p. 192), Park, Williams and Krumwiede After sterilization reaction slightly acid
(1924 p. 117). to litmus.
Use: Smith used the medium for the culti-
2280a. Koch's Nutrient Gelatin vation of Pseudomonas campestris Pam-
Constituents mel. He reported that the gelatin was
1. Any nutrient medium, slowly but distinctly liquefied. Slight
double strength 1000.0 cc. growth on this medium. The presence of
2. Gelatin solution, 5 to 6%. 1000.0 cc. . .
salt hindered the growth. Would not
Preparation grow on a medium alkaline to phenol-
Similar media were used as
(1) Place gelatin in distilled water and
phthalein.
dissolve by warming. A concentra- general culture media and also in water
tion of 5 to 6% gelatin recommended. analysis.
(2) Add this gelatin solution to an equal Variants

volume of any nutrient medium (a) Committee A. P. H. A. (1901) recom-
(double strength). mended the following method of
(3) Neutralize with sodium or potassium preparation:
hydroxide or phosphate. (1) Infuse 500.0 g. lean meat 24 hours
Boil and filter off precipitate. with 1000.0 cc. of distilled water in
(4)
Place in sterilized cotton stoppered refrigerator.
(5)
(2) Make up loss by evaporation.
Sterilization: Sterilize by boiling. (3) Strain infusion thru cotton flannel.
Use : General culture medium and isolation (4) Weigh filtered infusion.
of cholera bacillus. (5) Add 1.0% Witte's peptone and

Variants 10.0% gold label sheet gelatin.
(a) The author stated that larger quanti- (6) Warm on water bath, stirring till
ties of gelatin might be used, that peptone and gelatin are dissolved
the gelatin may be dissolved directly and not allowing the temperature
in the nutrient medium. to rise above 60C.
(b) Hueppe (1885) prepared the medium (7) Heat over boiling water (or steam)
as follows: bath for 30 minutes.

(1) Add 5 or 10% finely cut gelatin to (8) Restore loss by evaporation.
any desired nutrient medium. (9) Titrate after boiling one minute
(2) Allow to soak for half an hour. to expel carbonic acid.
(3) Heat to dissolve. (10) Adjust reaction to -1.0% by add-

(4) Neutralize by the addition of so- ing normal hydrochloric acid or
dium carbonate, or make slightly sodium hydrate as required.
alkaline to litmus. (11) Boil 2 to 5 minutes over a free
(5) Heat on the water bath for an hour flame constantly stirring.
and filter thru a damp filter paper. (12) Make up loss by evaporation.
(13) Filter thru absorbent cotton and (8) To each 100.0 cc. add 3.0 cc. of a
cotton flannel, passing the filtrate normal phosphoric acid and 2.0 cc.
thru the filter until clear. of 2.0% malachite green solution.
(14) Titrate and record the final Sterilization: Not specified.
reaction. Use: Isolation of typhoid bacilli from
(15) Tube, using 5.0 cc. in each tube feces.
in the case of gelatin. References: Loeffler (1906p. 289), Klimmer
(16) Sterilize 15 minutes in the auto- (1923 p. 213).
clave at 110 or for 30 minutes in
streaming steam on three successive 2283. Roux and Rochaix's Peptone Infusion
days. Gelatin
(17) Store in the ice chest in a moist Constituents
atmosphere to prevent evaporation. 1. Distilled water 1000.0 cc.
(b) Committee A. P. H. A. (1905) pre- 2. Beef 500.O g.
pared the medium as given in variant 3. NaCl 5.0 g.
(a), but sterilized for 5 minutes in 4. Peptone 10. g.
the autoclave at 120 C. or for 30 5. Sodium or potassium
minutes in streaming steam on each phosphate 2.0 g.
of 3 successive days. Cool quickly 6. Gelatin (15.0 to
on ice following heating. 18-0%) 150.0 to 180.0 g.
(c) Giltner prepared the medium as Preparation
follows: (1) Chop 500.0 g. of fat and tendon free
(1) Prepare meat infusion. beef into very small pieces.
(2) Dissolve 1.0% peptone and 15.0% (2) Add 1 liter of water and allow to
gelatin in a mixture of equal parts stand over night in the cold.
(1) and distilled water. (3) Press the juice thru a linen cloth
(3) Adjust the reaction to +1.0%. by means of a meat press.
(4) Sterilization not specified. (4) Make up the volume to 1000.0 cc.
References: Smith (1897 p. 480), Com- by the addition of water.
mittee A. P. H. A. (1901 p. 384), (1905 (5) Add 5.0 g. NaCl, 2.0 g. of sodium or
p. 107), Giltner (1921 p. 380). potassium phosphate and 10.0 g. of
peptone.
2282. LoefHer's Malachite Green Infusion
(6) Boil for an hour.
Gelatin
(7) Filter thru a linen cloth and then
Constituents thru paper.
1. Water 5000.0 cc. (8) Adjust the reaction slightly alkaline
2. Beef 4.0 lbs. to litmus by addition of KHCO3
3. Gelatin (15.0%) 750.0 g. solution, drop by drop.
4. Peptone (Witte 1.0%) 50.0 g. (9) Add 15.0 to 18.0% gelatin and heat
5. XaCl(0.5%) 25.0 g. on the water bath until solution is
6. Malachite green solution complete.
7. Phosphoric acid (10) Neutralize and make slightly alka-
Preparation line again to litmus.
(1) Chop four pounds clean lean beef. (11) Filter using a hot water funnel.
(2) Mix 5 liters tap water with (1). (12) Distribute in flasks or tubes.
(3) Add 15.0% = 750.0 g. gelatin, 1.0% Sterilization Sterilize on 2 or 3 successive
:
= 50.0 g. Witte peptone, 0.5% = days at 100 C.

25.0 g.NaCl. Use: General culture medium.
(4) Slowly heat to complete solution of Variants
gelatin. (a) Choquet's (Besson) prepared a me-
(5) Heat 45 minutes. dium as follows:
(6) Neutralize to litmus with sodium (1) Dissolve 5.0 g. peptone and 35.0 g.
carbonate. gelatin in 500.0 cc. beef juice.
(7) Heat and filter. (2) Heat in the autoclave.
(3) Filter in the autoclave or use a 4. Peptone (Witte's) 10.0 g.

hot water funnel. 5. Glucose 10.0 g.
(4) Tube. 6. Potassium phosphate
(5) Add magnesium phosphate (K2HPO4) 1.0 g.
Cas (PO 4) 2 and CaCO 3 to each tube. Preparation
(50.0 g. Ca3(P04)2, 5.0 g. mag- (1) Chop 500.0 g. beef liver into small
nesium phosphate and 10.0 g. pieces and add 1000.0 cc. water
CaCO were used for the entire
3 Weigh the infusion and container.
amount of medium.) (2) Boil blowly for 2 hours in a double
(6) Sterilization not specified. boiler, starting cold, and stirring it
(b) Besson prepared a similar medium occasionally.
as follows: (3) Make up the loss in weight by evap-
(1) Remove all fat and tendons from oration and strain thru a wire
beef and chop into small pieces. strainer.
(2) Allow 500.0 g. of (1) to macerate (4) Cool the filtrate to 50C. Add 3, and
with 1000.0 cc. of cold water for stir afew minutes until dissolved.
6 hours, or if one wishes to remove (5) Add 5 and 6.
the sugar, 12 hours at 37C. (6) Stir until the ingredients are dis-
(3) Place in an enamelled pot and solved, keeping the temperature
bring slowly to a boil. below 50C.
(4) Boil for 10 minutes. (7) After warming this mixture in a
(5) Throw on a thick cloth and press double boiler and stirring it for a few
the meat free from juice. minutes to dissolve ingredients, ti-
(6) Filter the juice thru moistened trate with N/20 sodium hydrate using
paper. phenolphthalein as an indicator, and
(7) Make up to 1000.0 cc. neutralize with normal sodium
(8) Add 10.0 g. Chapoteaut's peptone, hydrate.
5.0 g.NaCl, a pinch of sodium (8) Boil vigorously for 30 minutes in a
phosphate and 80.0 to 130.0 g. double boiler, and 5 minutes over a
gelatin (80.0 g. in winter, 130.0 g. free flame with constant stirring to
in summer). prevent the caramelization of the
(9) Heat slowly in an enamelled kettle. dextrose.
(10) After solution is complete, boil (9) Make up any loss inweight by evap-
for 2 or 3 minutes. oration and filter thru cotton flannel
(11) Make slightly alkaline by the and filter paper.
addition of soda. Sterilization: Sterilize in an autoclave for
(12) Heat at 115C. for 5 minutes. 15 minutes at 15 pounds pressure.
(13) Filter thru paper in the autoclave Use: Identification of certain bacteria.
or use a hot water funnel. Used also for cultivation of B. sporogenes.
(14) Tube. Reference Jackson and Muer (1911 p. 290),
:
(15) Sterilize at 110 for 20 minutes. (1911 p. 928).

References: Roux and Rochai.K (1911
2286. Fuhrmann's Glucose Infusion Gelatin
p. Ill), Besson (1920 pp. 38, 41).
Constituents
2284. Miiller's Indicator Infusion Gelatin
1. Horse meat infusion 1000.0 cc.
Same as medium 784, but solidified by the 2. Gelatin 100.0 g.
addition of gelatin. 3. Peptone (Witte) 20.0 g.
4. Dextrose 10.0 g.
2285. Jackson and Muer's Liver Infusion
5. NaCl 5.0 g.
Gelatin
Preparation
Constituents: (1) Prepare horse meat infusion.
1. Water 1000.0 cc. (2) Dissolve 2, 3, 4 and 5 in (1).
2. Liver, beef 500.0 g. (3) Pour in plates.
Use: Cultivation of Pseudomonas cerevi- (2) Prepare a 2.0% solution of congo red.
siae. The author reported that best (3) Add 4.0 cc. of (2) to every 50.0 cc. of
growth was obtained when the reaction (1), also add 0.83% brilliant green
was 1.0% N/1 alkali. and 0.5% glucose.
Variants: The author gave the following (4) Add 25.0 cc. of bromoform to 100.0 cc.
method of preparation: of sterile distilled water.
(1) Digest 1000.0 g. of horse meat with (5) Shake thoroly and allow to stand at
1 liter of water for 24 hours in the room temperature over night.
cold. Pipette some of the saturated solu-
(6)
(2) Boil for one hour in the steamer. tion into a sterile flask (do not dis-
(3) Cool and filter. Make up to 1 liter. turb the bromoform at the bottom of
(4) Add 40.0 g. of Witte's (siccum) pep- the flask, do not include any of the
tone, 20.0 g. glucose, and 10.0 g. of globules floating on the surface).
NaCl. (This is a stock bouillon.) (7) One cc. of (6) (saturated bromoform
(5) To 500.0 cc. of (4) and 100.0 g. gelatin. solution) is added to each 10.0 cc. of
(6) Neutralize and clarify.
(3) , (congo-red-brilliant-green-gela-
(7) Dilute with an equal volume of water. tin) just before use.
(8) Add N/1 acetic acid or N/1 NaOH to Sterilization: Not specified.
obtained the desired reaction. Use: Enrichment medium for typhoid
(9) Sterilization not specified. organisms.
Reference: Fuhrmann (1906 pp. 310, 316). Reference: Teague and Clurman (1916-17
p. 125).
2287. Choquet's Glycero Phosphate
Infusion Gelatin (Besson) 2289. Kowalski's Glycerol Lung Infusion
Constituents Gelatin
1. Beef juice 300.0 cc. Constituents
2. Gelatin (extra white) 35.0 g. 1. Water.
3. Peptone (Chapoteaut) 5.0 g. 2. Lung, calf.
4. Glycerol-phosphate of lime. 5.0 g.
.
3. NaCl.
Preparation 4 Potassium phosphate.
(1) Dissolve 2, 3 and 4 in beef juice. 5. NaSOi.
(2) Heat in the autoclave. 6. (NH4)2S04.
(3) Filter hot in the autoclave or use a 7. Sucrose.
hot water funnel. 8. Peptone.
(4) Tube. 9. Gelatin (10.0 to 15.0%).
Sterilization: Not specified. 10. Glycerol (8.0 to 10.0%).
Use: Cultivation of organisms from dental Preparation
caries. Grind one kilogram fresh
(1) calf lung.
Reference: Besson (1920 p. 40). (2) Add 2 liters water. Boil in glass
vessel 30 minutes.
2288. Teague and Clurman's Congo-Red
(3) Filter, pressing the meat.
Green Glucose Gelatin
Brilliant
(4) Dissolve 3, 4, 5, 6, 7 and 8 in (3).
Constituents Add 10-15.0% gelatin.
(5)
1. Infusion gelatin (5.0%) . . . 500.0 cc. Dissolve by heat.
(6)
2. Congo red (2.0%) 40.0 cc. Neutralize with
(7) equal parts of
3. Brilliant green 0.415 g. sodium and potassium hydrate.
4. Glucose (0.5%) 2.5 g. Make to
(8) 2.5 liters with distilled
5. Bromoform water.
Preparation Cool to 58C.
(9)
(1) E.xact method of preparation of 5.0% (10) Add the beaten whites of 4 eggs.
nutrient gelatin or composition not (11) Heat a few minutes.
given. It is to be prepared however
(12) Filter.
from meat infusion, not from beef (13) Add 8-10.0% glycerol to filtrate.
extract, and the reaction to be 1.0+.
Sterilization: Sterilize intermittently on 3 2292. Huntoon's Hormone Gelatin

successive days. Constituents
1. Water 1000.0 cc.
Variants : Dittrich solidified medium 1737
2. Heart, fresh beef (or steak) 500.0 g.
with gelatin instead of agar. 3. Peptone (Bacto) 10.0 g.
Reference: Kowalski (1890 p. 266). 4. Gelatin 10.0 g.
5. Salt 5.0 g.
2290. Uffelmann's Methyl Violet Infusion Egg, whole
6. 1
Gelatin
Preparation
Constituents: (1) Chop 2 and mix 1, 2, 3, 4, 5 and 6.
1. Infusion gelatin 1000.0 cc. Place in an enamel ware vessel or a

2. Citric acid
large coffee pot.
3. Methyl violet (2) Heat over a free flame with constant

Preparation
Prepare meat infusion peptone gela- meat infusion changes to browm at a
(1)
tin in usual way, making slightly temperature of about 68C. Do not
alkaline and fiter.
go beyond this temperature.
Add solution of citric acid in such (3) Adjust to slightly alkaline to litmus
(2)
amount that 10.0 cc. of medium re- with N/1 NaOH and then add 1.0
cc. per liter of medium.
quires 14.0 cc. of a solution of 5.3 g.
sodium carbonate in 1000.0 cc. water (4) Cover the vessel and place in an
to neutralize exactly. Arnold sterilizer or in a water bath
at 100 for 1 hour.
(3) Filter.
(4) Add to each 100.0 cc. of the filtrate, (5) Remove the vessel from the steri-
lizer and separate with a glass rod,
2.5 mg. of methyl violet that has been
rubbed up with 1 drop absolute alco- the firm clot which has formed from
hol and 1.0 cc. distilled water. the side of the vessel.
Sterilization: Sterilize in streaming steam (6) Return to the Arnold sterilizer at
100 for 90 minutes.
15 minutes.
Use: Isolation of typhoid bacillus. The (7) Remove the vessel and allow to
author reported that the medium elim- stand at room temperature for about
inated many non-typhoid organisms. 10 minutes in a slightly inclined
position.
Good growth of typhoid bacilli.
(8) Pipette off the fluid portion or
Reference: Uffelmann (1891 p. 855).
decant. If it is poured thru a fine
wire sieve, many of the fine pieces of
2291. Vincent's Glucose Glycerol Gelatin
meat clot may be caught. (Avoid
Constituents filtering thru cheese cloth, cotton
1. Infusion broth 500.0 cc. or other adsorptive materials.)
2. Gelatin (extra) 50.0 to 75.0 g. (9) Allow (8) to stand in tall cylinders
3. Glucose 5.0 g. for 15 to 20 minutes until the fat
4. Glycerol 5.0 g. present has risen to the surface and

Preparation removed.
(1) Prepare infusion broth from beef (10) Tube in 10.0 cc. lots.
and peptone. (Method not given.) (11) The medium may be further cleared

by thru glass wool, asbestos
filtering
(3) Neutralize. (Indicator not specified.)

wool, sedimentation or centrifuga-
Tube. tion.
(4)
Sterilization: Method not given. Sterilization : Sterilize in the steamer using
Use: Cultivation of anaerobes in water the intermittent method.
analysis. Use: Substitute for media containing ser-
Reference: Vincent (1907 p. 65). ous fluids. The author reported the me-
dium gave as good results as the average (11) Add 20.0 g. lactose or glucose.
grade of serum media. (12) To prepare litmus glucose gelatin
Reference: Huntoon (1918 p. 171). add 2.0% glucose and enough
Kubel-Tiemann neutral litmus to
2293. Rosenberg's Basal Extract Gelatin
color it a purple tint before steri-
Constituents lization.
1. Water 1000.0 cc. References: Rosenberg (1886 p. 452), Ban-
2. Gelatin (10.0%) 100.0 g. ning (1902 p. 426), Percival (1920 p. 47).
3. Peptone (Koch) (4.0%) 40.0 g.
2294. Burger's Basal Fuchsin Sulphite
4. Meat e.xtract (0.5%) 5.0 g.
Extract Gelatin
Preparation
(1) Dissolve 2, 3 and 4inl. Constituents
(2) Dissolve one of the added nutrients 1. Distilled water 900.0 cc.
in (1). 2. Meat extract (Liebig's) 10.0 g.
(3) Gelatin may be neutralized by the 3. Peptone (Witte Rostock) .... 10.0 g.
addition of Na^COs or used without 4. NaCl 5.0 g.
adjusting the reaction. 5. Gelatin 100.0 g.
Sterilization: Not specified. 6. NaaSOa 2.5 g.
Use: Bacterial count of water. Used also 7. Fuchsin, ale. solution 5.0 cc.
as a general culturemedium. 8. Soda (crystalline) 1.0 g.
Added nutrients: Rosenberg added 0.5% Preparation:

of any desired sugar. (1) Dissolve 2, 3 and 4 in 1 by heating in
Variants the steamer.
(a) Same as medium 1693, but using (2) Allow (1) to cool.
20.0 g. gelatin instead of 10.0 g. of (3) Add 100.0 g. of gelatin to the non-
The agar was used for those
agar. filtered nutrient medium.
organisms liquefying gelatin. (4) Allow to soak for an hour or to stand
(b) Percival prepared a medium as folin the ice box over night.
lows: (5) Dissolve the gelatin in the steamer
(1) Dissolve 5.0 g. Lemco meat ex- requiring about 30 minutes.
tract and peptone
10.0 g. Witte's (6) Neutralize with N/1 NaOH to lit-
in 1000.0 water at ordinary
cc. mus. Shake well after addition of
room temperature. NaOH.
(2) Add 100.0 g. gelatin (best sheet) (7) Make alkaline by the addition of
and soak for 30 minutes. 1.0 g. crystalline soda.
(3) Heat in a water bath until the (8) Add 10.0 g. of one of the added nu-
gelatin is dissolved. Shake from trients either in a solid state or in a
time to time. concentrated solution.
(4) Neutralize to phenolphthalein by (9) Cool to about 40-50C.
the addition of normal caustic soda. (10) Dissolve 1.5 g. of egg albumin in
(5) Add 10.0 cc. of normal HCl to each 25.0 cc. of luke warm water, and
1000.0 cc. of the medium. add to (9). Shake thoroly.
(6) Cool the medium to 40C. and (11) Boil in the steamer for about
slowly add the white of an egg 30 minutes.
well beaten up in a little water. (12) Filter hot, thru sterile paper at a
(7) Heat in the steam sterilizer or temperature of 50 or 60C. This
water bath. may be carried out in the steamer
(8) Filter the hot solution thru after the flame has been removed.
moistened hot filter paper into a May allow to filter over night.
sterilized flask. Use a hot water (13) Distribute in 100.0 cc. lots.
funnel for filtering. (14) When ready for use, melt sterile (13)
(9) Tube in 8 to 10.0 cc. quantities. and to each 100.0 cc. add 5.0 cc. of a
(10) Steam for 20 minutes on 3 suc- saturated filtered alcoholic solution
cessive days. of fuchsin and 02.5 g. of crystalline
pure sodium sulfite, either in solid Preparation:

or concentrated clear solution. (1 Boil 22.0 g. of ragit powder with 1 liter
(15) When material is dissolved mix well. of water.

Sterilization: Sterilize (13) in the steamer (2) Add slowly 100.0 g. gelatin and bring
for 20 minutes. Sterilize the final me- to the boiling point.
dium for 20 minutes in the steamer. (3) The reaction is slightly alkaline.
Use: To obtain bacterial counts in water. (4) Filter in the steamer.
Author reported that B. coli grew best on 3 successive days
on medium containing lactose. B. typho- minutes in streaming steam.
for 30
sus and B. paratyphosus A and B and Use: Cultivation of Bacterium alipha-
Gartner's enteritidis grew best on man- ticum, Bad. aliphaticutn liqucfaciens.
nitol or glucose. Shiga and dysentery- Added nutrients: The authors added 0.5%
bacilli developed best in glucose medium. glucose, lactose or other nutrients.
Cholera vibrio developed best in presence Reference: Tausz and Peter (1919 p. 49).
of sucrose. When one of the sugars were
2296. Buchan's Basal Litmus Extract
fermented by the organisms a red colony
Gelatin
developed. The medium should be color-
less when ready for use. On plating on a Constituents:
series of media of this type one may 1. Distilled water 850.0 cc.
determine the kind of organisms present 2. Peptone 20.0 g.
in the water as well as the relative number. 3. Lemco 10.0 g.
Added nutrients The author added 10.0 g.
: 4. Gelatin 100.0 g.
of lactose, mannitol or sucrose to (8). 5. Litmus
Variants Preparation
(a) Burger prepared a similar medium (1) Dissolve 2, 3, 4 and 10.0 g. of one of
using 20.0 g. Liebig's meat e.xtract, the added nutrients in 1.
20.0 g. Witte's Rostock's peptone, (2) Add 10.0 cc. of a 5.0% KOH solution.
10.0 g. NaCl, 200.0 g. gelatin, 2.0 g. (3) Tinge with litmus.
crystalline soda, 20.0 g. of the added Sterilization : Not specified.
nutrients, 10.0 cc. of a saturated Use: To study fermentation.
alcoholic fuchsin solutionand 5.0 g. Added nutrients: The author added 10.0 g.
NaoSOa instead of the amounts indi- ofany desired carbohydrate, alcohol, etc.
cated. Water under investigation Reference: Buchan (1910 p. 107).
may be added to the sterile medium.
2297. Bacto Nutrient Gelatin. (Dehydrated)
(b) Burger used the following amounts
of constituents in preparing a medium Constituents:
in the same manner as above. Dis- 1. Distilled water
tilled water 750.0 cc, Liebig's meat 2. Beef extract, Bacto 3.0 g.
e.xtract 25.0 g., Witte's Rostock pep- 3. Peptone, Bacto 5.0 g.
tone, 25.0 g., NaCl, 12.5 g., gelatin 4. Gelatin, Bacto 120.0 g.
250.0 g., saturated alcoholic fuchsin Preparation
solution 12.5 cc. NasSOj 6.25 g., (1) Dissolve 128.0 g. of Bacto Nutrient
crystalline soda 2.5 g., and 25.0 g. of Gelatin (dehydrated) in 1000.0 cc.
one of the added nutrients. Water distilled water by warming.
under investigation may be added to (2) If sterilized at 15 pounds for 20 min-
the sterile medium. utes pH = 6.6.
Reference: Burger (1916-17 p. 478). Sterilization: Sterilize for 20 minutes at
15 pounds pressure.
2295. Tausz and Peter's Basal Ragit Use: General culture medium. The au-
Gelatin
thors reported that the medium con-
Constituents: forms to the "Standard Methods"
1. Water 1000.0 cc. formula.
2. Ragit powder 22.0 g. Reference: Digestive Ferments Co. (1925
3. Gelatin 100.0 g. p. 10).
2298. Heinemann's Peptone Extract Gelatin (b) Bahr reported that Gartner's bacillus
Constituents: did not liquefy a medium prepared
1. Water (tap) 1000.0 cc. as follows
2. Meat extract 2.5 g. (1) Dissolve 200.0 g. gelatin, 10.0 g.
3. Peptone 10.0 g. peptone, 20.0 g. meat extract
4. Gelatin 100.0 to 120.0 g. (Cibil's) in 1000.0 cc. of water.
Preparation (2) Adjust so that 10.0 cc. of the me-
(1) Dissolve 2.5 g. meat extract in dium will titrate 0.3 with N/10
1150.0 cc. of tap water. (Added NaOH using phenolphthalein as an
150.0 cc. of water to be lost during indicator.
preparation.) (3) Sterilizationnot specified.
(2) Heat and when near boiling add 10.0 g. (c) Commiteee A. P. H. A. (1917) (1920)
peptone to (1).
gave the following method of prepa-
(3) When add 10.0 to 12.0% of the
boiling ration :
best Gold Label gelatin to (2). Add (1) Add and 5.0 g.
3.0 g. of beef extract
2 or 3 sheets of gelatin at a time, peptone to 1000.0 cc. of distilled
stirring constantly. water and add 100.0 g. gelatin,
(4) When solution is complete adjust the dried for 30 minutes at 105 C. before
reaction to alkaline to litmus or weighing.
neutral to phenolphthalein and then (2) Heat slowly on a steam bath to
add 0.5% normal HCl. 65C. until the gelatin is dis-
all
(5) Cool to 60C. and stir in the white of (May be soaked for 30
solved.
one egg dissolved in 30.0 cc. water. minutes before heating.)
(6) Heat over the flame on a piece of (3) Make up lost weight, titrate and
asbestos without stirring. Heat until if the reaction is not already be-
the egg albumin is completely coagu- tween +0.5 and +1.0, adjust to +1.
lated and forms a dry film on top. (4) Filter thru cloth and cotton until
(7) Add water to make up any loss in clear.
weight over 50.0 g. (5) Distribute in tubes in 10.0 cc.
(8) Filter thru paper or absorbent cotton quantities or in larger amounts as
previously moistened with hot water. desired.
(9) Tube in 10.0 cc. quantities. (6) Sterilize in the autoclave at 15
Sterilization Sterilize in the autoclave for pounds
:
(120C.) for 15 minutes
5 minutes at 120 C. or steam for 3 suc- after the pressure reaches 15
cessive days for 20 minutes each day. pounds.
Use: General culture medium. (d) Committee S. A. B. (1918) recom-
Variants mended the same medium as Com-
(a) Wherry used the following medium to mittee A. P. H. A. (1917) but clarified
determine effect of reaction and dry- with egg and adjusted to pH between
ness on liquefaction of gelatin by 6.6 and 7.4.
Cholera spirillum.
(e) Treece dissolved 12% gelatin, 2.0%
(1) Dissolve 200.0 Gold Label gela-
g.
peptone and 0.5% meat extract in
tin, 10.0 g. Witte's peptone and
water. The medium was used to
30.0 g. Liebig's beef extract in
determine fecal and non-fecal strains
1000.0 cc. water.
of the colon-aerogenes group. The
(2) Divide (1) into two 500.0 cc. lots. author reported that this medium
(3) To one lot add NaOH so that after correlated the fermentation of ado-
sterilization the final reaction is nitol. Positive results were indi-
-1-0.8.
cated by a line of 4 to 8 bubbles
(4) To the other portion add NaOH so extending down the line of isolation.
that the final reaction after steri- (f) Committee A. P. H. A. (1923) ad-
lization is +1.0. justed the medium to a faint pink
(5) Method of sterilization not given. with phenol red or to the required
tint with brom thymol blue instead gelatin in winter and 15.0% in
of titrating to +1.0% as in 1917. summer.)
(g) Committee A. P. H. A. (1925) pre- (5) Weigh the solution and the vessel.
pared the medium as follows: (6) Heat until solution is complete.
(1) Add 3.0 g. of beef extract, 5.0 g. (7) Neutralize the phenolphthalein.
peptone and 120.0 g. gelatin (un- (8) Boil 5 minutes and restore the weight
dried market product as stored in lost by the addition of distilled water.
the ordinary cupboard) to 1000.0 cc. (9) Test the reaction and readjust if
distilled water. necessary.

(2) Heat slowly on a steam bath to (10) Cool below 60C. and thoroly stir
65C. until the ingredients are dis- in an egg.
solved. (11) Boil until the albumin coagulates
(3) Make up the lost weight with dis- and floats in the clear fluid.
tilled water and adjust the reaction (12) Filter thru cotton supported on a
so that after the final sterilization coil of wire using a suction pump to
the pH value will be between 6.2 hasten filtration.
and 7.0. (13) Add a normal HCl

5.0 cc. (0.5%) of
(4) Bring to a boil, stirring vigorously. solution. In water analysis a +1.0

Make up the lost weight with dis- reaction is used and +0.5 for patho-
tilled water and clarify. genic bacteria.
(5) Distribute in the desired containers. (14) Tube.
(6) Sterilize in the autoclave at 15 Sterilization: Sterilize in the steamer for
pounds (120 C.) for 15 minutes, 30 minutes on 3 consecutive days or in
after the pressure has reached the autoclave at 110 for 15 minutes.
15 pounds. Use: General culture medium.
References: Heinemann (1905 p. 23), Variants
Wherry (1905 p. 318), Bahr (1916-17 (a) Stoklasa isolated radiobacter and
p. 21), Committee A. P. H. A. (1917 p. 96), Azotobacter chroococcuni on a me-
Committee S. A. B. (1918 p. 115), Ball dium containing 100.0 g. gelatin,
(1919 p. 77), Tanner (1919 p. 55), Com- 20.0 g. peptone, 5.0 g. NaCl and 5.0 g.
mittee A. P. H. A. (1920 p. 95), Levine Liebig's meat extract per liter of
(1921 p. Treece (1920 p. 9), Com-

109), water.
mittee A. P. H. A. (1923 p. 95), Park, (b) Day and Baker cultivated bacteria
Williams and Krumwiede (1924 p. 131), causing ropiness in beer on a medium
Committee A. P. H. A. (1925 p. 97). adjusted to +1.0, containing 0.5%
Lemco meat extract, 0.5% NaCl,
2299. Frost's Peptone Extract Gelatin and 12.0%
1.0% Witte's peptone
Constituents gelatin.
1. Distilled water 1000.0 cc. (c) Abel prepared the medium as follows:
2. Beef extract, Lie- (1) Dissolve 10.0 g. Lemco meat ex-
big's 3.0 g. tract, 10.0 g. Witte's peptone and
f:
3. Peptone (Witte) 5.0 g. NaCl in 1000.0 cc. water.
(1.0%) lO.Og. (2) Steam for 30 minutes.

4. NaCl (0.5%) 5.0 g. (3) Filter when cool.
5. Gelatin (Gold Label (4) Add 900 parts (3) to 100 parts gela-
sheet) (10.0 or 15.0%) . 100.0 or 150.0 g. tin and allow to soak and soften.
Preparation (5) Place in a steamer until solution

Weigh out 3.0 g. of beef extract such is complete (not longer than 30
(1)
as Liebig's. minutes in the steamer).
^
(2) Add a liter of distilled water. (6) Neutralize to litmus.
(3) Place in a cooking vessel. (7) Heat for 15 minutes.
Add 1.0% peptone (Witte) 0.5% (8) Readjust the reaction if necessary.
(4)
NaCl and from 10.0 to 15.0% the best (9) Add 1.5 parts of crystalline un-
gold label sheet gelatin. (Add 10.0% decomposed soda.
(10) Steam for 30 to 45 minutes, and 5. Gelatin 200.0 g. 250.0 g.

filter. Soda
6. (see step
(11) Tube in sterile tubes. (7) above) 2.0 g. 2.5 g.
(12) Steam once for 15 to 20 minutes, (f) Meier used the following medium for
(d) Burger made bacterial counts of bacterial counts of milk:
water on a medium prepared as (1) Dissolve 125.0 g. gelatin, 10.0 g.
follows: Liebig's meat e.xtract, 10.0 g.
(1) Dissolve 10.0 g. Liebig's meat ex- Witte's (siccum) peptone, and
tract, 10.0 g. Witte's Rostock pep- 5.0 g. NaCl in 1000.0 cc. water.
tone and 5.0 g. NaCl in 900.0 cc. (2) Neutralize litmus and then
to
distilledwater by heating in the add N/1 soda solution per
10.0 cc.
steamer (requires about one hour). liter medium.
(2) Allow (1) to cool. (3) Sterilization not specified.
(3) Add 100.0 g. gelatin to the non- (g) Besson prepared the medium as
filtered nutrientmedium. follows:
(4) Allow to soak for an hour or to (1) Dissolve 5.0 g. Liebig's meat ex-
stand in the ice box over night. tract in 1000.0 cc. of water.
(5) Neutralize with N/1 NaOH to (2) Dissolve 100.0 g. gelatin in (1).
litmus. Shake well after each (3) Boil for 3 minutes.
addition of NaOH. (4) Neutralize.
(6) Dissolve the gelatin in the steamer (5) Heat at 115 for 5 minutes.
requiring about 30 minutes. (6) Filter thru paper, in the autoclave
(7) Make alkaline by the addition of or use a hot water funnel.
1.0 g. crystalline soda. (7) Tube.
(8) Cool to about 40 or 50C. (8) Sterilize at 110 for 20 minutes,
(9) Dissolve 1.5 g. of egg albumin in (h) Wolf and Shunk reported that a
25.0 cc. of luke warm water and greater pH range may be obtained
add to (8). Shake thoroly. by adjustment of reaction after steri-
(10) Boil in the steamer for about lization before solidification using a
30 minutes. medium prepared as follows:
(11) Filter thru sterile paper at
hot, (1) Dissolve 100.0 to 150.0 g. gelatin,
room temperature of 50 or 60C. 3.0 g. Liebig's beef extract, 10.0 g.
This may be carried out in the Peptone (Armour's) and 5.0 g.
steamer after the flame has been NaCl in 1000.0 cc. of water by
removed. May allow to filter heating in autoclave.
over night. (2) Flask.
(12) Distribute in sterile flasks in (3) Sterilize at 10 pounds for 15
100.0 cc. lots or in 10.0 cc. lots in minutes.
sterile tubes under aseptic con- (4) Cool to about 40 C.
ditions. (5) With sterile pipette, pipette
(13) Sterilize in the steamer for about 10.0 cc. quantities into sterile test
20 minutes and cool quickly in tubes and add appropriate quan-
cold water. tities of strong acid or alkali
(e) Burger also prepared media in the to give the desired reaction.
same manner as indicated above using (Authors used HCl sp. gr. 1.20 or
the following amounts of constit- 39.11% and NaOH, sp. gr. 1.226 or
uents.Using these media, the water approximately 20.0%.)
under investigation may be added (6) Mix
the tube thoroly and cool.
directly to the sterile gelatin. Do
not sterilize.
1. Distilled water. . 800.0 cc. 750.0 cc, (i) Giltner prepared the medium as
2. Meat extract follows:
(Liebig's) 20.0 g. 25.0 g. (1) Add 150.0 g. gelatin to a liter of
3. Peptone (Witte tap water and heat on a water bath
Rostock) 20.0 g. 25.0 g. until solution is complete.
4. NaCl 10.0 g. 12.5 g. (2) Cool down to 60 C.
(3) Mix 10.0 g. of egg albumin in (4) To 900.0 g. of (2) add 100.0 g. of
100.0 cc. of water and add to (2). gelatin.
(4) Add 3.0 g. meat extract, 10.0 g. (5) Steam until solution is complete
peptone and 5.0 g. NaCl to (3). (not longer than 30 minutes).
(5) Cover the container, place in the (6) Add 4.0% NaOH solution until
autoclave and heat at about 15 blue litmus paper is no longer
pounds pressure for 45 minutes. turned red.
(6) Adjust the reaction to +1.5% by (7) Boil for 15 minutes in the steamer.
the addition of NaOH or HCl. (8) Add 1.5 g. of crystalline (not
(7) Filter while boiling thru
hot effloresced) soda per liter.
plaited filter paper, washed with (9) Boil 30 minutes.
500.0 g. boiling water. (10) Filter.
(8) Distribute as desired. (11) Tube in 10.0 cc. quantities.
(9) Autoclave for 20 minutes at (12) Sterilize by a single heating at 15
10 pounds pressure. to 20 minutes in steam.
(10) Cool in a running water bath im- References: Frost (1903 p. 6), Stoklasa
mediately, (1908 p. 489), Abel (1912 p. 19), Day and
(j) Stitt prepared the medium as follows: Walker (1912-13 p. 435), Burger (1916-17
(1) Place 3.0 g. Liebig's extract, p. 477), Meier (1918 p. 435), Besson (1920
10.0 g. peptone and 5.0 g. NaCl in p. 40), Wolf and Shunk (1921 p. 325)
a mortar. Giltner (1921 p. 35), Stitt (1923 p. 38),
(2) Dissolve the white of one or two Klimmer (1923 p. 193), Park, Williams
eggs in 1000.0 cc. of water. and Krumwiede (1924 p. 177).
(3) Add (2), little by little, to (1),
2300. Kohn's Peptone Extract Gelatin
until a brownish color is obtained.
(4) Heat (3) to 45 in a water bath. Constituents

(5) Dissolve about 120.0 g. of Gold 1. Distilled water 1000.0 cc.
Label or other good quality gelatin 2. Meat extract
in (4). (Liebig's) 6.0 g.
(6) Add normal NaOH to
sufficient 3. Peptone (Witte's) . . 10.0 g.
bring the reaction to +1. 4. NaCl 5.0 g.
(7) Place in the inner compartment of 5. K2HPO4 2.0 g.
a rice cooker. 6. Gelatin 100.0 or 120.0 g.
(8) Boil for 15 minutes. Preparation
(9) Filter thru paper in a hot
filter (1) Dissolve 2, 3, 4 and 5 in 1 by heating
funnel. The paper must be very on the water bath.
thoroly wetted with very hot (2) Add and dissolve 100.0 to 120.0 g.
water. gelatin in (1).
(10) Tube. (3) Neutralize to litmus with NaOH and
(11) Sterilize either in the Arnold on then add 1.5 g. crystalline soda
3 successive days or in the auto- (15.0 cc. of a 10.0% soda solution).
clave at 8 to 10 pounds pressure Sterilization: Not specified.
for 10 minutes. Use: Medium used for water analysis.
(12) Cool the tubes as quickly as Reference: Kohn (1909 p. 127).
possible.
(k) Klimmer gave the following method
2301. Buchan's Neutral Red Peptone
Extract Gelatin
of preparation:
(1) Dissolve 10.0 g. Liebig's meat Constituents
extract, 10.0 g. Witte's peptone, 1. Distilled water 850.0 cc.
and 5.0 g. NaCl in 1000.0 cc. water 2. KOH (5.0% solution) 10.0 cc.
by heating for 30 minutes in the 3. Gelatin 100.0 g.
steamer. 4. Lemco 10.0 g.
(2) Allow to stand until cool. 5. Peptone 20.0 g.
(3) Filter. 6. Neutral red
Preparation that liquefy gelatin. Oxalic acid was

2, 3, formed by these organisms. Wash a cul-
(2) Add neutral red to give a brilliant ture from the surface of the agar or gela-
color to (1). tin with H2SO4. Filter and evaporate.
Sterilization: Not specified. Oxalic acid crystals will form.
Use: To determine fluorescence production Variants: Banning prepared a medium by
by organisms found in ice cream. dissolving 20.0 g. glucose, 10.0 g. peptone,
Reference: Buchan (1910 p. 108). 10.0 g. meat extract and 70.0 g. gelatin
in 1000.0 cc. water. The reaction was
2302. von Caron's Nitrate Extract Gelatin
slightly acid.
Constituents References: Henneberg (1898 p. 18), Ban-
1. Water 1000.0 cc. ning (1902 p. 397, 426).
2. Peptone (1.0%) 10.0 g.
3. Beef extract (Lie- 2304. Gottheil's Glucose Extract Gelatin
big's) (1.0%) 10.0 g. Constituents
4. KNO3 2.0 g. 1- Water lOOO.O cc.
5. Gelatin (5.0 to 2. Gelatin loo.O g.
10.0%) 50.0 to 100.0 g. 3. Glucose 10.0 g.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 4. NaCl [.', 2*0
g!
Sterilization: Not specified. 5. Meat
extract (Liebig's) .... 8.0 g.
Use: Isolation of denitrifying bacteria. 6. Peptone (Witte) 12.0 g.
Bac. Stutzeri, Bac. filefaciens, Bad. deni- Preparation
trificans, Vibrio denitrificans, Bad. Dissolve 5 and 6
(1) 4, in 500.0 cc. of
nitrovorum, Bad. centropundatum. water.
Variants: Percival dissolved 0.5% Lemco or (2) Neutralize by the addition of a con-
other meat extract, 1.0% peptone, 0.0 or centrated Na2C03 solution.
0.5% NaCl, 0.3 g. NaNOa and 10.0% gelatin (3) Heat for a time in a sterilizer.
in water. The medium was neutralized
(4) Filter.
by the addition of Na2C03 and sterilizer (5) Soak 100.0 g. gelatin in 500.0 cc. of
on 3 successive days for 20 minutes. water for about 3 hours.
References: von Caron (1912 p. 70), Perci- (6) Add (4) to (5).
val (1920 p. 165). Allow to stand for 3 hours at 100C.
(7)
(8) Neutralize once more with Na2C03
2303. Henneberg's Glucose Extract Gelatin
and heat for a short time.
1. Water 1000.0 cc. (10) Add 10.0 g. glucose.
2. Dextrose 20.0 to 50.0 g. Sterilization: Sterilize on 3 successive days
3. Meat extract 10.0 g. in the steamer.
4. Peptone 10.0 g. Use: Gottheil cultivated organisms found
5. NaCl 2.0 g. in the soil and in the root and rhizomes of
6. Gelatin 50.0 to 100.0 g. leguminous plants. Bachman cultivated
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 obligate anaerobes on a similar medium.
in 1.
Variants: Bachman dissolved 16.0% gela-
Sterilization: Not specified. tin, 1.0% glucose, 1.0% Witte's peptone,
Use: Cultivation of acetic acid bacteria 0.8% meat extract, and 0.2% NaCl in
B. Pasteurianum, B. oxydans, B. aceti, water. The reaction was adjusted to a
B. Kiitzingianum. Banning used a sim- slight alkalinity by the addition of
ilar medium to study oxalic acid forma- Na.COa.
tion by Bad. industrium Henneberg, References: Gottheil (1901 p. 432), Bach-
Bad. oxydans Henneberg, Thermobac- man (1912-13 p. 7).
terium aceti Zeidler, Bad. acidi oxalic,
Bad. monasteriense, Bad. diabdicum, 2305. Buchner's Sucrose Extract Gelatin
Bad. dortmundense, and Bad. parvulum. Constituents
Agar was used to grow those organisms 1. Water 1000.0 cc.
2. Gelatin 100.0 g. tity of litmus added to give a light blue
3. Liebig's meat extract 5.0 g. color.
4. Peptone (puriss) 5.0 g. References: Zipfel (1911-12 p. 129), Besson

5. Sucrose 20.0 g. (1920 p. 59).
6. NasHPOi 5.0 g.
2308. Wurtz's Nutrient Gelatin
Preparation:
(1) Dissolve 2, 3, 4, 5 and 6 in 1. Constituents:
(2) Make slightly alkaline by the addition 1. Bouillon 1000.0 cc.
of soda. 2. Gelatin 70.0 g.

Use: Cultivation of cholera bacillus. (1) Add 70.0 g. gelatin to a liter of
Variants: Besson used 5.0 g. Ca3(P04)2 bouillon.

instead of Na2HP04. (2) Heat on a salt water bath until the
References: Buchner (1885 p. 368), Weis- gelatin is dissolved.
ser (1886 p. 325), Besson (1920 p. 40). (3) Adjust the reaction to a slight
alkalinity.
2306. Percival's Urea Extract Gelatin (4) Add the whites of 2 eggs beaten up in
Constituents some water, when the temperature
1. Water 1000.0 cc. of the gelatin reaches 50 C.
2. Beef extract (Lemco) 5.0 g. (5) Heat in the autoclave at 115 for one
3. Peptone (Witte) 10.0 g. minute.
4. Gelatin 100.0 g. (6) Filter thru Chardin paper.
5. Urea 20.0 g. (7) Distribute in tubes.

(2) If acid neutralize with (NH4)2C03.
Variants
(3) Tube. (a) Smith dissolved 10.0 to 15.0% of the
Sterilization: Method not given. best grade of sheet gelatin in bouillon.
Use: Cultivation of urea splitting organ- (b) Sergent cultivated Bacterium zopfii
isms, Urobacillus Pasteuri. The author on one of the following combinations:

reported that urea fermenting colonies Bouillon.. 1000.0 cc. 1000.0 cc. 1000.0 cc.
were surrounded with small white crys- NaCl .... 5.0 g. 5.0 g.
tals ofcalcium carbonate. Peptone.. 10.0 g. 10.0 g. 10.0 g.
Reference: Percival (1920 p. 224). Gelatin... 130.0 g. 80.0 g. 180.0 g.

(c) Lohnis prepared the medium as
2307. Zipfel's Basal Nutrient Gelatin follows:
Constituent: (1) Add 10.0% of the best Gold Label
1.Nutrient gelatin 1000.0 cc. gelatin to 350 cc. hot bouillon.
Preparation: (1) Prepare nutrient gelatin (2) When solution is complete neu-
and dissolve one of the added nutrients. tralize to litmus.
Sterilization: Not specified. (3) Steam for 45 minutes.
Use Zipfel cultivated nodule bacteria.

: (4) Readjust the reaction if necessary.
Added nutrients The author added one
: of (5) Beat the white of an egg thoroly.
the following: (6) Add (5) to (4) cooled to about
Leucine 2.0 or 3.0% 50C.
Tyrosine 0.05, 0.1 or 0.2% (7) Heat for 30 to 45 minutes in the
Cholesterin. . . . 0.02, 0.3 or 0.5% steamer.
Asparagin 2.0 or 5.0% (8) Filter thru folded filter paper
Nucleic acid. . . 0.2, 0.5, 1.0 or 2.0% using a hot water funnel.
Caffeine 0.1, 0.2, 0.3, 0.4 or 0.5% (9) Distribute as desired.
Variants : Besson added 2.0 to 4.0% of any (10) Steam for 10 to 15 minutes.
desired carbohydrate, alcohol, etc., to (d) Roddy gave the following method of
nutrient gelatin. Just before use the preparation:

medium was melted and sufficient quan- (1) Break up 100 to 300.0 g. of the best
French gelatin in 1000.0 cc. of 2. Neutral red (Sat. Aq. soln. Grubler's
bouillon. or other indicator).
(2) Allow to soak for 5 minutes. Preparation: (1) Add 4 drops of sterile
(3) Heat.
saturated watery solution of Grubler's
(4) Stir until solution is complete.
neutral red to 10.0 cc. of sterile melted
(5) Sterilize in the steamer for 15 min- gelatin at 40C.
utes on each of 3 successive days.
Sterilization: INlethod of sterilization of
(e) Bezangon prepared the medium as
gelatin or neutral red solution not given.
follows:
Use: Neutral red test for coli organisms.
(1) Dissolve 100.0 g. of gelatin (126.0
Author reported that B. coli caused a
to 150.0 g. in summer) in 1000.0 cc.
decolorization and fluorescence of this
bouillon.
Coolto50"C.
medium after 6 hours. Other investi-
(2)
gators used similar media for purposes
(3) Add the beaten white of an egg.
as indicated.
(4) Adjust the reaction to slightly
alkaline. Variants
(5) Heat for 15 minutes at 110C. (a) Calandra added 4 drops of a sterile
(6) Filter while hot. 1.0% watery solution of brilliant
Tube. cresyl blue to 10.0 cc. of sterile nu-
(7)
(8) Sterilize at 110C. for 15 minutes. trient gelatin. The gelatin was
(f) Giltner added 3.0% NaCl to nutrient colored bright green. The medium
gelatinand adjusted to 2.0%. The was used to differentiate colon bacilli
medium was used for the cultivation
from typhoid bacilli. He reported
of phosphorescent that B. coli did not cause decoloriza-
halophilic or-
ganisms. tion after 24 hours. Typhoid in con-
Cunningham prepared trast gave a light green layer on the

(g) the medium
as follows: upper surface. After several days,
(1) Add 10.0% Coignet's gelatin to however, the gelatin was completely
bouillon. decolorized if B. coli be present. A
Steam thin light green layer remained if
(2) for 1 hour to dissolve the
gelatin. typhoid bacilli be present.
(3) Adjust to a slight alkalinity using (b) Signorelli added 1.0 cc. of a 1.0%
turmeric paper as an indicator solution of dahlia to each 10.0 cc. of
(distinctly brown). neutral nutrient gelatin. He used
(4) Steam again for 45 minutes. the medium to show adsorption of
(5) Readjust the reaction if necessary. the dye by the cholera vibrio and
(6) Cool the medium to 50C. and add reported that liquefaction and de-
1-0% egg albumin mixed to a colorization took place at the same
thin paste in a few cc. of water. time. The bacteria were highly
(7) Steam for 45 minutes. colored.
(8) Filter hot thru gray filter paper (c) Signorelli added 1.0 cc. of a 1.0%
that has had boiling water poured solution of erythrosin and 1.0 cc. of
thru it. a 1.0% solution of safranin to 10.0 cc.
(9) Tube in 8.0 cc. quantities. of neutral nutrient gelatin. He re-
(10) Sterilize intermittently in steam. ported that the cholera vibrio ad-
References: Wurtz (1897 p. 28), Smith sorbed the dye, being highly colored
(1902 p. 86), Sergent (1906 p. 1015), and that the gelatin was liquefied.
Lohnis (1913 p. 15), Roddy (1917 p. 43), (d) Besson prepared a medium using
Worth (1919 p. 604), Bezangon (1920 Noeggerath's indicator solution.
p. 113), Giltner (1921 p. 370, Cunningham (1) Prepare saturated watery solution
(1924 p. 13). ofmethylene blue, gentian violet,
methyl green, chrysoidine and
2309. Heller's Indicator Nutrient Gelatin
fuchsin.
Constituents Mi.x
(2) 2.0 cc. of methylene blue
1. Nutrient gelatin. 4.0 cc. gentian violet, 1.0 cc. of
methyl green, 4.0 cc. of chrysoidine 2312. Wurtz's Litmus Lactose Gelatin
and 3.0 cc. of fuchsin. Constituents
(3) Add 200.0 cc. of distilled water to 1. Nutrient gelatin 1000.0 cc.
(2) and allow to stand several 2. Lactose (2.0%) 20.0 g.
hours. The color should be green- 3. Litmus
ish blue. If not obtain the origi- Preparation
nal color by adding either blue, Add 20.0 g. lactose to sterile neutral
(1)
green or red dye. nutrient gelatin.
(4) Sterilize at 100C. (2) Tube.
(5) Add drops of (4) to a tube
7 to 10 Add sufficient tincture of litmus to
(3)
of sterile melted nutrient gelatin. each tube to give a violet color.
References: Heller (1905 p. 118), Calandra (4) Pour sterile (3) into plates.
Signorelli (1912 p. 472),
(1910 p. 570), Sterilization: Sterilize (3) at 100C.
Besson (1920 p. 60). Use: Differentiation of Bad. colt. The
author reported that Bad. coli fermented
2310. Matzuschita's Glucose Gelatin lactose with the production of acid.
Typhoid bacilli colonies were colorless,
Constituents
colon colonies red.
Variants
2. Gelatin 100.0 g.
(a) Heinemann gave the following
3. Peptone 10.0 g.
method of preparation:
4. NaCl 5.0 g.
Dissolve 1.0% lactose in ordinary
(1)
5. Glucose 20.0 g.
10 or 12.0% gelatin.
Preparation: (1) Dissolve 2, 3, 4 and 5 in
1000.0 cc. bouillon.
tubes.
(3) Add 1.0% sterile litmus solution to
each tube before using (amount of
chicken tubercle bacilli. The author re-
solution not specified).
ported that the Mammalian and chicken
(b) Abbott prepared the medium as
types gave same kind of growth. A
follows:
white precipitate was formed in the nutrient gelatin so the
(1) Prepare
liquid gelatin (at 30C.). Other in-
requires
alkalinity is such that it
vestigators used similar media for pur- 1:20 normal H2SO4
0.1 cc. of a
poses as indicated below. neutralize 1.0 cc. of
solution to
Variants Indicator not specified.
medium.
(a) Rivas solidified medium 923 by the
(2) Add 2.0 to 3.0% lactose.
addition of gelatin. This medium
(3) Decant into test tubes.
was used to cultivate anaerobes. way (method
(4) Sterilize in the usual
(b) Frost added 1.0% glucose to nutrient
not given).
gelatin.
(5) Add sufficient sterile litmus tincture
(c) Worth cultivated members of the
to each tube to give a decided but
colon-typhoid group on nutrient
not intense blue color. Add the
gelatin of a reaction 0.4% or 1.0%
litmus under aseptic conditions.
acid to which was added 2.0% glu-
References: Wurtz (1897 p. 43), Heinemann
cose. He also prepared a medium Abbott
(1905 p. 127), Tanner (1919 p. 55),
adding calcium carbonate.
(1921 p. 142).
Rivas (1902 p. 836), Frost (1903 p. 64), 2313. Wurtz's Glycerol Gelatin
Worth (1919 p. 604).
Constituents
1. Nutrient gelatin 1000.0 cc.
2311. Ramond's Rubine Acid Lactose 60.0 g.
2. Glycerol (6.0%)
Gelatin
Preparation: (1) Add 6.0% sterile glycerol
Same as medium 1796 but solidified by the to sterile nutrient gelatin under aseptic
addition, of gelatin instead of agar. conditions.
Sterilization: Method not given. medium was then heated in the steam
sterilizer.
Reference: Wurtz (1897 p. 46). (c) Geilinger
cultivated urea splitting
2314. Holz's Phenol Gelatin organisms on nutrient gelatin con-
taining 1.0% urea.
Constituents: (d) Cunningham determined the de-
1. Nutrient gelatin.
composition of urea on nutrient
2. Phenol (5.0% solution).
12.0% gelatin containing 2.0% urea.
Preparation
The medium was sterilized by the
(1) Prepare nutrient gelatin in the usual
intermittent method in the steamer.
manner. References: Stutzer and Hartleb (1897
p.
(2) Neutralize with 10.0% NaoCOs solu-
403), Hoffman (1912 p. 387), Lohnis
tion, using litmus as an indicator. (1913 p. 95), Geilinger (1917 p. 246),
(3) Distribute sterile (2) in 10.0 cc. lots Cunningham (1924 p. 143).
by means of a burette.
(4) Heat once more. 2316. Park, Williams and Krumwiede's
(5) Add from 7 to 12 drops of 5.0% phenol Meat Gelatin
solution to each tube. (3 drops = Constituents
0.1 cc.)
1. Nutrient gelatin.
(6) Pour in plates, or prepare roll 2. Meat.
cultures. Preparation
Sterilization: Sterilize by repeated
(2) (1) Drop pieces of meat into tubes of
heatings.
nutrient gelatin.
Use: Isolation of typhoid bacilli. Sterilization: Method not given.
Variants: Migula added 0.5 cc. of a 4.0% Use: General culture medium.
phenol solution to each tube (10.0 cc.) Reference Park, Williams and Krumwiede
:
of slightly alkaline nutrient gelatin.
(1924 p. 125).
References: Holz (1890 p. 144), Migula
(1901 p. 24). 2317. Worth's Glucose Liver Gelatin
Constituents
2315. Stutzer and Hartleb's Urea Gelatin
Constituents: 2. Glucose (2.0%) 20.0 g.
1. Nutrient gelatin 1000.0 cc 3. Liver, rabbit
2. Urea (2.0%) 20.0 g. Preparation
Preparation
(1) Prepare nutrient gelatin and add 2.0%
(1) Add 2.0% urea to ordinary nutrient glucose.
gelatin.
(2) Adjust the reaction to -f-1.0% acid.
Sterilization: Method not given. (3) Add rabbit liver.
Use: Attempt to cultivate the bacterium Sterilization: Not specified.
of foot and mouth disease. Other in-
Use: Preservation of cultures.
vestigators cultivated soil bacteria and Reference: Worth (1919 p. 604).
urea splitters on similar media.
Variants 2318. Nastinkoff's Egg Yolk Nutrient
(a) Hoffman added 1.0% urea to 10.0% Gelatin
alkaline gelatin. The medium was Same as medium 1950 but solidified with
used for differential count of bacteria gelatin instead of agar.
from soil. He reported that if urea
was fermented the colonies were 2319. Pergola's Alkaline Blood Gelatin
surrounded by a halo of characteris- Constituents
tic biscuit shaped crystals.
(b) Lohnis studied urea decomposition 2. Blood, beef 150.0 cc.
on a medium prepared by adding KOH,
3. normal 150.0 cc.
1.0 cc. of a 15.0% aqueous solution
Preparation
of urea to tubes of gelatin. This (1) Collect beef blood in a sterile flask
containing glass beads. Shake to (5) May covered with a layer of

be
defibrinate the blood. but not necessary in order to
paraffin,
(2) Mix an equal volume of (1) and N/1 obtain growth. Incubate at 15 to
KOH solution. 37C.
(3) Prepare nutrient gelatin. It may be References: MuUer (1906 p. 519), Ito and
necessary to increase the gelatin Matsuzaki (1916 p. 558).
content of the medium to 20 or 30.0%

in order to obtain a solid medium 2321. Miiller's Serum Gelatin
after the addition of the alkaline
Constituents
blood.
(4) Neutralize (3) to litmus.
2. Serum, goat 1000.0 cc.
(5) To 700.0 cc. of the melted neutral
Preparation
nutrient gelatin add 30.0 cc. of sterile
(1) Prepare nutrient gelatin.
(2) and mix.
(2) Melt (1) and cool
about 60C.
to
(6) Distribute into sterile tubes or plates.
Sterilization: Sterilize (2) by heating in (3) Mix equal and goat serum
parts of (2)
that has been obtained under aseptic
the Koch steamer for 30 minutes. Steri-
conditions and heated to 60C.
lization of gelatin not specified.
Use: Enrichment and detection of cholera (4) Pour into sterile plates or sterile
tubes.
vibrio. The author reported that the
cholera vibrio liquefied the gelatin. Sterilization: Sterilization of gelatin not
Reference: Pergola (1910 p. 493). specified. See step (3) for sterilization
of serum.
2320. Miiller's Blood Gelatin
Use : Cultivation of fowl diphtheria bacilli.
Constituents Miiller reported that the gelatin was
1. Nutrient gelatin 1000.0 cc. liquified.
2. Blood, goat 100.0 cc. Reference: Muller (1906 p. 520).
Preparation
(1) Distribute nutrient gelatin in 240.0 2322. Beijerinck's Trypsinized Gelatin
cc. lots in sterile 400.0 cc. Erlenmeyer
Constituents
Allow about 25.0 cc. of blood to pass 1. Water 900.0 cc.
(2)
2. Gelatin (liquefied) 10.0 cc.
directly from the juglar vein of a
goat into melted gelatin, cooled to 3. (NH4)N03 5.0 g.
about 45C. 4. Potassium phosphate 5.0 g.

5. Gelatin 80.0 g.
(3) Mix well and pour into sterile Petri
dishes. Preparation
Sterilization: Method not specified. (1) Liquefy gelatin with pancreas extract
Use: Cultivation of fowl diphtheria bacilli. (method not given).
Ito and Matsuzaki cultivated Spiro- (2) Dissolve 10.0 cc. (1), 3, 4 and 5 in
chaeta icterohaemorrhagiae on a similar 900.0 cc. of water.
medium. (3) Neutralize, or adjust to a slight
Variants: Ito and Matsuzaki prepared acidity.

their medium as follows: Sterilization: Not specified.
(1) Prepare nutrient gelatin. Use: To study liquefaction of gelatin and

(2) Melt (1) and cool to 25-30C. pigment production by Chlorococcum.
(3) Add from 2 to 4 parts of guinea pig Beijerinck reported that chlorococcum
blood or human blood to one part of did not liquify the gelatin.
(2). The erythrocytes settle to the
bottom of the gelatin before solidifi-
cation giving it an opaque and deeper 2323. Jensen's Pepsinized Milk Gelatin
color.
(4) Inoculate with a drop of infected Same as medium 1112 but solidified by the
blood and mix well by stirring. addition of gelatin.
SUBGROUP III-B. SECTION 3 A Containing unknown constituent

2.
of
Basal or complete media containing animal origin only.
gelatinand other constituents of unknown Bj. Extracts or infusions not
employed.
chemical composition, but not digests. Ci. Tissues or body fluids used.
Ai. Containing unknown constituents of Nastinkoff's Egg Yolk Gelatin 2350
plant origin only. Deycke and Voigtlander's Albumi-
Bi. Yeasts or their derivatives employed. nate Gelatin 2351
Bobilioff-Preisser's Yeast C2. Secretions or excretions used.
Water
Gelatin 2324 Di. Milk or its derivatives used.
Beijerinck's Glucose Yeast Gelatin. 2325 Raskin's Whey Albumin Gelatin.... 2352
Will's Sucrose Yeast Gelatin Giltner and Ludum's Amniotic Fluid
2326
Beijerinck's Urea Yeast Gelatin. . . . 2327 Gelatin 2353
B2. Flowering plants or their derivatives Raskin's Whey Gelatin 2354
employed. Reinsch's Fat-free Milk Gelatin. . . . 2355
Ci. Non leguminous plants or derivatives Abbott's Milk Gelatin 2356
used. Heinemann's Whey Gelatin 2357
Dj. Grains or their derivatives used. Raskin's Casein Whey Gelatin
Will's Basal Wort Gelatin (Peif er) 2358
2328
Heinemann's Beer Wort Gelatin.. D2. Milk or derivatives not used.
its
. . 2329
Janke's Alcohol Beer Gelatin 2330 Piorkowski's Urine Infusion Gelatin
D2. Fruits or their derivatives used. (Scholz and Krause) 2359
Perold's Grape Juice Gelatin B2. Extracts or infusions employed.
2331
Dombrowski's Raisin Must Gelatin. 2332 Ci. Infusions specified.
Besson's Raisin Infusion Gelatin. Kutscher's Meat Infusion Gelatin.. 2360

. . 2333
Klimmer's Prune Infusion Gelatin.. 2334 Graham-Smith's Heart Infusion
Lohnis' Plum Extract Gelatin Gelatin 2361
2335
D3. Tubers or their derivatives used. Beijerinck's Asparagin Meat In-
fusion Gelatin (Ludwig) 2362
Eisner's Iodide Potato Gelatin
2336
Holz's Potato Gelatin Kopp's Tyroid Infusion Gelatin. 2363 . . .
2337
Goadby's Glycerol Potato Gelatin Wroblewski's Suprarenal Capsule
Infusion Gelatin 2364
.(Tanner) 2338
Zikes' Carrot Infusion Gelatin
Harde and Hauser's Fish Infusion
2339
Gelatin 2365
D4. Non leguminous plants or their
deriva- C2. Extracts specified.
vatives other than Dj to Dj used. Molisch's Meat Extract Gelatin .... 2366
Stutzer, Burri and Maul's Glucose
Hollborn's Meat Extract Gelatin. 2367
Mustard Seed Gelatin 2340
. .
A3. Containing unknown constituents other

Groenwege's Tomato Infusion than of plant or animal origin.
Gelatin 2341 Fremlin's Soil Infusion Gelatin 2368
Will'sCabbage Juice Gelatin 2342 Conn's Glucose Soil Infusion
Miiller-Thurgaw's Grape Vine In-
Gelatin 2369
fusion Gelatin 2343 Lohnis Cyanamide Soil Infusion
C2. Leguminous plants or derivatives used. Gelatin 2370
De Rossi's Glucose Leguminous Conn's Tartrate Soil Infusion
Plant Gelatin 2344 Gelatin 2371
Simon's Asparagin Legume E.xtract
Gelatin (Klimmer and Kriiger). 2345 . . .
2324. Bobilioff-Preisser's Yeast Water
Zipfel's Glucose Legume Infusion Gelatin
Gelatin 2346 Constituents
Kaufman's Jequirity Seed Gelatin. 2347 .
Stutzer, Burri and Maul's Glucose
2. Gelatm (10.0%) 100.0 g.
Alfalfa Gelatin 2348 Preparation :
Barthel's Basal Lupini Extract (1) Dissolve 10.0% gelatin in yeast water.
Gelatin 2349 Sterilization: Not specified.
Use: Cultivation of Oospora, fungi im- Vierling studied urease production by

perfecti. The authors reported that mycobacteria on similar media.
gelatin was liquefied. Variants
Reference: Bobilioff-Preisser (1916 p. 400). (a) Tausz and Peters cultivated Bac-
terium aliphaticum, Bacterium ali-
2325. Beljerinck's Glucose Yeast Gelatin phaticum liquefaciens, on the follow-
ing medium:
Same as medium 2081 but solidified by the Boil 100.0 pressed yeast in 500.0
(1) g.
addition of 20.0% gelatin instead of agar.
cc. water.
(2) Dissolve 50.0 g. of gelatin in (1).
2326. Will's Sucrose Yeast Gelatin
Constituents: (4) Sterilize on 3 successive days in
1. Yeast water 1000.0 cc. streaming steam.

2. Sucrose (6.0%) 60.0 g. (5) To determine the production of
3. Gelatin (10.0%) 100.0 g. urease, add 1.25 g. sterile urea to
Preparation the medium.
(1) Add 6.0% sucrose and 10.0% gelatin (b) Vierling studied urease production
to neutral yeast water. by mycobacteria on a medium
Sterilization: Not specified. prepared as follows:
Use: Cultivation of non-spore forming (1) Boil 200.0 g. pressed yeast with
saccharomyces. 1000.0 cc. of water.
Reference: Will (1908 p. 387). (2) Cool, decant and filter.
(3) Dissolve 25.0 g. urea and 100.0 g.

gelatin in the filtrate.
2327. Beljerinck's Urea Yeast Gelatin
(4) Sterilize (Method not given).
Constituents (5) Pour in plates.
1. Water 1000.0 cc. References: Beijerinck (1901 p. 39), Tausz
2. Y'east (pressed) 200.0 g. and Peters (1919 p. 49), Vierling (1920 p.
3. Urea 20.0 to 30.0 g. 205).
4. Gelatin
2328. Will's Basal Wort Gelatin
Preparation
(1) Prepare yeast water by boiling 200.0 Constituents
g. pressed yeast with 1000.0 cc. water. 1. Wort Gelatin (10.0%).
(2) Add 2 or 3.0% urea and gelatin (amount Preparation
not specified) to (1). (1) Prepare 10.0% wort gelatin.
Sterilization: Not specified. (2) Dissolve one of the added nutrients
Use: To show iridescence by urea splitting in (1).
organisms, Urococcus ureae and Uroba- Sterilization: Not specified.
,cillus pastenrii. Author reported that Use: Cultivation of mycoderma-like or-
when a urea splitting organism or a ganisms. Park, Williams and Krum-
particle of (NH4)2C03 was added to a wiede used a similar medium for the
properly prepared plate, a characteristic cultivation of molds.
precipitate was formed. This was amor- Added nutrients: The author added 0.7%
phous and stayed at first entirely on the asparagin or 1.0% ammonium tartrate.
upper surface of the gelatin in a thin Variants
sheet. This showed Newton's color rings. (a) Will used the 10.0% wort gelatin
As the layer grew it became thicker and without any additions.
the edge spread over a greater surface (b) Park, Williams and Krumwiede added
giving a color ring. After a time a white 2.0% of any desired carbohydrate to
precipitate formed in the depths of the beerwort, solidified by the addition of
gelatin, which assumed the shape of 10.0% gelatin.
plano-convex lens. Non-urea splitting References: Will (1900 p. 597), (1910 p. 3),
organisms did not show this. Tausz and Park, Williams and Krumwiede (1924 p.
Peters cultivated paraffin bacteria and 134).
2329. Heinemann's Beer Wort Gelatin 2330. Janke's Alcohol Beer Gelatin
1. Beerwort 1000.0 cc. 1. Lager beer 1000.0 cc.
2. Gelatin (10.0%) 100.0 g. 2. Gelatin (10.0 to
Preparation 12.0%) 100.0 to 120.0 g.
120 3. Alcohol (3.0%) 30.0 cc.

(1) Autoclave beerwort at for 5
Preparation
minutes.
(1) Evaporate lager beer to one-half its
(2) Cool.
original volume by steaming.
(3) Filter.
Dissolve 10.0% gelatin in the filtrate.
(2) Add water to its original volume.
(4)
(3) Dissolve 10.0% to 12.0% gelatin in
(5) Clarify with the white of egg.
(2).
Sterilization: Not specified. _

Use: General culture medium. thru folded filter.
(5) Filter
Variants Make up to the original volume by
(6)
(a) Nakazawa cultivated yeast on a the addition of water.
medium prepared by solidifying (7) Distribute in 10.0 cc. lots in Freuden-
12.0% unhopped beer wort with reich flasks.
10.0% gelatin. (8) Add 3.0% by volume of absolute
(b) Janke cultivated acetic acid bacteria alcohol.
from beers in a medium prepared as Sterilization: Sterilize on 3 successive days
follows: in the steamer.
(1) Dissolve 10.0 or 12.0% gelatin in Use: Cultivation of acetic acid bacteria
hopped lager beer wort. from beers.
(2) Clarify with egg albumin. Reference: Janke (1916 p. 6).
(4) Distribute in 50.0 cc. lots in flasks. 2331. Perold's Grape Juice Gelatin
(5) Sterilize the discontinuous method Constituents
in the steamer.
1. Water 1000.0 cc.
(c) Tanner gave the following method 2. Gelatin 150.0 g.
of preparation: 3. Grape juice 60.0 cc.
(1) Measure out 900.0 cc. of beer wort Preparation
in a sterile flask. (1) Dissolve 150.0 g. gelatin in 1 liter of
(2) Add 100.0 g. (10.0%) gelatin to (1). water.
(3) Bubble live steam thru to dissolve (2) Clarify the gelatin with egg white.
the gelatin. (3) To each 100.0 cc. of (2) add 6.0 cc. of
(4) Cool to 60C. grape juice.
(5) Clarify with egg. Sterilization: Sterilize for one hour in the
(6) Filter. steamer.
(7) Tube. Use: Cultivation of bacteria from wine.
(8) Sterilization [not specified. Reference: Perold (1909 p. 18).
(d) Klimmer cultivated yeast and molds
on the following medium: 2332. Dombrowski's Raisin Must Gelatin
(1) Obtain beer wort from a brewery. Same as medium 2109 but solidified by the
(2) Add gelatin (amount not given). addition of 10.0% gelatin instead of 1.5%
(3) Boil and filter. agar.
(5) Sterilize. 2333. Besson's Raisin Infusion Gelatin
References: Heinemann (1905 p. 128), Constituents

Nakazawa (1909 p. 530), Dombrowski 1. Water 1000.0 cc.
(1910 p. 380), Janke (1916 p. 6), Tanner 2. Raisins 250.0 g.
(1919 p. 56), Klimmer (1923 p. 207), Park, 3. Gelatin 100.0 g.
Williams and Krumwiede (1924 p. 134). 4. Sodium phosphate
Preparation 2. Potato 500.0 g,

(1) Soak 250.0 g. of dried raisins in a 3. Gelatin
liter of water for several hours. 4. KI (1.0%) 10.0 g.
(2) Boil. Preparation
Pass thru a coarse sieve.
(3) (1) Prepare a gelatin using 500.0 g.
Filter thru paper.
(4) potatoes and 1 liter of water.
(5) Dissolve 100.0 g. gelatin and a pinch Amount of gelatin to be added or
of sodium phosphate in the filtrate. method of preparation not given.
(6) Boil for 2 or 3 minutes. (2) Add N/1 NaOH to neutralize to
(7) Make slightly alkaline by the litmus.
addition of soda. (3) Filter and flask.
(8) Heat at 115 for 5 minutes. (4) Add 1.0% KI to each flask of sterile
(9) Filter thru paper, in the autoclave or (3).
use a hot water funnel.
(10) Tube.
(3) not given.
Sterilization: Sterilize at 110 for 20
Use: Differentiation of coli and typhoid
minutes.
bacteria. The author reported that B.
coli grew rapidly and luxuriantly. Colo-
nies were brown. Typhoid organisms
2334, Klimmer's Prune Infusion Gelatin grew slower. After 48 hours colonies
were small and glistening resembling
Constituents:
drops of water.
1. Water 500.0 cc.
Variants
2. Prunes 100.0 g.
3. Gelatin
(a) Grimbert gave the following method
of preparation:
Preparation
Boil 100.0 g. of dried prunes in 500.0 (1) Grate 500.0 g. potatoes and allow
(1)
to macerate with one liter of water
cc. of water,
for 3 or 4 hours or better allow to
(2) Filter.
stand over night in the cold.
(3) Add gelatin (amount not given).
(2) Decant and filter.
(4) Boil.
Filter. (3) Autoclave the filtrate for 10
(5)
minutes,
(4) Filter,
Use: General culture medium. (5) Add 15.0 g, of gelatin for each
Reference: Klimmer (1923 p. 206). and melt

100.0 cc. of the filtrate,
by heating on the salt water bath.
2335, Lohnis' Plum Extract Gelatin (6) Cool to 55C. and add the white of
Constituents an egg suspended in a little water.
Water (7) Adjust the reaction so that 5.0 cc.
1. 1000.0 cc.
Plums of lime solution is required to
2. 1000.0 g.
3. Gelatin (10.0%) 100.0 g.
neutralize 10.0 cc. medium to
Preparation phenolphthalein. (An acidity
Boil 1000.0 g. of plums with 1000.0 cc. equivalent to 1.0 g. H2SO4 per
(1)
liter.)
of water twice.
Add 10.0% gelatin to the juice of (1) (8) Heat in the autoclave for 10
(2)
without neutralization. minutes at 110C. to clarify.
(9) Filter.
Use: Cultivation of molds. (10) Tube in 9.0 cc. lots.
Reference: Lohnis (1918 p. 88). (11) Sterilize in the autoclave at 110

for 15 minutes.
2336. Eisner's Iodide Potato Gelatin
(12) When ready for use add 1.0 cc. of
Constituents a sterile 10.0% KI solution to each
1. Water 1000.0 cc. tube.
(b) Migula prepared the medium as (1) Mash 500.0 peeled and washed
g. of
follows: potatotes, and press thru a fine

(1) Prepare potato infusion from 1 cloth.
liter of water and 500.0 g. of (2) Allow the juice to settle.
potato. (3) Filter.
(2) Boil (1) with nutrient gelatin (4) Cook for one hour.
(amount not specified). (5) Add 10.0% gelatin to (4).
(3) Add 2.5 to 3.0 cc. of 0.1 normal (6) Add 2.5 cc. of 1/10 normal NaOH
NaOH to each 10.0 cc. of (2). and 1.0% KI.
(4) Filter. (7) Sterilization not specified.
(method not given).
(5) Sterilize (e) Bezanôn gave the following method
(6) When ready for use, pour into a of preparation:
flask, add 1.0% KI and the water (1) Peel and grate 500.0 g. of potatoes.
under investigation. (2) Add (1) to a liter of water and
(7) Pour into plates. allow to soak for several hours.
(c) Smith prepared the medium as (3) Allow to settle.
follows: (4) Decant and filter.
(1) Grate 500.0 g. of pared potatoes (5) Autoclave for 10 minutes.
and place the pulp in the refrig- (6) Add 150.0 g of gelatin and melt on
erator in a porcelain dish over a salt water bath.
night. (7) Adjust the reaction to a slight
(2) Express the juice from the pulp. acidity.
(3) Filter several times thru a layer of (8) Sterilize at 150C.
absorbent cotton or thru animal (9) Filter.
charcoal. (10) Flask in 100.0 cc. lots.
(4) The should now be titered

filtrate (11) When ready for use add 1.0 g. of
with decinormal sodium hydroxide KI and distribute in tubes.
solution to determine its reaction (12) Final sterilization not given.
and the amount of water which References: Eisner (1896 p. 29), Grimbert
will be required to be added to (1896 p. 723), Migula (1901 p. 23), Smith
reduce its acidity to the standard. (1902 p. 78), Ball (1919 p. 82), Bezangon
In this quantity of water is now (1920 p. 340).
boiled amount of gelatin
the
required to make 10.0% proportion 2337. Holz's Potato Gelatin
of the gelatin when the potato
juice shall have been added, the Constituents
reaction the gelatin being
of 1. Potatoes.
corrected to neutral point after 2. Gelatin.
it has been dissolved (and before Preparation
adding the potato juice) by means (1) Thoroly wash potatoes.
of normal sodium hydroxide solu- (2) Cut out the eyes and all rotten
tion. This done and the bulk of parts of the potatoes.
the fluid corrected for evapora- (3) Peel and wash the potatoes in tap
tion to that necessary to properly water.

dilute the potato juice the latter (4) Rub the potatoes to a pulp on an
isslowly added to the gelatin and ordinary kitchen grater.
mixed, and the whole boiled for (5) Filter the juice thru a clean towel
5 to 10 minutes. and press the pulp free from juice.

(5) Filter. (6) Place the juice in a flask, plug with
(6) Tube. cotton and allow to stand for 24
(7) Sterilize by the intermittent hours at 10C.
method. (7) Filter, the juice has turned brown.
(d) Ball gave the preparation of the (8) Heat in the steamer for 30 minutes
medium as follows: and filter again.
(9) Add 50.0 g. gelatin to 400.0 cc. of the (1) Peel clean potatoes and grind in
filtrate, and heat for 45 minutes in the grinding machine.
the steamer. (2) Press out the juice by means of a
(10) Tube in 5.0 or 10.0 cc. lots in sterile hand press.
test tubes. (3) Add bone black and heat and
(11) The medium is clear and brown. finally filter while hot.
The reaction is acid. 10.0 g. re- (4) Add 10.0% gelatin and prepare the
quires 1.6 cc. N/1 alkali to neutralize potato water gelatin in the usual
to litmus. manner. (Method not given.)
Sterilization Sterilize on 3 successive days
: (Carrots may be handled in the
for 15 minutes each day in streaming same way. Heat the carrot juice
steam. in the steamer before filtering.)
Use: Isolation of Bacillus typhosus and (d) Eisner (Tanner) gave the following
Bacillus coli communis. nethod:
Variants (1) Peel and grate 500.0 g. of potato.
(a) Holz added 0.05% phenol to inhibit (2) Grind (1) and soak in distilled
molds, liquefiers and other forms. water for 12 hours.
(b) Smith prepared the medium as (3) Strain and allow to stand.
follows: (4) Filter to clear.
(1) Grate 500.0 g. of pared potato and (5) Make up to one liter.
place the pulp in the refrigerator (6) Dissolve 15.0% gelatin in (5).
in a porcelain dish over night. (7) Adjust the reaction to acid (indi-
(2) Express the juice from the pulp. cator not specified).
(3) Filter several times thru a layer of (8) Sterilize (method not given).
absorbent cotton or thru animal (e) Eisner (Besson) prepared the medium
charcoal.
as follows:
(4) The filtrate should now be titered Thoroly
(1) wash, grate and peel
with decinormal sodium hydroxide potatoes.
solution to determine its reaction
(2) Soak 500.0 g. of (1) in a liter of
and the amount of water which will water for 4 hours.
be required to be added to reduce Pass thru a sieve.
(3)
its acidity to the standard. In
(4) Allow to settle over night.
this quantity of water is now
(5) Decant.
boiled the amount of gelatin
Make up the decanted liquid to
(6)
required to make 10.0% proportion
1000.0 cc.
of the gelatin when the potato
(7) Dissolve 150.0 to 200.0 g. of gelatin
juice shall have been added, the in (6) by heating gently.
reaction of the gelatin being
(8) Boil for several minutes.
corrected to neutral point after
(9) Add soda solution until the re-
it has been dissolved (and before
action isbut only slightly acid.
adding the potato juice) by means Heat at 115 for 5 minutes.
(10)
of normal sodium hydroxide solu-
(11) Filter thru paper, in the autoclave
tion. This done and the bulk of
or use a hot water funnel.
the fluid corrected for evaporation
(12) Tube.
to that necessary to properly
dilute potato juice, the latter is
References: Holz (1890 p. 159), Smith
slowly added to the gelatin and
(1902 p. 78), Will (1907 p. 435), Tanner
mixed, and the whole boiled for 5
(1919 p. 60), Besson (1920 p. 40).
to 10 minutes.
(5) Filter.
2338. Goadby's Glycerol Potato Gelatin
(6) Tube.
(Tanner)
(7) Sterilize by the intermittent
method. Constituents:
(c) Zikes (Will) prepared a medium as 1. Glycerol potato broth 1000.0 cc.
follows: 2. Gelatin 100.0 g.
Preparation (4) Add 10.0% gelatin.

(1) Prepare glycerol potato broth. (5) Clarify and filter.
(2) Add 100.0 g. of gelatin to (1). Sterilization: Not specified.

(3) Boil. Use: Cultivation of Phytibacter lyco-
(4) Adjust the reaction. persicum (causing tomato rot).
(5) Filter. Reference: Groenewege (1917 p. 24).
2342. Will's Cabbage Juice Gelatin
Reference: Tanner (1919 p. 60). Constituents
1. Cabbage juice.
2339. Zikes' Carrot Infusion Gelatin
2. Gelatin.
Same as variant (c) medium 2337 but Preparation
using carrots instead of potatotes. (1) Press the juice from cabbage leaves.
(2) Solidify (1) by the addition of 10.0%
2340. Stutzer, Burri and Maul's Glucose
gelatin.
Mustard Seed Gelatin
Constituents Use: Cultivation of mycoderma.
1. Water 1000.0 cc. Reference: Will (1910 p. 3).
2. White mustard seed 400.0 g.
Glucose
2343. MuUer-Thurgau's Grape Vine
3. 20.0 g.
Infusion Gelatin
4. Gelatin 100.0 g.
Preparation Same as medium 1188 but solidified by the
(1) Germinate white mustard seed at addition of gelatin.
30C. for 3 days. The sprouts are
2344. de Rossi's Glucose Leguminous Plant
from 1 to 1.5 cm. long.
Gelatin
(2) Add 1 liter of water to 400.0 g. of (1).
(3) Steam at 100C. for 2 hours. Constituents

(4) Filter while hot. 1. Water 1000.0 cc.
(5) Mix 500.0 cc. of filtrate with an 2. Leguminous plant
equal volume of water. 3. Gelatin
(6) Dissolve 100.0 g. gelatin and 20.0 g. 4. Glucose (2.0%)
of glucose in (5). Preparation
(7) Finish the preparation in the usual (1) Prepare a gelatin from the extract of
manner. (Method not specified.) leguminous plant leaves. (Method
(8) Adjust the reaction to a slight acidity. not given.)
(9) Distribute in 8.0 cc. lots. (2) Add 2.0% glucose.
Sterilization: Not specified. (3) Distribute in tubes.
Use: Cultivation of Bacillus radicicola Sterilization: Method not given.
and other nodule bacteria. Use: To study nitrogen fixation.
Reference: Stutzer, Burri and Maul (1896 Reference: de Rossi (1909, 1910 p. 271).
p. 667).
2345. Simon's Asparagin Legume Extract
2341. Groenewege's Tomato Infusion Gelatin (Klimmer and Kriiger)
Gelatin
Constituents
1. Water 100.0 cc. 2. Gelatin 110.0 g.
2. Tomato 20.0 g. 3. Legume extract 2.0 cc.
3. Gelatin (10.0%) 10.0 g. 4. Asparagin 2.0 g.
(1) Boil 20.0 g. of the fresh material of 6. Malic acid

ripe tomatoes in 100.0 cc. of water. Preparation
(2) Filter. (1) Prepare the legume extract by pour-
(3) Neutralize the filtrate to litmus by ing hot water over finely chopped
the addition of NaoCOs. straw of the legumes, mix well,
macerate for 2 days and then boilan 2349. Barthel's Basal Lupini Extract
hour. Pour off the liquid, filterand Gelatin
evaporate until it contains 10.0%
Constituents
dry material.
1- Water goQ.O cc,
2. Gelatin lOO.O g.
(3) Add 2.0 cc. of (1) to (2).
3. Sucrose 5.0 g.
(4) Neutralize by the addition of NaOH 4. Lupine extract 2.5 cc.
using litmus as an indicator.
Preparation
(5) Add malic acid to give a distinct acid
reaction.
Use: Cultivation of nodule bacteria from
Sterilization: Sterilize the fractional
leguminous plants.
method.
Variants: Klimmer prepared the medium
Use: To study formation of bacteroids by
using citric acid to adjust the reaction
Bacillus radicicola and other legmnes.
instead of malic acid.
Added nutrients: The author used the
References: Klimmer and Kriiger (1914 p.
following materials:
258), Klimmer (1923 p. 228).
(a) 0.1, 0.3 or 0.5%, caffeine ground up
2346. Zipfel's Glucose Legume Infusion with water and gum arable.
Gelatin (b) 0.1, 0.3 or 0.5 g. guanine ground up
with a water and gum arable.
little
Same as medium 2140 but solidified with
(c) 0.1, 0.3 or 0.5 g. guanidine from a
150.0 g. gelatin instead of 30.0 g. of agar.
watery solution. Acidify slightly by
2347. Kaufman's Jequirity Seed Gelatin the addition of tartaric acid.
(d) 0.1, 0.3 or 0.5% pyridine from a
Same as medium
2132 but solidified with
watery solution.
15.0% gelatin instead of 1.5 to 2.0% agar.
(e) 0.1, 0.3 or 0.5% chinoline from an
2348. Stutzer, Burri and Maul's Glucose alcoholic solution.
Alfalfa Gelatin Reference; Barthel (1921 p. 638).
Constituents
1. Water
2350. Nastiukoff's Egg Yolk Gelatin
1000.0 cc.
2. Alfalfa 400.0 g. Same as medium 2144 but solidified with
3. Glucose 20.0 g. 80.0 to 100.0 g. gelatin instead of 15.0 to
4. Gelatin 100.0 g. 20.0 g. agar.
5. Na2C03 1.0 g.
Preparation
2351. Deycke and Voigtlander's Albuminate
(1) Pour 1 of water over 400.0 g.
liter
Gelatin
green alfalfa without roots.
(2) Add 20.0 g. glucose. Same as medium 2034 but solidified with
(3) Steam for 90 minutes. gelatin instead of agar.
(4) Filter.
(5) Add 100.0 g. gelatin to the filtrate
2352. Raskin's Whey Albumin Gelatin
and proceed to prepare a sterile medi- Constituents
um in the usual manner. (Method 1. Whey 1000.0 cc.
not specified.) 2. Albumin 100.0 g.
(6) Do not neutralize but add 1.0 g. 3. Gelatin 60.0 to 100.0 g.
Na2C03 (Reaction slightly acid). Preparation
(7) Tube in 8.0 cc. lots. (1) Stir thoroly with a glass rod, the
Sterilization Not specified
: albumin of fresh eggs in a flat bot-
Use: Cultivation of Bacillus radicicola tomed dish, and add, drop by drop,
and other nodule bacteria. concentrated NaOH until a solid
Reference: Stutzer, Burri and Maul (1896 opaque gelatinous material is
p. 666). obtained.
(2) Cut this solid egg albumin in small 2354. Raskin's Whey Gelatin
pieces and place in distilled water.
Same as medium 2161 but solidified with
Do not allow the alkaline albumin to 12.0% gelatin instead of 1.75% agar.
stand or it will liquefy in several
hours.
2355. Reinsch's Fat-free Milk Gelatin
(3) Shake thoroly and pour off the water.
Continue this washing until the wash Constituents
water is only slightly alkaline. 1. Water 100.0 cc.
(4) Place the coagulated albuminate in a 2. Milk 500.0 cc.
flask of distilled water, plug with 3. Gelatin 20.0 g.
cotton and heat in the steamer for 15 Preparation
to 30 minutes. This dissolves the (1) Dissolve 20.0 g. of gelatin in 100.0
albumin. cc. water.
(5) Filter. This is to be a saturated (2) Place 500.0cc. of fresh cow milk in a
solution of albumin. separatory funnel and add 1.0 g.

(6) Add 10.0% (6) to medium 2274 instead NaOH (2.5 cc. solution of 400.0 g.
of 1.0% peptone. NaOH in a liter).
(7) Neutralize with soda. (3) Shake well and allow to stand at
(8) Filter until clear thru paper in a hot about 18C. for 48 hours.
water funnel. (4) Remove the nearly transparent milk
Sterilization: Not specified. from the bottom of the funnel and
Use: Cultivation of pathogenic bacteria. add it to a second separatory funnel.
Variants (5) Add 250.0 cc. ether and shake well.
(a) The author added 0.5% NaCl. (6) Allow to stand for 48 hours.
(b) Feifer gave the following method of (7) Place the opalescent liquid now in a
preparation large sterile flask, plug with cotton
(1) Heat fresh milk to 60 to 70C. and heat to 50C.
(2) Dissolve 60.0 to 70.0 g. gelatin in (8) Place under the receiver of a water
1000.0 cc. of (1). suction pump for 3 or 4 hours until
(3) Boil until the casein is completely all the ether is evaporated.
coagulated. (9) Add about 0.2% NaOH in order that
(4) Strain thru a linen towel. the acid gelatin does not cause a
(5) Pour in a suitable container so that precipitate of casein.
the fat may raise. (10) Add two to three parts of (9) to one
(6) Remove the fat that comes to the part (1).
top. (11) Distribute in sterile test tubes.

(7) Add 1.0% of a sodium albuminate Sterilization: Not specified.
powder solution and soda to Use: General culture medium. The author
neutralize. reported that the medium was too alka-
(8) Method of sterilization not given. line to support the growth of most
References: Raskin (1887 p. 359), Feifer bacteria.
(1888 p. 568). Reference: Reinsch (1892 p. 32).
2353. Giltner and Ludlum's Amniotic Fluid 2356. Abbott's Milk Gelatin
Gelatin
Constituents
Constituents
1. Milk 1000.0 cc.
1. Amniotic fluid.
2. Gelatin (10.0 to
2. Gelatin.
12.0%) 100.0 to 120.0 g.
Preparation
Preparation: (1) Solidify milk by the addi-
(1) Solidify amniotic fluid with gelatin.
tion of 10.0 to 12.0% gelatin.
Use: General culture medium. Sterilization: Not specified.
Reference: Giltner and Ludlum (1916 pp. Use: General culture medium.
91-92). Reference: Abbott (1921 p. 139).
2357. Heinemann's Whey Gelatin Use: Cultivation of pathogenic organisms.

References: Raskin (1887 ji^43), Abst. by
Constituents
Peifer (1888 p. 568).
1. Whey 1000.0 cc.
2. Gelatin (10.0%) 100.0 g. 2359. Piorkowski's Urine Infusion Gelatin
Preparation: (1) Solidify clarified whey by (Scholz and Krause)
the addition of 10.0% gelatin.
Constituents
1. Urine.
2. Meat water gelatin.
Variants
Preparation:
(a) Bobilioff-Preisser cultivated Oospora
(1) Collect urine under aseptic conditions
on a similar medium. They reported
from a healthy individual. Specific
that the gelatin was liquefied.
weight of the urine to be 1.015 to
(b) Meier made bacterial counts of milk
1.020. It is slightly alkaline or
and whey on a medium prepared as
neutral in reaction.
follows:
(2) Inoculate with a loop of Micrococcus
(1) Dissolve 110.0 g. gelatin and 5.0 g.
urea (liquefac. or non liquefac.)
NaCl in 500.0 cc. water.
from a glycerol agar culture.
(2) Mix equal parts of whey and (1).
Incubate for 24 to 48 hours at 22C.
(3)
(3) Neutralize by the addition of
(4) After this time the reaction is slightly
KOH. Add KOH until turmeric
alkaline.
paper is turned quite weakly
(5) Filter.
brownish red.
(6) Distribute in tubes in 5 to 10.0 cc.
(4) Sterilization not specified
lots.
(c) Klimmer prepared the medium as
(7) Add to each tube of sterile (6) 5.0 to
follows:
10.0% meat water
10.0 cc. of a sterile
(1) Coagulate the casein from milk by
gelatin (exact method of preparation
heating at 40 C. in the presence of
not given) under aseptic conditions.
rennet.
Sterilization: Sterilize (6) in the steamer at
Separate the whey from the casein.
(2)
100C. on 3 successive days for 10 minutes.
(3) Solidify by the addition of gelatin.
Method of sterilization of gelatin not
given.
References Heinemann (1905 p. 128), Bob-
:
Use: Diagnosis of typhoid fever.
ilioff-Preisser (1916 p. 400), Meier (1918
Reference: Scholz and Krause (1900 p. 432).
p. 435), Tanner (1919 p. 55), Klimmer
(1923 p. 172). 2360. Kutscher's Meat Infusion Gelatin
Constituents
2358. Raskin's Casein Whey Gelatin
Meat Infusion
1. 1000.0 cc.
(Peifer)
2. Gelatin (10.0%) 100.0 g.
1. Water 150.0 cc. (1) Prepare meat infusion.
2. Casein (8.0%) 12.0 g. (2) Dissolve 10.0% gelatin in 1 by boiling
3. Whey 350.0 cc. over a free flame.
4. Gelatin 42.0 g. (3) Add soda solution until the reaction
Preparation is only slightly alkaline.
(1) Prepare an 8.0% solution of pure (4) Filter.
fat-free casein. Sterilization: Method of sterilization not
(2) Prepare a filtered mixture of whey given.
with 12.0% gelatin. Use: Isolation and cultivation of Spirillum
(3) Mix 150.0 cc. of (1) with 350.0 cc. of undula majris. Also used by other
(2). investigators in making bacterial counts.
(4) Heat to 60C. for 15 minutes. Variants
(5) Distribute into sterile containers. (a) Fuller and Johnson made bacterial
Sterilization : Not specified. counts in water analysis on a medium
prepared by dissolving 120.0 g. of (3) Add (2) to moistened linen and press
gelatin in 1000.0 cc. of meat infusion the fluid thru.
(contains no peptone or salts), (4) Prepare a 20% watery gelatin solution
(b) Meier used the following preparation containing 1.0% NaCl.
for the bacterial count of milk: (5) Mix equal volumes of (4) and sterile
(1) Boil 500.0 g. of fat and tendon free (3).
beef in 1 liter water. Sterilization: Filter (3) thru a clay filter to
(2) Filter. sterilize. Sterilization of gelatin not

(3) Dissolve 10.0 g. gelatin and 5.0 g. given.
NaCl in the filtrate. Use: Cultivation of colon typhoid bacteria.
(4) Neutralize by the addition of Reference: Kopp (1895 p. 81).
KOH. Add KOH
until turmeric
2364. Wroblewski's Suprarenal Capsule
paper is turned quite weakly
Infusion Gelatin
brownish red.
(5) Sterilization not specified. Constituents
References Kutscher (1895 p. 615), Fuller
: 1. Water 500.0 cc.
and Johnson (1899-1900 p. 85), Meier 2. Suprarenal capsule (ox) 100.0 g.
'
(1918,-p. 435). 3. Gelatin 60.0 g.
4. NaCl 3.0 g.
2361. Graham-Smith's Heart Infusion Preparation
Gelatin
(1) Remove the suprarenal capsule from
Same as medium 1342 but solidified with an ox immediately after death.
gelatin. (2) Chop into small bits and rub to a
pulp.
2362.'^Beijerinck's Asparagin Meat Infusion
To 100.0 g. of add 200.0 cc. of
(3) (2)
Gelatin (Ludwig) water.
Constituents (4) Make (3) alkaline with soda and boil
1. Sea water meat infusion 1000.0 cc. for 2 hours. (Filtration not spe-
2. Glycerol 10.0 g. cified.)
3. Asparagin 5.0 g. (5) Dissolve 60.0 g. of gelatin and 3.0 g.
4. Gelatin 80.0 g. NaCl in 300.0 cc. of water.

Preparation (6) Mix equal quantities of (5) and (4).
(1) Exact method of preparation of meat (7) Sterilize. (Method not given.)
infusion not given. To be prepared (8) Filter 2 or 3 times to clarify.
from sea water. (9) Sterilize again (method not specified).

(2) Dissolve 2, 3 and 4 in (1). Sterilization: Sterilize (9) method not
Sterilization : Method not given. given. Store at 36C. for several days to
Use: Cultivation of photogenic bacteria. test sterility.
{Ph. Pfugeri, Ph. phosphorescence, Ph. Use: Cultivation of Bad. coli, Bad.
indicum and Ph. luminosum). cholerae asiat., anthrax, prodigiosus and
References: Ludwig (1890 p. 617), Beijer- pyocyaneus.
inck (1890 p. 617). Reference: Wroblewski (1896 p. 529).
2363. Kopp's Thyroid Infusion Gelatin 2365. Harde and Hauser's Fish Infusion
Gelatin
Constituents
1. Water 1000.0 cc. Same as medium 1235 but solidified with
2. Thyroid glands, sheep 500.0 g. 15.0% gelatin.
3. Gelatin 100.0 g.
2366. Molisch's Meat Extract Gelatin
4. NaCl 5.0 g.
(1) Free sheep
thyroid glands from fat 1. Water 1000.0 cc.
and chop the glands into small pieces. 2. Meat
extract 0.5 g.
(2) Extract the

glands for 3 hours with 3. Gelatin (4.5%) 45.0 g.
an equal weight of sterile water. Preparation: (1) Dissolve 2 and 3 in 1.

Sterilization: Not specified. 2. Gelatin (12.0%) 120.0 g.

Use: Cultivation of iron bacteria, Clado- 3. Soil infusion (20.0%) 200.0 cc.
ihrix dichotoma Cohn. 4. Glucose (0.1%) 1.0 g.
Variants Zikes used 0.5% meat extract.
: Preparation
References: Molisch (1910 p. 31), Linde (1) Prepare soil infusion by heating soil
(1913 p. 373), Zikes (1915 p. 543), Besson for one hour at one atmosphere pres-
(1920 p. 40). sure, mix with twice its weight of
water, allow to stand over night cold,
2367. HoUborn's Meat Extract Gelatin boil 30 minutes and filter. Dilute to
Constituents contain 0.12 to 0.14% total solids, if
1. Water 1000.0 cc. necessary.
2. Meat extract 16.0 g. (2) Dissolve 12.0% gelatin, 20.0% soil
3. Gelatin 100.0 g. extract and 0.1% glucose in water.
4. K3PO4 1.0 g. (3) Add NaOH to adjust the reaction to
Preparation +0.5 to phenolphthalein.
(1) Dissolve 2, 3 and 4 in 1. Sterilization : Not specified.
(2) Make weakly alkaline by the addition Use: Bacterial count of soils.
of NaOH. Variants: Conn prepared the medium as
Sterilization: Method of sterilization not follows:
given. (1) Heat soil in an autoclave at 20 to 25
Use: Cultivation of organism causing Alo- pounds pressure.
pecia areata (baldness) Trichophyton (2) Mix (1) with an equal volume of
todens. distilled water and allow to stand in
Reference: Hollbom (1895 p. 110). the cold for 12 hours.
2368. Fremlin's Soil Infusion Gelatin
(4) Make up the water lost by evapora-
Constituents tion.
(5) Filter.
2. Gelatin (10.0%) 100.0 g. Dilute with distilled water to tV
(6)
Preparation its natural strength (900.0 cc. distil-
(1) Prepare soil infusion. led water 100.0 cc. extract).
(2) Dissolve 10.0% gelatin in (1). (7) Dissolve 120.0 g. Gold Label Gelatin
Sterilization: Method not given. in 1000.0 cc. of (6).
Use: Cultivation of nitroso bacteria. (8) Clarify by the use of egg white.
Variants: Lohnis prepared the medium as (9) Adjust to 0.5% normal acid using
follows: phenolphthalein as indicator.
(1) Heat 1000.0 g. good rich garden
of
(10) Add 1.0 g. of glucose.
soil with a liter of tap water for 30 Tube.
(11)
minutes in the autoclave under pres- (12) Sterilization not given.
sure of 1 atmosphere or boil with 2
References: Conn (1910 p. 425), (1911-12
liters of water over a free flame.
p. 71), (1914 p. 512), (1916 p. 722), Tanner
(2) Pour off the turbid liquid.
(1919 p. 55).
(3) Mix talc with the liquid.
2370. Lohnis Cyanamide Soil Infusion
Gelatin
necessary.
(6) Solidify by the addition of gelatin, Constituents
(7) Sterilize. (Method not given.) 1. Water 1000.0 cc.
References: Fremlin (1903 p. 373), Lohnis 2. Soil 1000.0 g.
(1913 p. 101). 3. Calcium cyanamide (0.2%). 1.6 g.
4. Asparagin (0.01%) 0.08 g.
2369. Conn's Glucose Soil Infusion Gelatin
5. Glucose (0.01%) 0.08 g.
Constituents 6. K2HPO4 (0.05%) 0.4 g.
1. Water 1000.0 cc. 7. Gelatin (10.0%) 80.0 g.
Preparation 2. Gelatin (12.0%) 120.0 g.

(1) Heat good rich garden soil
1000.0 g. of 3. Soil infusion (20.0%) 200.0 cc.
with a liter of tap water for 30 minutes 4. Glucose (0.1%) 1.0 g.
in the autoclave under pressure of 1
5. Ammonium tartrate (0.1%). 1.0 g.
atmosphere or boil with 2 liters of
Preparation
water over a free flame.
(1) Prepare soil infusion by heating soil
(2) Pour off the turbid liquid.
for one hour at one atmosphere pres-
(3) Mix with the liquid.
talc
sure, mix with twice its weight of
water, allow to stand over night cold,
boil 30 minutes and filter. Dilute,
necessary.
to contain 0.12 to 0.14% total solids,
(6) Dissolve 3, 4, 5, 6 and 7 in (5),
if necessary.
(7) Tube. The reaction must be dis-
tinctly alkaline. (2) Dissolve 12.0% gelatin, 20.0% soil
Sterilization: Not specified. infusion, 0.1% glucose and 0.1%
Use: Cultivation of cyanamide decom- ammonium tartrate in water.
posers. (3) Add NaOH to adjust the reaction to
Reference: Lohnis (1913 p. 108). +0.5 to phenolphthalein.
2371. Conn's Tartrate Soil Infusion Gelatin

Constituents Use: Bacterial count in soil.
1. Water 1000.0 cc. Reference: Conn (1910 p. 425).

GROUP IV. REVERSIBLY SOLID MEDIA OTHER THAN AGAR
OR GELATIN
Ai. Additional constituents, if any, of (3) Heat to boiling again and filter thru
known chemical composition. cotton or a usual filter in the steamer.
Marpman's Chondrin Medium 2372 (4) In order to hasten filtration, a 1 or
Lehmann's Caragheen Medium 2373 2.0% extract may be prepared and
Giltner's Butter Medium 2374 then concentrated to the desired
A2. Containing additional constituents of strength after filtration.
unknown chemical composition. Sterilization: Not specified.
Bi. Onlyone additional constituent of Use: Agar substitute for solidification of
unknown chemical composition. liquid media. "Caragheen" is a

Mortensen's Gum Arabic Media 2375 derivative Chrondrus crispus.
of
Schluter's Basal Isinglass Medium 2376 . 5.0% "caragheen" without any addi-
Marpmann Peptone Plant Tissue tion of nutrients supported the
Medium 2377 growth of parasitic and saprophytic
Matzuschita's Peptone Konbu fungi and air forms.
Medium 2378 Reference: Lehmann (1919 p. 426).
Fucus Medium 2379

Puccinelli's Infusion
Jacobi's Peptone Fucus Medium... 2380 .
2374. Giltner's Butter Medium
Jacobi's Infusion Broth Fucus Constituents:
Medium 2381 1. Butter.
Celli's Bouillon Fucus Medium 2382 Preparation
(1) Melt butter fat at about 100C.
2372. Marpman's Chondrin Medium (2) Allow the casein to settle.
Constituents (3) Decant the clear fat.

(4) Place in about 8.0 cc. sterile test
1. Water.
tubes.
2. Cartilage or ear muscles.
Preparation
Sterilization: Sterilize by the intermittent
ripe pieces of cartilage or ear
method.
(1) Boil
Use: To study fat decomposition. Author
muscle in water under an atmosphere
reported that other kinds of fat might be
pressure in an autoclave, or in small
similarly treated.
amounts in a pressure flask which
stand in 100.0% solution of Na2S04.
(2) Filter thru a filter paper.
Sterilization: Not specified. 2375. Mortensen's Gum Arabic Media
Use: Substitute for gelatin. Mortensen's solidified media 207, 329 and
Reference: Marpman (1891 p. 123). 560 with gum arable (method not given.)
2373. Lehmann's Caragheen Medium 2376. Schluter's Basic Isinglass Medium

2. "Caragheen" 5.0 g. 2. Isinglass

Preparation 3. Peptone (siccum) 1-25 g.
(1) Boil 5.0 g. of "caragheen" in 100.0 4. NaCl 1.25 g.
cc. of distilled water for about 30 to Preparation:

60 minutes. Stir. (1) Prepare a decoction of isinglass in
(2) Cool and make up the loss of water by water by boiling. (Exact method not
adding distilled water. given). Filter.
765
(2) Dissolve 3 and 4 in (1). (3) Boil for one hour in the steamer
(3) Dissolve one of the added nutrients (4) Filter.
in (2) in any desired concentration. Add 3 and
(5) 4 to the filtrate.
(4) Distribute into test tubes, plug and (6) Boil.
slant.
Sterilization: Method not specified. Sterilization: Sterilize in the steamer on
Use: To study growth of bacteria on acid from 2 to 5 successive days for 15 to 30
media. minutes. Incubate for 2 days at 37C. to
Added nutrients: test sterility.
Lactic acid Acetic acid Use: Cultivation of spore forming bacilli,
Tartaric acid HCl Clostridium hutyricum, Bacillus oedma-
Citric acid Alum tis maligni, Bacillus anthracis sympto-
Reference: Schluter (1892 p. 592). matici, Bacillus sporogenes, Bacillus
2377. Marpmann's Peptone Plant Tissue hotulinus. The Konbu is a Laminaria
likeJapanese sea weed.
Medium
Reference: Matzuschita (1902 p. 2S7).
Constituents
1- Water 700.0 cc. 2379. Puccinelli's Infusion Fucus Medium
2. Glycerol 40.0 g.
Constituents
3. Sphaerococcus confervoides . . 30.0 g.
4. Peptone (liquid Koch)
1. Meat infusion 200.0 g.
20.0 g.
2. Fucus crispus 6.0 g.
Preparation
Preparation
(1) Macerate 30 parts Sphaerococcus
(1) Method of preparation of meat
confervoides with 2 parts HCl and 1
infusion not given. Neutralize.
literwater for two hours.
Wash with water until the washings (2) Wash well, 6.0 g. of Fucus crispus in
(2)
water.
no longer turn blue litmus paper red.
(3) Boil (2) for one hour in (1) in a water
(3) Add to the residue of (2) 700 parts
bath or steamer.
water, 40 parts glycerol, 20 parts
peptone (liquid Koch) and 2 parts (4) Filter thru a funnel heated by a
flame or water funnel.
beaten egg-white.
(5) Distribute in tubes and sterilize.
(4) Boil for 20 minutes in a steam
cylinder.
Sterilization : Method not given.
(5) Neutralize.
Reference: Puccinelli (1890 p. 405).
(6) Filter thru a syrup filter, prepared
from a balloon flask with a filter
2380. Jacobi's Peptone Fucus Medium
layer of buck-skin and felt.
Use: General culture medium. Agar 1- Water 1500.0 cc.
substitute. 2. Peptone (Kemmerich's) .... 7.5 g.
Reference: Marpmann (1891 p. 123). 3. Peptone (siccum) 15.0 g.
4. Fucus 37.5 g.
2378. Matzuschita's Peptone Konbu
Preparation
Medium
(1) Dissolve 2, 3 and 4 in 1 by heating
Constituents over a free flame.
1. Water 1000.0 cc. (2) Make up to the original volume by
2. Konbu 100.0 g. addition of water.
3. Peptone, Koch 10.0 g. (3) Press thru a towel, for some of the
4. Glucose 20.0 g. fucus will not dissolve.
Preparation Add Na2C03
(4) or sodium phosphate
(1) Cut 100.0 g. of dry Konbu in small until the reaction is slightly alkaline.
pieces and mix with one liter of water. Pour into a
(5) flask and steam until the
(2) Allow to stay in the water for 24 albuminous material still to be
hours in the cold. removed, is completely separated.
(Usually two hours if sodium phos- (7) Pour into a flask and steam until the
phate is used and longer if Na2C03-) albuminous material still to be
(6) Filter thru cotton, using compressed removed is completely separated.
air to effect a fast filtration. (Usually 2 hours if sodium phosphate
Distribute into small sterile flasks. is used and longer if Na2C03.)
(7)
Finally distribute sterile (7) into (8) Filter thru cotton, using compressed
(8)
sterile test have been
tubes that air to effect a fast filtration.
autoclaved for 2J hours. (The tubes (9) Distribute into smaller sterile flasks.
(10) Finally distribute sterile (9) into
are contained in an enamel container
in the autoclave. The inner tem- sterile tubes that have been
test
perature reaching about 150C.) autoclaved in the autoclave for 2^

Sterilization: Heat in streaming vapor for hours. (The tubes are contained
two hours. in an enamel container in the auto-
Use: General culture medium. clave. The inner temperature reach-
Reference: Jacobi (1888 p. 540). ing about 150C.)

Sterilization: Heat (9) in streaming vapor
2381. Jacobi's Infusion Bjoth Fucus for two hours.
Medium Use: General culture medium.
Constituents Reference: Jacobi (1888 p. 540).
2382. Celli's Bouillon Fucus Medium
2. Peptone (siccum) 15.0 g.
3. NaCl 7.5 g. Constituents:
4. Fucus 37.5 g.
Preparation: 2. Fucus crispus (5.0%) 50.0 g.
(1) Method of preparation of meat Preparation

infusion not given. (1) Prepare a 5.0% solution of
Fucus
Add 2, 3 and 4 to (1). crispus in bouillon. (Author also
(2)
(3) Heat over a free flame until the used water instead of bouillon.)
materials are dissolved. (2) Add for each 10.0 cc. medium, 1.0 cc.
(4) Make up to the original volume by of N/10 NaOH solution or 4 to 5.0 cc.
addition of water. saturated solution of Na2C0j.

(5) Press thru a towel for some of the (3) Distribute in plates.
Fucus will not dissolve. Sterilization: Method not given.
(6) Add Na2C03 or sodium phosphate Use: Isolation and cultivation of amoeba.
until the reaction is slightly alkaline. Reference: Celli (1896 p. 537).
GROUP V. INITIALLY LIQUID MEDIA BECOMING PERMA-
NENTLY SOLID. SOLIDIFYING AGENT ORGANIC
Key to the Subgroups of Group V 2383. Matzuschita's Basal Rice Flour
Ai. Solidifying agent of plant origin. Medium

Subgroup V-A (Med. 2383 to 2397) Constituents
A2. Solidifying agent of animal origin. 1- Water 1000.0 cc
Bi. Solidified by blood or its derivatives. 2. Rice flour
Subgroup V-B (Med. 2398 to 2430) Preparation
B2. Solidified by materials other than blood (1) Boil water containing rice flour
or its derivatives (eggs, etc.) (amount not given).
Subgroup V-C (Med. 2431 to 2465) (2) To the paste formed, either add 2.0%
glucose or 6.0% glycerol or use plain
without any additions.
SUBGROUP V-A Sterilization: Sterilize in the autoclave.
Solidifying Agent of Plant Origin ^'11^.^!')^'! ,

"^.,,.^7:^"^^ ^^d
^ chicken tubercle bacilli. Author reported
Basal or >.
T> J-
1 -R/r 1 .
Complete Media .L
Contain- that best growth of mammalian type on
ing Materials of Plant Origin, glycerol medium. Its colonies were
Initially Liquid but Becom- orange yellow. Chicken type gave whit-
ing Permanently Solid ^^^ S!'^^' grayish black or yellowish
colonies.
Ai. Additional materials, if any, of known Added nutrients: The author added 2.0%
chemical composition. glucose or 6.0% glycerol.
Matzuschita's Basal Rice Flour Variants:
Medium 2383 (a) The author used the basal medium
Hefferan's Starch Medium 2384 without any additions.
Lloyd, Clark and McCrae's Gluten (b) Buchanan cultivated Monascus pur-
Medium 2385 purens from silage on a medium
Baginsky's Basal Starch Medium. 2386 . .
prepared from a thick paste prepared
Uschinsky's Asparaginate Starch from rice flour and tap water.
Medium (Smith) 2387 References Matzuschita (1899 p. 128),
:
Hefferan's Peptone Starch Medium. 2388 Buchanan (1910 p. 103).

Kita's Bran Medium 2389
Kita's Rice Medium 2390 2384. Hefferan's Starch Medium
Kita's Soy Bean Medium 2391 Constituents
As. At least one additional constituent of Water...
1 -1 .,
unknown chemical composition.
.,
1. innn n cc.
i-UUU.u ^
2. Starch (10.0%) 100 g
Bi. Peptone present. Preparation
Hefferan's Peptone Rice Flour d) Cook 10.0% starch with water
Medium 2392 (2) Pour into petri dishes.
Lloyd, Clark and McCrea's Bouillon SterilizationSterilize by the discontinuous
:
Corn Flour Medium 2393 method in the steamer

Lohnis' Bouillon Milk Rice Medium 2394 . Use Cultivation of red pigment producing
:
B2. Peptone not present. organisms, B. prodigiosus, B. ruber bal-

Sanguineti's Yeast Extract Starch ticus, B. kilienis, B. ruber miquel,
B.
Md^^^ 2395 rutilus, B. amyloruber. Author reported
Harras' Tissue Starch Medium 2396 growth. Good growth was obtained
Smith's Milk Rice Medium 2397 when 1.0% peptone was added.
768
Variants: Besson prepared a similar me- 6. K2HPO4 2.5 g.

dium as follows: 7. CuCU 0.01 g.
(1) Mix 10.0 g. of potato starch in 180.0 8. MgS04 0.01 g.
g. of water and add 5.0 g. of pre- 9. Starch (potato)
cipitated chalk. Preparation

(2) Distribute in flasks or petri dishes. (1) Dissolve2, 3, 4, 5, 6, 7and8inl.
(3) Sterilize at 115C. (2) Dry potato starch.
References: Hefferan (1903-04 p. 520), (3) Add 10.0 cc. of (1) to each test tube
m (1920 p. 57). containing 3.0 g. of (2).
(4) Stir the mixture with a glass rod until
2385. Lloyd, Clark and McCrea's Gluten the starch has dissolved.
Medium (5) Place the tubes in a slanted position
at 75 to 85C. for 20 minutes to 3
Constituents:
hours for 5 or 6 successive days. It
1.Water.
2. Gluten flour.
may be necessary to add 2.0 cc. dis-
tilled water to each tube to make up
Preparation
for evaporation after the second
(1) Mix tap water and gluten flour so
heating. The plugs must fit well.
that a thick, but not sticky paste is
Sterilization : See step (5) above.
formed.
Use: To study diastase action. Author
(2) Place in petri dishes.
reported that other starches may be used
instead of potato starch. Sugars and high
115C.
alcohols may be added, 500 mg. per 10.0
Use: Cultivation of Flugge's Mesentericus
cc. of medium. B. coli commune Ps.
group.
hyacinthi, Sarcina aurantiaca and others
Reference: Lloyd, Clark and McCrea
grew very well on this medium if sugars
(1920-21, p. 383).
were added. Smith also used the medium
to cultivate plant parasites.
2386. Baginsky's Basal Starch Medium
Variants
Constituents:
(a) Smith added 1.0 g. portions of starch
1. Water 400.0 cc.
to each 10.0 cc. of medium instead of
2. Starch 20.0 g.
3.0 g. The starch was prepared as
Preparation
follows:
(1) Boil 20.0 g. sugar free amylum with (1) Scrub one-half bushel of large
400.0 cc. water until a starch paste is
smooth potatoes and dig out the
formed.
black spots.
(2) Distribute in 50.0 cc. lots in previ-
(2) Soak fbr 45 minutes in a 1:1000
ously sterilized Erlenmeyer flasks.
mercuric chloride solution.
(3) Add one of the added nutrients. Sterilize the hands by thoroly
(3)
Sterilization: Sterilize in flowing steam.
washing in the mercuric chloride
Use: To study fermentations. solution.
Added nutrients: The author added 1.0 g. Rinse the tubers in sterile water,
(4)
peptone or 10.0 cc. g. a solution of 1.0 g.
pare deeply and run thru a grating
(NH4)2S04 and 1.0 g. or potassium phos-
machine.
phate in 100.0 cc. water to each flask.
(5) Throw into several liters of dis-
Reference: Baginsky (1888, p. 434-462).
tilled water and work the pulp
thoroly with the hands to liberate
2387. Uschinsky's Asparaginate Starch
as much of the starch as possible.
Medium (Smith)
(6) Filter thru several layers of gauze.
Constituents (7) Allow the starch to settle and
1. Water 1000.0 cc. decant off the brownish fluid.
2. Ammonium lactate 5.0 g. (8) Stir up the sediment in a little
3. Sodium asparaginate 2.5 g. water and force the starch thru a
4. Na.SOi 2.5 g. moderately meshed towel with
5. NaCl 2.5 g. gentle rubbing.
MICROORGANISMS
(9) Allow the starch to stand in the bran with 10.0 g. of water in a 400.0
icebox for about a week in distilled Erlenmeyer flask.
cc.
water (3 liters per jar or beaker). Reference: Kita (1913 p. 446), (1914
p. 354).
Siphon off the water twice a day at
first, and afterwards once a day, 2390. Kita's Rice Medium
stirring up the starch thoroughly Constituents
each time fresh water is added. 1. Water.
(10) Drain the starch very free from 2. Rice.
water, scoop up with sterile spoons Preparation
or spatulas and place in sterile (1) Prepare one of the following com-
petri dishes. binations:
(11) Dry in the blood serum oven at (a) 100.0 g. rice + 100.0 g. water.
56C., the cover being raised on (b) 25.0 g. rice + 10.0 cc. water.
corks an inch to let the moisture (c) 10.0 g. rice + 5.0 cc. water.
out. (d) 100.0 g. rice + 50.0 cc. water.
(b) Smith prepared a similar medium as (2) Place in Erlenmeyer flasks.
follows Sterilization: Not specified.
(1) Dissolve 30.0 g. to 40.0 g. glycerol, Use: Cultivation of Japanese molds, As-
5.0 to 7.0 g. NaCl, pergillus Okazaki, Aspergillus
0.1 g. CaCl., candidus,
0.3 to 0.4 g. MgS04, 2.0 to 2.5 g.
Aspergillus albus, Aspergillus tamarii
K2HPO4, 6.0 to 7.0 g. ammonium Pseudorhizopus Aspergillus glaucus and
lactate, to other molds.
3.0 4.0 g. sodium
asparaginate in 1000.0 cc. of
Variants The author steamed 25.0 of rice
water. :
g.
The glycerol may
be omitted. with 15.0 cc. of water in a 400.0 cc. Erlen-
(2) Add 1.0 g. of potato starch to each meyer flask.
10.0 cc. of (1) in a test tube, and Reference: Kita (1913 p. 446), (1914 p. 353).
proceed as above.
2391. Kita's Soy Bean Medium
References: Smith (1899 p. 102), (1905 p.
196), Tanner (1919 p. 61).
Same as medium
2390 but the author used
fat-free soy bean flour instead of rice.
2388. Hefferan's Peptone Starch

Reference: Kita (1914 p. 356), (1913 p. 446).
Medium
Same as medium 26 with the addition of 2392. Hefferan's Peptone Rice Flour
1.0% peptone. Medium
Constituents
2389. Kita's Bran Medium 1- Water 100.0 cc.
Constituents 2. Rice flour 35 q g.
1. Water. 3. Peptone 1 q
g'
2. Wheat bran. Preparation

Preparation (1) Cook 35.0% rice flour and 1.0% pep-
Add 25.0 g. wheat bran tone with water.
(1) to 10.0 g. water
or 10.0% wheat bran to 20.0 g. water. (2) Pour into petri dishes.
SterilizationSterilize by the discontinuous
Details of preparation not given. :
Place in Erlenmeyer flasks. method.

(2)
Use: Cultivation of red pigment producing
bacteria B. prodigiosus, B. ruber bal-
Use: Cultivation of Japanese molds, As- ticus, B. kilienis, B. ruber miquel, B.
pergillus Okazaki, Aspergillus candidus, rutilus, B. amyloruber.
Aspergillus albus, Aspergillus tamarii
Reference: Hefferan (1903-04 p. 520).
Pseudorhizopus, Aspergillus glaucus and
other molds. 2393. Lloyd, Clark and McCrea's Bouillon
Variants Corn Flour Medium
(a) The author used rice bran instead of Constituents
wheat bran. 1. Bouillon 1000.0 cc.
(b) The author steam.ed 10.0 g. of rice 2. Corn flour starch 70.0 g.
Preparation (2) Distribute as a layer in test tubes

(1) Exact composition of broth not given. or flasks.
(2) Neutralize (1) using phenol red as an (3) Sterilize 30 minutes at 100C. on 3
indicator. days.
(3) Add starch to (2). (4) The paste may be distributed in
(4) Shake well. petri dishes and sterilized at 115C.
(5) Tube. for 20 minutes.
(6) Gelatinize by boiling, shaking until References: Lohnis (1913 p. 88), Besson
complete.
gelatinization is (1920 p. 58), Tanner (1919 p. 72), Harvey
Sterilization: Sterilize in the autoclave at (1921-22 p. 95).
20 minutes at 115C.
Use: Cultivation of Fliigge's Mesentericus 2395. Sanguineti's Yeast Extract Starch
group. Author especially recommended Medium
this medium for stab cultures.
Reference: Lloyd, Clark and McCrea Constituents
(1920-21 p. 383). 1. Yeast extract 500.0 cc.
2. Starch 15.0 g.
2394. Lohnis' Bouillon Milk Rice Medium Preparation
(1) Preparation of yeast extract not
Constituents: given.
1. Bouillon 50.0 cc. Distribute yeast extract in 500.0 cc.
(2)
2. Rice meal 100.0 g. lots in 1500.0 cc. flasks.
3. Milk 150.0 cc.
(3) Add 15.0 g. of starch to each flask.
Preparation: (1) Mix 1, 2 and 3.
(4) Boil for 30 minutes on a salt water
Sterilization: Sterilize on each of three bath.
successive days. Sterilization: Autoclave for 30 minutes at
Use : Cultivation of molds. 120 C.
Variants :
Use: To study fermentation of starch by
(a) Besson prepared the medium as Aspergillus orizae, Mucor alternans,
follows: Aimjlomyces Rouxii.
(1) Thoroughly mix 100.0 g. of rice Reference: Sanguineti (1897 p. 267).
powder with 50.0 cc. of peptone
bouillon and 150.0 cc. of milk. 2396. Harrass' Tissue Starch Medium
(2) Distribute in layers 1 to 2 centi-
meters thick in petri dishes. Constituents
Heat at 115C. for 20 minutes. 1. Water.
(3)
Besson noted that this medium had 2. Liver or brain.
been described by Soyka. 3. Starch.
(b) Harvey prepared a medium as Preparation

follows: (1) Pass liver or brain through a meat
Prepare a paste from 10.0 parts rice grinder.

(1)
flour, 7 parts infusion broth (see 779 (2) Prepare a paste with starch in the
(bb)) and 2 parts milk. usual manner. (Method not given.)
(2) Distribute as a layer in test tubes (3) Mix (1) and (2) thoroughly (amounts
or flasks. not specified).
Heat over boiling water to solidify (4) Distribute in petri dishes.
(3)
the paste. Sterilization: Sterilize in streaming steam.
(4) Sterilize 30 minutes at 100C. on Use: Cultivate obligate anaerobes. Bac.

3 days. butyricus, Bac. botulinus, the black leg
(c) Harvey prepared a similar medium maligenens oedema bacillus,
bacillus, the
as follows: and other obligate anaerobes. This me-
(1) Prepare a paste from 2 parts rice dium gave a quite solid medium enabling
flour, 1 part infusion broth (see one to obtain pure cultures.
779 (bb)) and 3 parts milk. Reference: Harrass (1906 p. 2339).
2397. Smith's Milk Rice Medium Ci. Carbohydrates present.

Cobbetts' Glucose Serum Medium.. 2404
Constituents:
Costa's et al. Litmus Glucose
1. Milk 100.0 cc.
Serum 2405
2. Rice 10.0 to 20.0 g.
Rankin's Potassium-Sulphocyanide
Preparation: Place one or two grams of
(1)
Glucose Serum 2406
rice into each tube containing 10.0 cc. of
Dubois' Glycerol Glucose Serum.... 2407
milk.
C2. Carbohydrates not present.
Marx's Glycerol Ragit Serum 2408
Use: Cultivation of chromogenic plant Picker's Glycerol Phosphate Serum. 2409
parasites. Klimmer cultivated skin mi- Picker's Citrate Glycerol Serum 2410
crophytes on a similar medium. Proca, et al. Pyrogallic Acid Serum. 2411
Variants: Klimmer prepared a similar Shmamine's Nucleate Serum 2412
medium as follows: A2. At least one additional constituent of
(1) Thoroughly mix 100.0 g. of rice unknown chemical composition.
powder with 250.0 cc. of skimmed Bi. Carbohydrates added.
cow's milk.
Ci. Additional organic carbon of known
(2) Heat in a porcelain dish, stirring
chemical composition added.
constantly.
Conradi and Troch's Extract Broth
(3) Pour into cylindrical tubes.
Serum Medium 2413
(4) When cool, shove the solidified rice
Hall and Stone's Glycerol Serum
milk mixture from the tubes and cut
Medium 2414
into dices.
Greenspon's Veal Infusion Serum
(5) Place the discs into petri dishes.
(Stitt) 2415
(6) Add 8 drops of milk to each disc.
Bacto Loeffler's Blood Serum
(7) Sterilize for 1 to 1.5 hours.
(Dehydrated) 2416
Klimmer gave Krai the credit for describ-
Loeffler's Glucose Infusion Serum 2417 . .
ing this medium.

Rankin's Cyanide Glucose Serum... 2418
References: Smith (1905 p. 48), Klimmer Ball's Glucose Bouillon Blood Al-
(1923 p. 207). bumin Medium 2419
Crowe's Glucose Agar Blood
SUBGROUP V-B Medium 2420
C2. Additional organic carbon of known
Basal or Complete Media Solidified
chemical composition not supplied.
by Blood or Derivatives, Initially
B2. Carbohydrates not added.
Liquid but Becoming Per- Yardon's Bouillon Serum (De-
manently Solid siccated) 2421
constituents, any, Thalmann's Infusion Broth Serum

Ai. Additional if of
known chemical composition. Medium 2422
Picker's Brain Infusion Serum
Bi. Additional organic constituents not
Medium 2423
employed.
Picker's Potato Juice Serum
Frothingham's Coagulated Blood
2398
Medium 2424
Serum
Gentian Violet Serum. 2399 Vanned' s Nutrose Serum Medium. 2425 .
Proco, et al.,
Lorrain Smith's Alkali Serum Pergola's Egg Yolk Serum Medium. 2426
(Harvey) 2400 Greenspon's Glucose Citrate Serum
Duval's Trypsinized Serum 2401 Medium 2427
Carrel and Burrows' Plasma Me- Duval's Egg Serum Medium 2428
dium 2402 Eberling's Serum Tissue Plasma
Ball's Coagulated Blood Medium. . . 2403 Medium 2429
B2. Additional organic constituents Wolbach and Schesinger's Tissue

employed. Plasma Medium 2430
2398. Frothingham's Coagulated Blood (5) Coagulate by heating between 90

Serum and 100C.
(d) Gliicksmann cultivated diphtheria
Constituents: 1. Serum
bacilli on a medium prepared as
Preparation
follows:
(1) Collect blood in large jars which can
(1) Collect beef blood under aseptic
be closed tightly. Close the jars.
conditions from a slaughter house
(2) When the blood has begun to clot,
into a sterile four liter cylinder.
I pass a sterile glass rod around the
(2) Allow to stand in the ice box for 4
clot between its surface and the wall
or 5 days at 1 to 8C.
of the jar. This permits the clot to
(3) Pipette the serum under aseptic
sink.
conditions into sterile test tubes in
(3) Close the jars and place in an ice box
4.0 cc. lots.
for 24 to 48 hours.
(4) Solidify by heating in a slanted
(4) Draw off the serum with a sterile
position at a temperature of 70 to
pipette and distribute into sterile
72C. for about one hour.
test tubes.
(5) Incubate 2 or 3 days to test
(5) Place in a blood serum sterilizer and
sterility.
heat to 68 or 70C. for one hour on
(e) Smith cultivated tubercle bacilli on
five successive days.
the following medium:
(6) Place the tubes in the apparatus for
(1) Bleed a dog under chloroform and
solidifying blood serum and heat to
draw the blood from a femoral
75 or 80C. until the serum is co-
artery under aseptic conditions
agulated.
thru sterile tubes directly, or into
Sterilization: Sterilization given with sterile flasks.
method of preparation. Draw the serum from the clots with
(2)
Use: Culture medium for parsitic forms. sterile pipettes and either distrib-
Variants: The following methods of prep- ute at once into sterile test tubes
aration have been given by the following or else store with 0.25 to 0.3%
investigators: chloroform.
(a) Frothingham heated the serum col- (3) Slant the tubes and heat to 75 to
lected as indicated above, at 90 to 76C. to coagulate, requiring about
95C. for an hour or more, and then three hours. No other steriliza-
steamed on each of 2 successive days tion or heating is required. If
for 15 to 30 minutes. chloroform has been added it is
(b) Michel cultivated diphtheria bacilli necessary to place the tubes for an
on the following medium: hour or longer in a water bath at
(1) Obtain horse or beef serum under 55-60C. or under the receiver of an
sterile conditions. air-pump to drive off the antiseptic.
(2) Distribute into sterile test tubes. (4) Keep in cold closed space and in an
(3) Solidify in a Koch's apparatus at a inclined position before in-
temperature of 85C. oculation.
(c) Wurtz. (5) Use a ground glass stopper instead
(1) Collect blood from the slaughter of the ordinary cotton plug to
house under aseptic conditions in close the tubes.
petri dishes, Erlenmeyer flasks or (f) Thoinot and Masselin.
jars. (1) Obtain blood under as nearly
(2) When the blood has clotted pipette aseptic conditions as possible in
off the serum by means of a sterile large glass jars.
pipette. (2) Store in a cool place for 24 to 48
(3) Heat for 3 hours at 58C. on each of hours to allow the blood to clot.
three successive days to sterilize (3) Remove the serum from the clot
(Tyndall method). by means of a sterile pipette.
(4) Distribute as desired. (4) Distribute into sterile tubes.
(5) Solidify in a Koch inspissator at 68 (k) Ball.

to 70C.
(1) Collect blood under aseptic con-
(g) Heinemann. ditions at the slaughter house,
if
Coagulate dog's blood serum in
(1)
possible, in large tall sterile flasks.
slanted tubes in the Koch inspis- Place on ice for 48 hours.
(2)
sator for three hours at Ib^C. Draw
(3) out the serum by means of
(h) Schereschewsky cultivated Syphilis
sterile pipettes into test tubes.
spirochaete on the following medium Do not shake the jar.
(1) Draw off normal horse serum, by Place the tubes in an inspissator
(4)
means of a syphon under aseptic in a slanted position.
conditions into thin walled sterile Heat to 65 to 68 until coagulation
(5)
tubes about 10 to 15 cm. long. occurs.
Allow the serum to run down the (6) Remove the tubes and sterilize by
wall of the tube so as not to mix the the fractional method.
serum with air. Keep for 3 or 4
(7) days in the incubator
(2) Seal the tubes with cotton or cork at 58C. and discard those tubes
and gum. showing growth. These tubes are
(3) Heat in a water bath at 57-58 C. for transparent and straw colored.
one hour. (8) The serum may be prepared by
(4) Raise the temperature slowly to coagulating at a temperature short
70 C. of the boiling point temperature
(5) When the serum has solidified, not specified. Sterilize by exposing
incubate at 37C. over night to the tubes to a temperature of about
test sterility.
90C. on each of three successive
(i) Sowade used the following medium to days for 5 minutes. These tubes
cultivate syphilis spirochaete: are opaque and white.
(1) Collect horse blood in long measur- (1) Harvey.
ing cylinders. (1) Collect ox or sheep blood at the
(2) Place in the ice box until the serum slaughter house in a sterile blood
separates. jar.
(3) Distribute into test tubes to a (2) Allow the blood to coagulate.
depth of 12.0 cm. (3) Detach the clot.
(4) Heat in a water bath on 3 successive (4) Place in the ice chest.
days for two hours at 58C. (5) Pipette off the serum with a sterile
(5) On the third day slowly raise the pipette.
temperature until the serum (6) Keep the serum in the ice chest till
reaches a gelatinous consistency. required.
Raise the temperature slowly or an (7)Distribute a portion of the serum
intensive turbidity will take place. in quantities of 5.0 cc. in test tubes.
(6) Medium is honey yellow, trans- (8) Sterilize 2 days 30 minutes at 60 C.
parent and reacts slightly alkaline. (9) Coagulate in a slanting position in

(j) Abbott.
an inspissator, or over steam at
(1) Obtain blood serum from the temperatures varying from 65 to
slaughter house or 90C. according to the degree of
anti-toxin
manufacturers. transparency required.
(m) Harvey.
(2) Tube in sterile tubes and plug.
(1) Collect ox or sheep blood at the
(3) Slant.
slaughter house in a sterile blood
(4) Place in a dry air sterilizer and
jar or in a bucket.
slowly raise the heat to 80-90 C.
(2) Allow the blood to coagulate before
Keep at this temperature until the
removing it.
serum has solidified.
(3) Separate the clot from the sides of
(5) Steam on three successive days for the containing vessel with a sterile
20 minutes at 100 C. glass rod.
(4) Place in a cool place for 24 hours. (3) Close the test tube with a rubber
(5) Transfer the separated serum with a cork.
sterile pipette in quantities of 5.0 (4) Heat one hour at 60C. in a water
cc. to sterile test tubes. bath.
(6) Sterilize 30 minutes at 58C. on (5) Heat, 24 hours later one hour at
each of 8 successive days or 20 70C. in a water bath.
minutes at 100C. three days. (6) Heat, 24 hours later at 70C. until
(7) Test sterility by incubation for 48 the medium becomes syrupy.
hours. (7) Keep in the ice chest till required
(n) Harvey. for use.
(1) Prepare: chloroform 2; blood serum (r) Pitfield.

100. (1) Obtain dog, sheep or cow blood
(2) Keep in a well stoppered bottle in under aseptic conditions.
the dark. (2) Pipette off the serum with a sterile
(3) Distribute in sterile test tubes with pipette.
sterile precautions. (3) Distribute into sterile test tubes.
(4) Coagulate in a slanting position in (4) Coagulate by heat.
an inspissator or over steam at (s) Jones cultivated an organism re-
temperatures varying from 65 to sembling Bacillus actinoides from
90C. according to the degree of pneumonic rat lungs on a medium
transparency required. prepared as follows:
(o) Harvey. (1) Coagulate horse serum in tubes.
(1) Coagulate serum in a slanting (2) Remove a small portion of the lung
position in an inspissator or over of an infected rat under sterile
steam at temperatures varying precautions.
from 65 to 90C. according to the (3) Add 0.5 cc. of sterile calf serum
degree of transparency required. water to the water of condensation.
(2) Add 0.85 sterile salt solution to each (4) Push the piece of tissue down the
test tube, slope to cover the tube and into the liquid at the
medium. bottom.
(3) Sterilize 60 minutes at 115C. (5) Seal the tubes with sealing wax.
(4) Pour off the salt solution at the time (t) Vardon prepared a desiccated
of use. medium as follows: He reported that
Note: The addition of salt solu- the dried serum when dissolved in
tion allows for satisfactory sterili- water and brought to its original
zation and keeps the medium moist volume can be used for all purposes
till required, where serum is used in media.
(p) Harvey cultivated spirochaetes on (1) Measure out a quantity of serum.
the following medium: (2) Pour into shallow trays.
(1) Fill the serum into tall test tubes. (3) Dry at a temperature not exceeding
(2) Heat in the upright position at 50C.
65C. Note: Coagulation of the serum
(3) Remove as soon as the serum begins should not occur in the process
to set. of drying. The drying process may
Note: The heat retained in the be continued overnight by placing
tube will complete the coagulation. the trays in a large incubator.
A soft, almost transparent coagu- To use powder.
lum is formed. (4) Dissolve by shaking the requisite
(q) Harvey prepared a medium as follows amount, calculated from amount
for the cultivation of spirochaetes: of dry material obtained from the
(1) Prepare: horse serum 3; distilled liquid, of (3) in cold water.
water 1. (5) Distribute when dissolved in
(2) Distribute in quantity to nearly amounts of about 7.0 cc. into test
fill test tubes. tubes.
MICROORGANISMS
(6) Inspissate the medium in a sloping

2400. Lorrain Smith's Alkali
position at a temperature of 75C. Serum
(Harvey)
for 60 minutes on three successive
days. Constituents
(7) Incubate tubes 24 hours and reject

1. 10.0%NaOH 1.5,,
any which are not sterile. 2. Ox serum 100.0 cc.
Preparation
(u) Park, Williams and Krumwiede cul-
(1) Add 10.0% NaOH to 1000.0
15.0 cc. of
tivated spirochaetes on a medium ox serum.
cc. of
prepared as follows:
(2) Coagulate in an inspissator
(1) Tube serum in tall tubes. or over
steam at 65 to 90C. according to
(2) Warm the tubes to 65C. the
degree of transparency required.
(3) Place a few of the tubes at a time Use: General culture medium.
inwater at 75C. Reference: Harvey (1921-22 p.
78).
(4) Tilt these tubes every few seconds
and remove as soon as they start to 2401. Duval's Trypsinized Serum
set. The heat in the tube com-
pletes the coagulation. Constituents
They reported that the serum may be 1. Serum (human).

Preparation
diluted with saline or broth and
treated in the same way. (1) Pour human blood serum into sterile
petri dishes
and inspissate for three
References Frothingham (1895 pp. 55, 57),
:
hours at 70C. (Egg albumin
Marmier (1895 p. 569), Michel (1896 or
nutrient agar with or without
p. 261), Wurtz (1897 p. 33), Glucksmann 1.0%
tryptophane may be treated in the
(1897 p. 436), Smith (1898 p. 456), Thoinot
same manner.)
and Masselin (1902 p. 42), Heinemann
(2) Cut an excised leprous nodule into
(1905 p. 131), Schereschewsky (1912 p. thin slices, two to four millimeters
1335), Sowade (1912 p. 797), Abbott in
breadth and 0.5 to 1.0 millimeters in
(1921 p. 135), Ball (1919 p. 78), Harvey
thickness and distribute over
(1921-22 pp. 78-81), Pitfield (1922 the
p. 119), surface of the coagulated serum
Jones (1922 p. 363), Vardon (1923-24
p! (albumin or agar).
431), Park, Williams and Krumwiede
(3) The medium is now seeded. Bathe
(1924 p. 125).
the surface with a 1.0% sterile
trypsin
solution, taking care not to
2399. Proco, et al., Gentian Violet Serum submerge
the piece of leprous tissue.
Add
Constituents: sufficient trypsin solution to com-
1. Water. pletely cover the medium.
2. Serum. (4) Incubate in a moist chamber at 37C.
3. Gentian violet. for a week or ten days. Remove the
4. NaCl. plates from time to time and add
more
Preparation trypsin as is necessary.
(1) Dissolve 0.1 centigrams of gentian Sterilization : Method not given.
violet in 300.0 cc. of physiological Use: Isolation and cultivation of B.
salt leprae.
solution. The author reported that the colonies
at
first were grayish white,
(2) Mix equal parts of tyndallized serum but after several
days they assumed a distinct
and (1). orange
yellow tint. Transfers may
(3) Coagulate at 80 C. be made
from this medium to slants or
Sterilization: Sterilization other
given in the plates.
preparation.
Use: Cultivation of Syphilus spirochaete
References: Proco, Danila and Stroe 2402. Carrel and Burrows'
(1912 Plasma Medium
p. 896), Harvey (1921-22 p. 81).
Constituents: 1. Blood plasma
MICROORGANISMS 777
2404. Cobbett's Glucose Serum Medium

Preparation:
(1) Obtain the blood from the animal Constituents
whose tissue are to be cultivated or 1. Serum 1000.0 cc.
another animal of the same species, 2. Glucose 20.0 g.

under aseptic conditions. Collect Preparation:
to
the blood in sterile tubes cooled (1) Collect beef blood
from a slaughter
0C., coated with sterile paraffin. house and store the blood in a cool
(2) Cork the tubes immediately and place to allow the serum to separate.
place in large tubes filled with ice
and serum add 2.0 g. of
(2) To 100.0 cc. of
centrifuge for 5 minutes, and place glucose and 1.75 cc. of 10% NaOH
box at 0C.
in a small ice solution.
(3) Remove the supernatant plasma with (3) Distribute into test tubes (or Petri
It is
sterile paraffin coated pipettes. dishes).
generally used immediately. (4) Slant the tubes and place in the auto-
to be cultivated In order to avoid air bubbles
(4) Take the tissue clave.
directly from the living animal
or
in the medium close the stop cock of
immediately after death. Cut the the autoclave before all the air is
tissue in small pieces and
transfer
removed. This gives a high pressure.
either to a cover glass or larger con- Sterilization: Sterilize for 20 minutes at
tainer. The cutting must be done 120C.
quickly for desiccation kills the
Use: Diagnosis of diphtheria. Author re-
ported that diptheria colonies were

tissue.
(5) Cover the tissue with the plasma. After a few days
flat, gray or colorless.
Sterilization: See preparation. surface became irregular. Different
sera
Author re-
Use: Cultivation of tissue.
may require different amounts of alkali.
ported that citrated blood might be used. Old laboratory strains grew less luxuri-
Sufficient blood was added to a 1.0% antly than did freshly isolated strains.
sodium oxalate solution making the Variants: The author prepared a similar
pre-
solution 0.1%. At the time of use medium as follows:
cipitate the sodium oxalate
quantitively
(1) To 100.0 cc. of
horse serum add 2.0 g.
from the plasma by the addition of CaCU. dextrose, and 1.25 to 1.3 cc. of 10.0%
This does not give as good results as does NaOH solution.
pure plasma. Distribute into tubes or Petri dishes.
(2)
Variants: Carrel prepared the medium as
Heat to 90C. on two successive days
(3)
follows: to sterilize.
(1) Dilute plasma with about 2/5 of its Reference: Cobbett (1898 p. 395), (1898
volume of dist. water.
p. 362).
(2) In order to accelerate the coagulation
embryonal extracts or Serum
of the plasma, 2405. Costa's et al. Litmus Glucose
serum may be added.
of tissue Constituents
(3) Pour (1) over small pieces 100-0 cc.
to be cultivated. 1. Serum (horse)
2. Glucose
Reference: Carrel and Burrows (1911 p.
3. Litmus
391), Carrel (1912 p. 393).
4. H2SO4
2403. Ball's Coagulated Blood Medium Preparation
(1) Prepare a
solution of 30 parts glucose
Constituents: 1. Blood. to 100 of water.
Preparation: (1) Coagulate blood (not the Mix 10.0 cc. of sterile (1), 30 drops of a
(2)
serum only) in test tubes. sterileconcentrated solution of tinc-
sterile one
Sterilization: Not specified. ture of litmus, 3.0 cc. of a
to one hundred H2SO4 and 100.0 cc.
of sterile horse serum.
Reference: Ball (1919 p. 80).
MICROORGANISMS
(3) Coagulate in the autoclave for 75 Sterilization: Tube and sterilize the m
minutes at 75 to 80 or in a hot air steamer for an hour on three successive
oven at the same temperature and days at a temperature of from 180
time. to
190F.
Sterilization: Method of sterilization of Use: Show presence of B. diphtheriae
glucose, litmus or in
H2SO4 solutions not throats. Author reported that
B. diph-
given.
theriae always gave a characteristic
Use: Diagnosis of diphtheria. red
Author re- colony. He recommended that
ported that diphtheria colonies were red the me-
dium be used in the discharging
Variants: Harvey prepared of
the litmus patients.
tincture as follows:
Reference: Rankin (1912 p. 63).
(1) Grind up litmus in a mortar.
(2) Add 5 volumes 90 per cent alcohol. 2407. Dubois' Glycerol Glucose
Serum
(3) Boil on a water bath.
Constituents:
(4) Decant the supernatant fluid. 1- Serum 100.0 cc.
(5) Add 6 parts distilled water to the 2. Glucose 7.0 g
residue. 3.Glycerol 2.O g^
(6) Boil.
Preparation:
(7) Allow to cool. Tube
(1) non-sterilized serum containing
(8) Divide into two portions.
7 parts glucose and 2 parts glycerol
(9) Render one portion slightly red with per 100 in 2.0 cc. quantities.
dilute sulphuric acid.
(2) Solidification not specified.
(10) Add to this reddened portion the Sterilization Not specified.
:
other untreated portion littleby Use : Cultivation of Koch's bacilli.

little until the mixture becomes Reference: Dubois (de Nancy)
blue again.
(1896 p
204).
(11) Filter thru paper when cool.
(12) Distribute into test tubes. 2408. Marx's Glycerol Ragit Serum
(13) Sterilize at 110C.
Constituents
(14) Keep for use.
1- Water 1000.0 cc.
References: Costa, Troisier and Dauvergne
2. "Ragit" serum 130. g.
(1917 p. 80), Harvey (1921-22 p. 81). 3. Glycerol 50.0 cc.
2406. Rankin's Preparation
Potassium-Sulphocyanide
Glucose Serum (1) Pour 13.3 g. "Ragit" serum in a large
mortar.
Constituents
(2) Add 100.0 cc. water in small lots.
1- Water 100.0 cc. (3) When the serum has become en-
2. Sheep serum 300.0 cc. tangled by mixing with a pestil, add a
3. Glucose 40e few drops of water and rub the water
4. 1.0% aqueous neutral red
in the powder.
solution 4 cc (4) As soon as the mixture has become
5. 50.0% potassium sulphocy-
thick add more water.
Always rub
anide solution 8.0 cc. in a circle as mixing milk and flour
if
Preparation
in cooking. Continue this until all
(1) Obtain fresh serum from sheep blood.
the 100.0 g. has been added.
Serum should contain no red blood
(5) Add 5.0 cc. of glycerol to the mixture
cells.
of 13.3 g. "Ragit" serum and water.
(6) Distribute into tubes or plates.
(1). (Author used bouillon or equal
(7) Solidify by placing the tubes or
parts of water and bouillon and water,
plates in flowing steam.
but water gave equally good results.)
Sterilization: Place the medium in
(3) Add 1.0% glucose, 4.0 cc. of a 1.0% the
autoclave for 15 minutes (pressure not
neutral red solution and 8.0 cc. of
a specified).
50.0% solution of potassium-sulpho- Use: Cultivation of diphtheria bacilli.
cyanide solution.
Author reported that growth was nearly
OF MICROORGANISMS 779
CULTURE MEDIA FOR CULTIVATION
80-0 cc
Loeffler's 2. Blood serum
as good on this medium as on 0.25 g
3. Magnesium citrate
serum. 0.3 g
4. (NH4)2SO.
Reference: Marx (1913 p. 250).
Potassium phosphate 0.5 g
5
2409. Picker's Glycerol Phosphate Serum 6. MgS04 0-25 g
7. Glycerol 2.0 g
Constituents
1000.0 cc Preparation
1. Blood serum 6 and 7 in 1,
ÔS- (1) Dissolve 3, 4, 5,
2. KHoPO^ 80.0 cc. of serum.
20.0 g. (2) Add
3. Glycerol
(3) Solidify (method not given).
Preparation: Method not given.
Sterilization:
(1) Add 0.5% KH2PO4 to blood serum.
Add 2.0% glycerol to (1).
(2)
Variants: The author omitted the MgS04,
Solidify (method not given).
used 0.2 g. (NH4)2S04 instead of 0.3 g.
(3)
of tubercle bacilli. and added 0.6 g. mannitol.
Use: Cultivation
bacilli Reference: Ficker (1900 p. 510).
Author reported that the tubercle
grew without the glycerol. In concentra- 2411. Proca, et al., Pyrogallic Acid Serum
checked
tion higher than 2.0% glycerol
Constituents
the growth.
1. Distilled water.
Variants
2. Serum.
(a) Thoinot and Masselin prepared a
3. Pyrogallic acid.
similar medium
as follows
Preparation
(1) Autoclave
glycerol at 115C. and
pyrogallic acid
parts by weight of (1) to (1) Dissolve 1.0 g.
(2) Add 6 to 8 NaOH in 100.0 cc. distilled
2.0 g.
100 parts of sterile serum.
water.
(3) Mix well.
serum (beef or horse) add
(2) To 10.0 cc.
(4) Tube.
l.Occ. of (1).
(5) Solidify at a
temperature between
80.
(3) Coagulate at
75 to 86C. serum in the
similar mediiim by Sterilization: Sterilize the
(b) Harvey prepared a
100 steamer using Tyndall's method.
adding 7 parts sterile glycerol to spirochetes.
and Use: Cultivation of syphilus
parts sterile ox serum, tubing
Variants: Harvey specified that the pyro-
coagulating in the steamer or inspis- stored several
gallic acid solution be
sator at 65 to 90C.
weeks before and inoculated the
use,
(c) Harvey gave the
following method of
solidified medium by passing
a pipette
preparation: the
glycerol and 95 parts ox containing a culture material between
(1) Mix 5 parts
medium and the wall of the tubes, seal
serum. incubate.
the tubes hermetically and then
(2) Heat 30 minutes at 56C. in a water (1912
References Proca, Danila and Stroe
bath on each of two successive
:
p. 895), Harvey (1921-22 p.

80).
days.
Coagulate in the inspissator or
(3) 2412. Shmamine's Nucleate Serum
steamer at 65 to 90C.
before use by in- Constituents
(4) Test sterility 200.0 cc.
1. Serum
cubating 48 hours.
Thoinot 2 Sodium salt of nucleic
References: Ficker (1900 p. 510), 1.0tol.5g.
and acid
and Masselin (1902 p. 48), Roux
Preparation
Rochaix (1911 p. 128), Besson (1920 p.
(1) Obtain horse blood under aseptic
Dopter and Sacquepee (1921 p. 139),
53),
conditions if possible.
Harvey (1921-22 pp. 78, 79).
(2) Draw off the clear serum by means of
2410. Picker's Citrate Glycerol
Serum sterile pipettes.
Allow to stand in the ice box for two
(3)
Constituents
20.0 cc. or three days.
1. Distilled water.
MICROORGANISMS
(4) This serum should be very clear, 2. Meat extract . ; la .
-^^*-"-^^paê 3. Peptone (Witte).
Zt
lZ77r: .:::: ;:::;
(5) Dissolve 10 to 1.5 g. sodium salt of 5.'

Caliumacid malate 60 f
nucleic acid (Bohrenger) in 200.0
of sterile (4) by shaking.
cc. 6. Glucose .J'
'"'^^
7. ge.um (beef)
(6) Pass carbonic acid from a Kipp ap-
8. Potassium tellurite (1 0%
paratus thru (5) for 2 or 3 minutes.
solution)
This tends to clear the medium. .
pr-
(7) Distribute into tall test tubes about (T) D 'sîU 004 ' .,- ,
f full and heat in a

three successive days for one hour
water bath on
m
rZ
2
Ar\r} I'no/ 1
'
each day at 60C. ^-^^^ g^^^-

f ^^
^f,
(8) After removal from the water bath ^^ ^^'^ ^^^ * ^ ^^"^' ^'''^ '*""^^
t .
place into ice cold water. ... ,
t^"" inn'o""' ,
(9) At the time of the fourth sterilization ^^ ?" '^^^*"'' ^^^ ^'^
l" '''t
solidify
the serum by gradually "' "^^^ Potassium tellurite
g'j^.f
^ ^^"
raising the temperature to 70
C fr,\ d^
You can obtain three different degrees ^^ '"^
^^*" "^'^^^^ ^^^^ bibulous
of hardness of the serum. By heating Paper mside covers.
at a low temperature one obtains ^'^^ Solidify at 85-90C.
a
soft, clear and transparent medium. Sterilization: Sterilize
by repeated heating
By heating at a high temperature one (temperature not given),
obtains a hard solid nearly opaque ^ê: Isolation of diphtheria bacilli,
medium. Then there is a medium Author reported that diphtheria colonies
between the two extremes. were coal black due to the reduction
of
Sterilization: Sterilize (5) by the fractional tellurium.
method in the steamer. Further sterili- Reference: Conradi and Troch (1912
zation
pp
is obtained in step (7) under the 1652-1653).
preparation.
Use: Cultivation of spirochetes. 2414. Hall and Stone's Glycerol Serum
Variants Futaki, et al., studied the motility
:
Medium
of Spirochaetamorsus niuris (cause of rat
bite fever) on a medium Constituents:
prepared as
follows: 1- Water 1000.0 cc.
(1) Shake 0.5 to 0.75 g. of sodium nucleate ^- ^^Ptone (Witte) 10.0 g.
with 100.0 cc. horse serum until the ^- ^^^^ 5.0 g.
nucleate has dissolved. * Glucose lO.o g.
(2) Pass CO2 thru the medium for 2 or 3 ^- ^^y^^rol 5O.0 g.
minutes until the medium is trans- ^' ^^"' blood 3000.0 cc.
parent. Preparation:
(3) Heat on three successive days for ^^^ Dissolve 2, 3, 4 and 5 in 1.
about an hour at 60C. (2) Mix one part of (1) with 3 parts of 6.
(4) On
the 4th day heat to 65C. for 30 ^^^ Filter thru Berkefeld filter,
minutes, when a fluid and coagulated ^^^ ûbe.
portion is formed. (5) Slant.
References: Shmamine (1912 p. 313), Futaki, Sterilization: Sterilize in autoclave at 10
Takaki, Taniguchi and Osumi (1917 pounds pressure for 30 minutes.
p.
^'- Use: Cultivation of diphtheroid bacillus
2413. Conradi Pêisz-Nocard from equine and bovine
and Troch's Extract Broth f
abscesses. Author reported that several
Serum Medium
strains of B. diphtheriae grow on
^ ^. will
Constituents: this medium.
^' ^^^^^'^

1000-0 cc. Reference: Hall and Stone (1916 p. 196).
MICEOOEGANISMS 781
CCLTOEE MEDIA FOB CULTIVATION OF
following coagulation is rec-
Infusion Serum minutes
2415 Greenspon's Veal
(<zr,u\ ommeneded.
^"^ ^ Use: Cultivation of diphtheria
bacilli.
Constituents: Reference: Digestive Ferments Co. (1925

cc.
1 Glucose veal infusion broth. 24.0 .
^ ^^^
"
1.0 cc.
2. Sodium citrate (50.0% soln.) .
Blood
2417. Loeffler's Glucose Infusion
3 Serum, pig, sheep or
human.. 75.0 cc.
^ .. Serum
Preparation
(1) Add 1.0 cc. of
a 50.0% sodium citrate Constituents:
solution to 75.0 cc. of clear pig,
sheep 300.0 cc.
^ g^^^^ ^^^^.g^)
human serum and sufficient glucose 100.0 cc.
or ^ Meat infusion
veal infusion broth, or glucose
meat 1-0 S-
^ Peptone
extract broth to bring the
volume to 0.5 g.
^ ^^^^
100.0 cc. 5. Dextrose 10 g.
(2) Adjust the reaction to pH = 6.4 by

preparation:
citric acid
the addition of 3.0% ^^^ Prepare meat infusion in the usual
blue
solution using di-brom-thymol manner (exact method not given).
as an indicator. Dissolve 1.0 g. peptone, 0.5 g. NaCl
(2)
cc.
(3) Tube. _
and 1.0 g. glucose in 100.0 (1)
(4) Coagulate in an inspissator. ^

making a bouillon.
Sterilization: Sterilize by the fractional ^^^ Obtain horse serum under
sterile
method on 3 successive days. conditions.

diphtheria bacilli. sterile
Use: Cultivation of
^^^ ^^^.^ 3 p^^.^^ (3) ^^^^ ^ne part
citric acid
The author reported that the ^2).
inhibited the growth of organisms
from Koch's apparatus, heating
^^^ Solidify in
the mouth. to 90C.
Use Cultivation of diphtheria bacilli.

:
2416. Bacto Loeffler's Blood Serum have sug-

Variants The following authors
:
of prep-
(Dehydrated) gested the following methods
^ i-i *. aration.
Constituents:
hel (1896)
1. Distilled water infusion in the usual
300.0 cc P^^^ m;at
^''\l)'prepare
2. Serum, beef blood. _
(
^^^^^^ ^^^ ^.^^^^^
Glucose infusion broth 100.0 cc.
3.
^.^^^^^^ ^^ ^ ^^^^^^^^ ^^ ^
Preparation: glucose in 100.0
(^^) Dissolve 80 g. of
.
.
Bacto t
Loeffler's m ' NaCl and 10
JNa^^^ anu i.u g.
g. g,
SI!
re\ttl?in'r:,anteci position (4, ^t^ts <3, wit. one pa.
in Koch's apparatus,
(5) Solidify
<4, Sot rilTaTand t.e door of the
autoclave before turning on the
,,, , ^-ff/X
(WO^
steam. Run the pressure to 15 O) Smr^^h ^
Preparation
pounds as quickly as possible and (1)
glucose to (I,,
llZt (2, i'dl"l.O of
Stprilization-
::"sur'e^r20^.::;les"o:to 5
rrrsreforeo minute.
over 15 pounds pressure.
When coagulation
Bo
is
not use
com-
C3, -- -fe
j-^
conditions
^ u^dr alt:
^.
^
Coagulate in the inspissator.
;;:t open lowest'part and replace
le (4)
maintaining (0 f-^t 903^

the entrapped air with steam, ^^^_
constant pressure during the change. ^^^
% !fabattoir.
.hlttoir
for 20 tamer
, at an
Sterilization at 15 pounds pressure
782 CULTURE MEDIA FOR CULTIVATION OP
MICROORGANISMS
(2) Allow the blood to stand un-
(3) Sterilize (2) by heating at 100
disturbed for 15 to 30 minutes or C.
either in a water bath
until the clot has firmly or in a
attached steamer for 2 minutes on
itself to the sides of the three
vessel. successive days.
(3) Cover the vessels and remove to
the laboratory. (4) To 90 parts (1) add 10 parts
(3).
This makes the sugar concentra-
(4) Separate the clot from the sides of
tion 1.0%.
the vessel by means of a sterile
knife, or a glass rod. (5) Pour (4) into sterile petri dishes
Place the
vessel in the ice chest. m thin layers, and solidify.
(5) Allow to stand for 48 hours, and Roth reported that all diphtheria
pipette or siphon off the serum. bacilli fermented glucose and levu-
(6) Place in tall cylinders
lose. Some pseudo diphtheria bacilli
(graduates)
and allow to stand 24 hours. fermented glucose and levulose.
(7) Separate the straw colored serum (f) Abel (1912).
from the sediment. (1) Dissolve 1.0% peptone, 0.5%
NaCl
(8) Add 0.5 cc.
chloroform and pre- and 1.0% glucose in slightly
serve in tightly stoppered flasks. alkaline meat infusion.
(9) Mix one part of 1.0% glucose
infu-
(2) Add 3 or 4 parts serum to 1 nart
sion broth, with 3 parts (1).
(8).
(10) Tube in sterile test tubes (about 3 (3) Mix well and tube.
cm. deep), and place in a sloping (4) Solidify at a temperature between
position in an inspissator 90 and 95C.
or
steamer and heat to 95C. for (g) Drigalski and Bierast (1913).
1
hour on 3 successive days, (1) Mix 600 cc. beef serum, 174 cc.
1%
(d) Heinemann (1905). dextrose bouillon, 26 cc. bile.
(1) Collect fresh o.x blood in sterile (2) Pour into petri dishes in amounts
museum jars. of about 16 cc.
(2) Set in an ice chest until the (3) Solidify at 90-95C.
serum
has separated. (4) Sterilize fractionally on 3 succes-
(3) Filter the serum if necessary. sive days as with Loeffler serum.
(4) Take 3 parts (3) and mix thoroly (h) Eastwood (1916) cultivated menin-
with 1 part of infusion broth gococci on the following medium:
containing 1.5% glucose. Medium
(1) is composed of 3 parts
(5) Tube. ox-blood serum, one part bouillon
(6) Place in a Koch inspissator two with 1.0% of glucose, galactose,
or
three rows deep. Place malatose and sucrose respectively,
about
25.0 cc. of water with the tubes. and 1 in 10,000 of neutral red is
(7) Incline the inspissator to added as an indicator. Details
the
proper angle so as to slant the of preparation not given,
serum. (i) Shimer (1916).
(8) Heat the water in the apparatus to Add or 1.6 cc. of a 1.0% potas-
1.4, 1.5
boiling and boil 5 minutes.
Then sium tellurate solution to Loeffler's
allow to cool.
serum,
(9) Repeat (8) on the two following
(j) Roddy (1917).
days.
(1) Preparation of glucose bouillon
(e) Rothe (1907).
not given.
(1) Mix 4 parts sterile beef serum with
(2) Mix 1 part of (1) with 3 parts of
1 part sterile sugar free nutrient
calf's or lamb's blood serum.
bouillon (method of preparation
(3) Tube.
or composition not given).
(4) Place in the serum sterilizer.
(2) Prepare 10.0% solution of levulose,
(5) Heat at 57C. for 1 hour each
glucose, maltose, mannitol or day for 6 days and then at 70 C. 1
sucrose.
hour each day for 2 days or heat in
MICROORGANISMS 783
tion is complete. Do not heat

the Arnold steam sterilizer at
over 95 C.
80C. 1 hour each day for 3 days.
Sterilize in the Arnold on each of
n^ Tanner nQ1Q^
(k)
rr.
(1919).
(6)
,
, ,, .
3 successive days for 20 minutes.
Collect beef blood at the abatto.
(1) ^^^^^J^^^ZIZI (1887), Michel (1897
in clean --^--^ ^^ p^^^^,
^^ ^
p"261), Smith (1902 p. 104), Frost (1903
the ice box until clot is formed^ P
clear P^^^^ Heineman (1905 p. 126), Rothe
(2) Siphon or decant off the )>
^^^^^ ^^^^ ^^^ ^^^^^^.^ ^^^^^
'^- Kolle Wasser-
xi, / , n 2471 Abel (1912 p. 25),
(3) Add one part glucose broth (prep-
aration not given) to three parts
P;
f
rnanî^'^f^^
^^p^^^
U), Srigal'ski
^^^^J^^
and Bierast
^^^^^ ^^ ^^^^
^^7"^-
Shimer (1916 p. Ill), Roddy ^1917 p. 43),
(4) Tube. Ball (1919, p. 80),
. , ^ ,

Tanner (1919
919 P p. 69),
(5) Coagulate (method not given). ;
^^^^_^^ ^J
Besson ( P
(6) Sterilize m
the autoclave at about ^,^ ^ ,,^ (1921
112C. or m
the Arnold usmg the P-
'^^\^J^ ^,,^, p. 367). Abbott
P^
intermittent method if desired. ^^^^^ ^^.^^
J39), ^^^^^ ^^^^^^^ ^^^^^ ^
(1) Harvey (1921-22). K
Klimmer (1923 p. 221, 222),
a923 p^ 4
Harvey added 2.0% or 3.0% chloro- vviiimui
J^^^^P.^^^ ^^^24
pre- -târk,
form to the serum as a
servative. ^ 124).
p.
(m) Park, Williams & Krumwiede (1924)^

^^^^^^^ S^^^
-^^ô- ^'" ^
(1) Obtain beef or sheep blood from
the slaughter house, and place constituents:
in a sterile tall cylindrical vessel. ^ Bouillon lOO-^ ^^
If the coagulum adheres

to the 2' 300.0 cc.
(2) Serum, sheep
sides of the vessel, loosen with a ^ Glucose 2.0 g.
sterile glass rod. _
Potassium-sulphocyanide. . . . 4.0 g.
4.
(3) Allow to stand for 24 hours on ice. ^'

Neutral red (5.0% solution
Pipette or siphon off the serum. 8.0 cc.
'
(4) aqueous)
If bloody, place on ice and
allow
(5) preparation:
the corpuscle to settle out. Method of preparation of bouillon
^^^
(6) Mix 3 parts beef or sheep serum ^^^ gi^gn
and 1 part nutrient broth (neutral ^2) Mix 3 parts good blood serum of the
to litmus at a pH = 6.8 to 7.0) to
^^^^p ^^^ i part (1).
which 1.0% dextrose has been and 5 to (2).
(3) Add 3, 4
added. (4) Tube.
(7) Tube. Sterilization: Coagulate
and sterilize
(8) Slant the tubes in an apparatus (^^^ct method not given).

where the temperature can be j^-i^ ^f diphtheria. Author re-
u b
slowly raised to between 80 and aivhtheriae always pro-
Po^t^^ t
90C. Do not heat above 95C. ^^.^^ ^.^^^ ^^
until the coagulated^
medium is
d^st nguished from
cocci colonies.
for 20 aisuufe
(9) Sterilize in the Arnold
Vanants^:^^^^^^
minutes on each of 3 successive ^^^^^^
^^^^^ ^^^ ^^^^^^.^^
ParkJNviUiams and Krumwiede

(n) ^^^^|-7::rp;sition and method of
preparation of nutrient broth not

(ilTid 0.5% glucose to heart infusion
broth (see medium 794).
(2) Ubtam
(2)^0bTain fresh
ire sheeph blood serum
(2) Mix 3 parts horse serum or plasma
.,, /n
.t (1). free from blood cells,
with 11 part 0.5%
^^ ^^^ ^^^^^ ^^^ 25.0% (1),
glucose, 1.0% of 1.0% watery

^ If"^:
neutral red solution and 1.0%
l5!H::tto80to90^C.untilcoagula-
MICROORGANISMS
potassium sulphocyanide. (CaCl2 ilize by the fractional method in the

may be added also if desired). steamer.
(4) Adjust to faint alkalinity. Use: General culture medium.
(5) Distribute into test tubes. Reference: Ball (1919 p. 84).
(6) Sterilize and coagulate (method not
specified). 2420. Crowe's Glucose Agar Blood
Medium
Coplans reported that B. diphtheriae Constituents
colonies after 18 hours were pink
Nutrient agar
1.
250.0 cc.
and a bluish pink tint diffused out 2. Blood, defibrinated (bullock). 750.0
cc.
into the
medium. Medium originally 3. Glucose (1.0%)
iq.q g.
yellow. 1.0% KCNS sufficed for this
Preparation
purpose. If 2.0% be added B. Hof- Strain about a liter of defibrinated
(1)
manni inhibited. If more than 2\% bullock blood thru muslin.
be added B. diphtheria and Torulae
(2) Place 750.0 cc. of (1) in a water bath
are inhibited.
at 50C.
(b) Coplans used 0.5 to 2.0% ofPotas- Melt 250.0
(3) cc. of peptone (or trypsin)
sium-ferri-cyanide instead of 1.25 g. agar of the usual reaction.
of potassium-sulpho-cyanide as in (4) Mix (2) and (3) and add 1.0% glucose.
variant above. (0.5 to 2.0% KCNS (5) Keep at 50C. while plates or tubes
may be added). are filled. At least 0.25 of an inch
is
(c) Coplans used l.Og. potassium-sulpho- required for each plate.
cyanide instead of 1.25 g. as in variant Place in a steam sterilizer at 65 to
(6)
(a) above and added 0.5 to 2.0
g. 70C. for an hour or two and then at
boric acid. (The KCNS may be 80-90C. on each of 3 successive days.
omitted.) Sterilization: See step (6) above.
(d) Coplans added 1.0 g. Potassium Use: Differentiation of streptococci.
ferrocyanide instead of 1.0% potas- Reference: Crowe (1921 p. 486).
sium sulpho-cyanide as in variant
(a). (1.0 g. potassium sulpho-cy- 2421. Vardon's Bouillon Serum
anide may also be added if desired). (Desiccated)
(e) Harvey added 0.5% glucose, 1.0% Constituents
potassium sulpho-cyanide, and 0.5% 1. Bouillon 100.0 cc.
of a 2.0% neutral red solution to 2. Horse serum 300.0 cc.
infusion broth, see variant (bb) 779. Preparation
One part of this mixture was added (1) Mix part bouillon with 3 parts
1
to 3 parts sheep serum and co-
serum.
agulated.
(2) Pour the mi.xture into shallow trays.
References: Rankin (1911 p. 273), Coplans (3) Dry at a temperature not e.xceeding
(1911 pp. 287, 282), Harvey (1921-22 50C.
p. 81). Scrape the dried film
(4) off the trays and
store for use.
2419. Ball's Glucose Bouillon Blood Albumin (5) Dissolve the required amount of (4)
Medium in cold water. (Amount required to
be calculated from the amount of
Constituents
serum and bouillon started with and
1. Glucose bouillon 85.0 cc.
2. Blood albumin (commercial).. 15.0
amount of powder obtained).
g.
(6) Distribute when dissolved in amounts
Preparation
about 7.0 cc. into test tubes.
of
(1) Prepare glucose bouillon.
(7) Inspissate the medium in a sloping
(2) Dissolve 15 parts commercial blood
position at a temperature of 75C.
albumin in 85 parts (1).
for 60 minutes on three successive
(3) Tube.
days.
(4) Inspissate in a slanting position.
(8) Incubate tubes 24 hours and reject
Sterilization: Remove the tubes and ster- any which are not sterile.
Sterilization: Not specified. Sterilization: Sterilization effected during
Use: Cultivation of diphtheria bacilli. preparation.

Variants: The serum may be substituted Use: Isolation of the gonococcus. The
by a solution of dessicated serum. author reported that after 16 hours,
Reference: Vardon (1923-24 p. 431). gonococci colonies were round, dull
typical colonies. The addition of sugar
2422. Thalmann's Infusion Broth Serum did not increase growth.
Medium Variants Klimmer mixed one part bouillon
:
with three parts serum and coagulated at

Constituents
2000.0 cc. 65 to 80 C.
1. Distilled water
2. Beef 1000.0 cc. References: Thalmann (1900 p. 831), Klim-
3. Peptone (siccum) 20.0 g. mer (1923 p. 200).
4. NaCl 10.0 g.
5. Serum (beef or hog) 2423. Picker's Brain Infusion Serum
Preparation Medium
(1) Cut lean beef into small pieces in a
Constituents:
meat cutting machine. Distilled water 500.0 cc.
1.
(2) Add a double weight of distilled
2. Brain 500.0 g.
water to (1).
3. Serum 500.0 cc.
(3) Boil for i hour, stirring continually 30.0 cc.
4. Glycerol
with a glass rod.
Preparation
(4) Make up the loss of water and filter
(1) Pass fresh brain (500.0 g.) thru a meat
thru a filtering cloth.
chopping machine.
(5) Add 1.0% peptone (siccum), and
(2) Add an equal weight of (500.0 g.)
0.5% NaCl. distilled water.
(6) Boil. and heat slowly to a boil.
(3) Stir
(7) Make up the volume of water lost.
Boil slowly for i hour.
(4)
(8) Cool (in a closed container) and
(5) Filter thru a filtering cloth until the
filter.
filtrate assumes a pulpy character.
(9) Distribute in 300 to 500.0 cc. portions
Press the coagulated mass free from
in clean flaskswith patented sealers.
liquid.
(10) Sterilize in streaming steam for one
(6) Mix equal parts of sterile (5) and
hour.
serum.
(11) Bring to boil on a concentrated salt
(7) Add 3.0% glycerol.
solution bath and boil for 45 minutes
(8) Tube.
shaking often.
(9) Solidify in a serum oven.
(12) Take 30.0 cc. of (11), add a drop of
Sterilization: Steam the filtrate from (5)
alcoholic phenolphthalein solution
for two hours. Sterilization of other
and add N/1 sodium solution until a
materials not specified.
red coloration is formed.
(13) Estimate the amount of (11) and
calculating from (12) add I to f the Author reported that beef lungs, testicles,
amount of sodium solution required spleen, liver, kidney, udder, pancreas
for neutralization. were treated in like manner but none

Place in warm water. gave as good results as brain. The lungs,
(14)
spleen, liver and pancreas of man were
(15) Filter.
equal portions of serum (beef or used, but brain proved to be the best
(16) Mi.x
hog, but hog serum preferred) and medium.
sterile (15). Variants: Klimmer prepared the medium
(17) Tube. as follows:
Place in a serum oven in a slanted (1) Cut fresh brain into small pieces and
(18)
position and heat on the first and add an equal weight of distilled water.
second day for 2 hours at 70C. and (2) Heat to boiling, stirring constantly.
on the third day for one hour at (3) Filter thru a filtering cloth, and
100 C. squeeze the meat free from juice.
(4) Sterilize for two hours (temperature 2426. Pergola's Egg Yolk Serum Medium
not given).
Constituents:
(5) Add an equal volume of sterile solu-
1. Ox serum 50.0 cc.
tion of 3.0% glycerol in serum to (4)
2. NaCl (0.8% soln.) 50.0 cc.
under aseptic conditions.
3. Potassium tellurite (1.0%
(6) Mix thoroly.
soln.) 2.0 cc.
(7) Solidify quickly.
4. Egg yolk 1
References: Ticker (1900 p. 593), Harvey
Preparation: Details of preparation not
(1921-22 p. 98), Klimmer (1923 p. 224).
given in the abstract.
2424. Picker's Potato Juice Serum Medium Sterilization: Not given.
Use: Diagnosis of diphtheria.
Constituents
Reference: Pergola (1918 p. 101 taken
1. Potato juice 100.0 cc.
from 1919 p. 57).
2. Serum (beef) 200.0 cc.
3. Glycerol (3.0%) 6.0 cc.
2427. Greenspon's Glucose Citrate Serum
Preparation
Medium
(1) Exact method of preparation of
potato juice not given. Constituents
(2) Mix one part (1) with two parts beef 1. Glucose veal infusion broth
serum. to 100.0 cc.
(3) Add 3.0% glycerol. 2. Serum, pig, sheep or human . . 75.0 cc.
(4) Reaction may be neutral or slightly 3. Sodium citrate (50.0% soln.) 1.0 cc.
alkaline or acid. Preparation

(5) Solidify in a serum oven. (1) Add 1.0 cc. of a 50.0% sodium citrate
Sterilization: Not specified. solution to 75.0 cc. of clear pig, sheep
Use: Cultivation of tubercle bacilli. Au- or human serum and sufficient glucose
thor reported that best growth was ob- veal infusion broth to bring the
tained on neutral or slightly alkaline volume to 100.0 cc.
medium. (2) Adjust to pH 6.4 by the addition of
Reference: Ficker (1900 p. 509). 3.0% citric acid.
(3) Tube.
2425. Vannod's Nutrose Serum Medium (4) Coagulate in the Koch inspissator.
Constituents Sterilization: Sterilize the fractional
1. Distilled water 40.0 to 50.0 cc. method on each of three successive days.
2. Serum (hog) 15.0 cc. Use: Isolation and cultivation of diph-
3. Glycerol 3.0 cc. theria bacillus.
4. Nutrose 1 .0 cc. Variants: Beef extract broth may be used
Preparation instead of veal infusion.
(1) Obtain hog blood from the abattoir. Reference: Greenspon (1922 p. 32).
(2) After 24 hours syphon the serum

from the blood cells.
2428. Duval's Egg Serum Medium
(3) Filter the serum thru a Berkefeld Constituents
candle to free it from red corpuscles. 1. Distilled water 200.0 cc.
(4) Add 40, 45 or 50.0 cc. distilled water 2. Human blood 100.0 cc.
to 15.0 cc. of the filtered serum and 3. Glycerol 10.0 cc.
mix thoroly. 4. Eggs 12
(5) Add 3.0 cc. of glycerol and 1.0 cc. of Preparation
nutrose to each flask of (4). (1) Mix 200.0 cc. distilled water, 100.0
(6) Heat by a flame, and shake until cc. of human blood serum and 10.0
boiling. cc. of glycerol.
Sterilization: Sterilize three successive (2) Carefully separate the whites of 12
times in steam at 102C. eggs from the yolks and drop each
Use: Cultivation of gonococci. separately and aseptically into a
Reference: Vannod (1907 p. 13). flask containing sterile (1).
(3) Shake thoroly. 2430. Wolbach and Schlesinger's Tissue

(4) Tube carefully. Plasma Medium
(5) Heat the tubes in a slanted position
Constituents
at a temperature not exceeding 70C. 1. Plasma, guinea pig.
until coagulation has taken place. 2. Brain (testicle) guinea pig.
(6) Paraffin the cotton plugs to keep the
Preparation
medium moist. Obtain guinea pig plasma by bleeding
(1)
Sterilization: Sterilize (1) by the inter-
a guinea pig directly from the heart.
mittent method in the Arnold sterilizer.
The anasthetization of the guinea
Use: Isolation of tubercle bacilli. pig must be such as to have no reflex
movements of the animal, but still
have a vigorous heart action.
2429. Ebellng's Serum Tissue Plasma
(2) As soon as collected (in a sterile
Medium
paraffined tube) the blood is placed
Constituents in an ice and salt mixture and chilled
1. Distilled water 90.0 cc. for a period of about 3 minutes.
2. Plasma, chicken 10.0 cc. (3) Centrifuge surrounded by an ice and
3. Serum, chicken salt mixture for a period of 3 to 5
4. Embryonic tissue juice minutes.
5. Acetic acid 1.0% (4) Return to the salt and ice mixture
Preparation (temperature just above the freezing
(1) Dilute 10.0 cc. of normal adult point).
chicken plasma with 90.0 cc. of sterile (5) Collect the supernatant plasma by
distilled water. Shake thoroly in an means of chilled sterile paraffined
Erlenmeyer flask, while adding 1.0 cc. pipette and transfer to another sterile
of a 1.0% acetic acid solution drop chilled paraffined tube, and keep in
by drop. this tube in the ice and salt bath at
Allow to precipitate partially in the
5 to 7C. If the plasma freezes,
(2)
cold for about an hour. thaw out slowly. It may still be
used.
(3) Shake the contents of the flask and
(6) Obtain the tissue bran or testicles for
pour into centrifuge tubes 25.0 cc.
cultivation with aseptic precautions,
in each tube.
and keep immersed in sterile Ringer's
(4) Centrifuge for 10 minutes and decant
solution until the plasma is prepared.
the supernatant fluid.
(7) Cut into pieces 0.5 to 1.0 cmm. in
(5) Invert the tubes over a sterile piece
size under Ringer's solution.
of filter paper for complete drainage.
(8) Transfer to sterile coverslips and
(6) Make up the precipitate contained in cover immediately with a drop of
the tubes to 2.5 cc. with sterile dis- sterile plasma (5).
tilled water.
(9) As soon as the plasma has clotted, the
(7) When (6) is thoroly mixed it has the coverslips are inverted into hollow
appearance of rich milk. ground slides and sealed with sterile
(8) Mix i part of (7) with I chicken serum vaseline.
and one volume of embryonic tissue Sterilization: Method not given.
extract, by drawing them up and Dermacentroxenus
Use: Cultivation of
expelling them from a bulb pipette.
rickettsi (causing Rocky Mountain Spot-
Such a preparation has a pH value ted Fever) and Rickettsia prowazeki
between 7.0 and 7.3 and coagulates (causing typhus). To make transplants,
in about 1 minute.
remove the bits of tissue from the plasma,
Sterilization: Method not given. wash a few minutes in sterile Ringer's
Use: Cultivation of connective tissue. solution, transfer to fresh sterile cover-
Reference: Ebeling (1921 p. 643). slips and add fresh sterile plasma.
(Authors have kept tissue cultures from Corper's, et al.. Gentian Violet Egg
the testicles and brain of guinea pigs in Medium 2447
active growth for four to six generations Twort's Bacteria Infusion Egg
over a period of four to six weeks). Use Medium 2448
guinea pig brain for growing typhus Harvey's Trypsinized Heart Egg
organism and guinea pig testicles for the Medium 2449
cultivation of Rocky Mountain Spotted Rosenthal and Schulz's Meat In-
Fever microorganism. For injection into fusion Egg Medium (Zimmer-
animals the tissue cultures are removed mann) 2450
from the coverslips, placed in Ringer's by egg white.
A2. Solidified
solution and teased into pieces small Thoinot and Masselin's Egg Albumin
enough to pass thru a number 18-gauge Medium 2451
hypodermic needle. Barthel's Egg Albumin Medium 2452
Reference: Wolbach and Schlesinger (1923, Dal Pozzo's Transparent Egg
p. 233). Albumin 2453
Rosenthal and Schulz's Alkaline Egg
SUBGROUP V-C Albumin (Zimmermann) 2454
Barthel's Milk Egg Albumin
Solidifying Agent Egg or Egg Medium 2455
Derivatives, etc. Brown and Orcutt's Veal Infusion
Egg Albumin Medium 2456
Basal or Complete Media Solidified by
A3. Solidified by yolk of egg.
Materials other than Blood or its Deriv-
Pergola's Tellurite Egg Yolk
atives (Egg or Egg Derivatives,
Medium 2457
etc.) Initially Liquid but Becom-
Smith's Egg Yolk Medium 2458
ing Permanently Solid
McCoy and Chapin's Egg Yolk
Ai. Solidified by whole egg. (Francis) 2459
Bi. Additional constituents, if any, of Dorset's Egg Yolk Medium (Heine-
known chemical composition. mann) 2460
Dal Pozzo's Lapwing Egg Medium . 2431 Nastinkoff's Egg Yolk Medium
Ball's Boiled Egg Medium 2432 (Rechtsamer) 2461
Abel's Egg Medium 2433 Dal Pozzo's Egg Yolk Medium 2462
Smith's Egg Medium 2434 Lubenau's Bouillon Egg Yolk
Dorset's Egg Medium (Heinemann) 2435 Medium (Schoenburg) 2463
Modified Dorset's Egg Medium Phisalix's Potato Egg Yolk Medium 2464
(Brown and Smith) 2436 A4. Solidified by other materials.
Dorset's Egg Medium (Abel) 2437 Sputum Medium
Steffen's 2465
Soparkar's Glycerol Egg Medium... 2438 McCann's Ovarian Cyst Fluid
Lubenau's Glycerol Egg Medium Medium 2466
(Abel) 2439
2431. Dal Pozzo's Lapwing Egg Medium
Twort and Ingram's Basal Glycerol
Egg Medium 2440 Constituents
Bj. At least one additional constituent of 1. Water 1000.0 cc.
unknown chemical composition present. 2. Egg (lapwing) 100.0 cc.
Roddy's Glucose Bouillon Egg Preparation
Medium 2441 (1) Wash lapwing's eggs with a 1% sub-
Putnam and Gay's Milk Egg limate solution. (One egg will make
Medium 2442 about 4 or 5 tubes of medium.)
Petroff's Gentian Violet Egg (2) Open the egg under aseptic conditions
Medium 2443 and allow the white of the egg to flow
Harvey's Sperm Egg Medium 2444 into a sterile flask.
Harvey's Olive Oil Egg Medium 2445 (3) Mix the egg albumin with its volume
Lubenau's Glycerol Bouillon Egg of sterile water.
Medium (Park and Krumwiede) 2446
. . .
(4) Distribute into sterile test tubes.
(5) Coagulate in the same manner as the (3) Add several drops of sterile water
coagulation of serum, (details of to each piece.
method not given) at a temperature (4) Sterilize (method not given).
of about 70C. References: Ball (1919 p. 84), Bezangon
Sterilization: Method of sterilization of (1920 p. 121), Besson (1920 p. 54), Harvey
water or containers not given. (1921-22 p. 85), Klimmer (1923 p. 223).
Use: Cultivation of saprophytic and para-
sitic forms. 2433. Abel's Egg Medium
Variants The author added a sterile solu-
:
tion of glycerol, dextrin, or any other Constituents

carbohydrate to the medium. 1. Egg.
Reference: Dal Pozzo (1888 p. 526). Preparation
(1) Carefully wash and brush the shell
2432. Ball's Boiled Egg Medium of an egg.

(2) Wash in a 5.0% sublimate solution
Constituents and in sterile water.
1. Egg.
(3) Dry with sterile wool.
Preparation Make a small opening in the apex of
(4)
(1) Boil eggs. the egg.
(2) Remove the shell over a small portion.
(5) After inoculation close the opening
Sterilization: Not specified. with sealing wax or sterile

A Compilation of Culture Media - Color (Danh Luc MT Nuoi Cay VSV)

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SOCIETY OF AMERICAN BACTERIOLOGISTS

Volume I. General Systematic Bacteriology.

Volume II. A Compilation of Culture Mediafor

+ 969 pages. $15.00

PREPARED AT THE REQUEST OF

Made in the United States of America

Published June, 1930

All rights reserved

Composed and Printed at the

1. Nitrogen present as free N2 only.

Section 3 (Med. 30-56) 11

3. Nitrogen supplied as nitrites. Section 6 (Med. 86-95) 28

Section 7 (Med. 96-106) 31

1. Chemical composition of all constituents known.

Section 3 (Med. 324-327) 91

Section 5 (Med. 366-479) 102

(1) Additional constituents inorganic.

(2) Incomplete or "basal" solutions with additional organic

(5) Additional materials of plant origin. Chemical composi-

(6) Additional material of animal origin of unknown chemical

(2) Containing animal derivatives.

1. Nitrogen present only as free N2. Section 1 (Med. 1402-1410) 411

2. Nitrogen supplied as ammonium salts.

b. Organic nitrogen supplied. Section 2 (Med. 1496-1519) 436

2. Composition of some of the added constituents not known.

known composition. Section 3 (Med. 1530-1596) 443

b. Agar media containing commercial digests together with ma-

plant origin but

of ash or soil origin

liquefiable gelatin media containing

1 Gelatin media with other constituents of known composition.

KEY TO REVERSIBLY SOLID MEDIA OTHER THAN AGAR OR

solidified by materials of animal

XI. KEY TO SUB-GROUP OF MEDIA INITIALLY SOLID.

in the following pages was made possible

Institute, Journal of Hygiene, Journal of Pathology and Bacteriology, and all

Smith's Sucrose Peptone Agar."

PRIMARY CLASSIFICATION OF MEDIA

GROUP I. LIQUID MEDIA

Preparation magnesium citrate 0.05%

SUBGROUP I-B Ci. Sodium salts present.

Inorganic basal solutions of known 2. Physiological Salt Solution (Normal

Physiological Salt Solution. (Normal Sterilization: As desired.

3. Molar Salt Solution 4. MgS04 0.3 g.

4. Ashby's Basal Solution (Hoffmann

the following materials or combinations

phthalein. (sucrose 5.0 g.

Constituents Sterilization: As desired.

media, various nitrogen or carbon sources 3. CaCOs 0.5 g.

being added. 4. NaCl 0.5 g.

Reference: Park, Williams and Krumwiede 5. FeS04 some

(2) Distribute in flat bottomed flasks.

Preparation: (1) Dissolve 2, 3 and 4 in 1 soil. More nitrogen was assimilated

Adjustment of reaction not given.

Sterilization: Autoclave at 120C. Time

growth using urea. 3. Na2S04 2.0 g.

1.0 g. of one of the following organic 5. KH2PO4 1.0 g.

materials to each 50.0 cc. lot of medium: Preparation

9. Gerlach and Vogel's Basal Solution garis, B. prodigiosus and B. subtilis.

(b) Egg albumin prepared by method of 12. Percival's Basal Solution

Constituents: (2) Add 10.0 g. of one of the added nu-

tion: peptone (Okuney) 10.0 g.

Added nutrients: 16. Sohngen's Basal Solution