Pub - Microbiology and Biochemistry PDF

Microbiology And Biochemistry
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"This page is Intentionally Left Blank"
MICROBIOLOGY
ANn
BIOCHEMISTRY
Dr. Madan Lal Bagdi
MANGLAM PUBLICATIONS
DELHI-II0053 (INDIA)
Published by:
MANGLAM PUBLICATIONS
L-2111, Street No. 5, Shivaji Marg, Near Kali Mandir,
J.P. Nagar, Kartar Nagar, West Ghonda, Delhi -110053
Phone: 9968367559,9868572512
Email: manglam.books2007@rediffmail.com
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Microbiology And Biochemistry
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First Edition: 2009

ISBN 978-81-906785-0-6
All rights reserved no part ofthis work may be reproduced, stored

in a retrieval system, or transmitted in any form or by any means,
electronic, mechanical, phtocopying, recording or otherwise, with-
out the prior permission in writting from publisher of this book.
PRINTED IN INDIA
Published by D. P. Yadav for Manglam Publications, Delhi-110053,

Printed at Sachin Printers, Moujpur Delhi-53
Preface
The present title Microbiology and Biochemistry is an

authoritative text book compilated for under-graduate and
post-graduate students of various Indian Universities offering
this subject. It would· be equally useful as a text in courses
in molecular biolo,gy, pharmacology and certain other
desciplines of biology. All kinds ofmicroorganisms have been
touched to create an impression about the divl!rsity. The
scope and practices of using different micro-orgabisms have
been shown which may attract future generation. The
enormous prospect of application of microbiology and
biochemistry have been indicated
The author expresses his thanks to all those friends,
colleagues, and research scholars whose continuous
inspirations have initiated him to bring this title.
The author wishes to thank the Manglam Publications,
printer and staff mambers for bringing out this book.
Constructive criticisms and suggestions for iniprovement
of the book will be thankfully acknowledged.
Author
Contents
1. Introduction ......................................................... 1-35
1.1 Host-Parasite Relations ........................................ 2
1.2 Diagnosis of Parasitic Infections ............................. 4
1.3 Laboratory Procedures ......................................... 5
1.4 Procedures for Intestinal Parasites ........................... 6
1.4.1 Collection and Handling of Fecal Specimens ... 6
1.4.2 Gross Examination of Feces ...................... 8
1.5 Procedures for
Microscopic Examination ..................................... 8
1.5.1 Calibration and Use of an Ocular Micrometer. 9
1.5.3 Direct Wet Mount.. ............................... lG
1.5.4 Concentration Procedures ........................ 12
1.5.5 Permanent Stains .................................. 17
(ii) Unpreserved specimens with
PV A fixative .................................. 18
(iii) PV A fixative-preserved specimens ......... 18
1.5.6 Egg Counts ......................................... 21
1.5.7 Duodenal Material ................................ 21
1.5.8 Sigmoidoscopic Material ......................... 12
1.5.9 Abscess Material ................................ , . 22
1.5.10 Cellophane Tape ................................... 23
1.5.11 Examination of Cellophane Tape ................ 23
1.5.12 Culture for Amoebae ............................ ,. 23
1.5.13 Larval Maturation ................................. 24
1.5.14 Adult Worms ....................................... 25
1.6 Blood and Tissue Parasites ................................... 25
(i)
(ii) CONTENTS
1.7 Collection and Handling of Blood Specimens .............. 7fj
1.7.1 Tissue ............................................... Z7
1.7.2 Aspirates of Bone Marrow or Spleen ........... 28
1.7.3 Fluids ............................................... 28
1.7.4 Skin Snips .......................................... 28
l. 7.5 Concentration Procedures for Blood ............. 28
1.7.6 Membrane Filter Concentration for Filariae .. 19
1.7.7 Saponin Lysis Concentration for Filariae ...... 19
1.8 Staining Procedures ........................................... 30
l.8.1 Giemsa Stain Procedure .......................... 30
1.8.2 Gram-Weigert Stain Procedure .................. 30
1.8.3 Culture Procedures for Blood and Tissue Para-
sites ................................................. 31
1.9 Urine ............................................................ 32
1.10 Sputum .......................................................... 32
1.11 VAginal Material ............................................. 32
1.12 Referral of Materials ....................... ~ ................. 32
1.13 Safety ........................................................... 33
1.14 Quality Assurance ............................................. 35
2. Origin of Microbiology .......................................... 36--51
2.1 Beginnings of Microscopy .................................... 37
2.2 The First Microscopes ........................................ 38
2.3 Microorganisms and
the Origin of Life .............................................. 42
2.4 "Diseases" of Wines .......................................... 46
2.5 Pasteurization .................................................. 47
2.6 Pasteur on Specificity of Disease ........................... 48
2.7 Pasteur of Spontaneous Generation .......................... 48
2.8 Modem Style ................................................... 49
2.9 Chemical Evolution ........................................... 49
3. Microbiology of Fungi ....••.......•.....••.......•....•........• 52--82
3.1 Characterization ............................................... 52
3.2 Collection and Storage of Specimens ....................... 53
3.3 Direct Exainination ........................................... 53
3.4 Culture and Isolation .......................................... 56
3.5. Identification ................................................... 63
CONTENTS (iii)
3.5.1 Aureobasidium Spp ................................ 70

3.5.2 Cladosporium Spp ................................. 70
3.5.3 Curvularia Spp ..................................... 71
3.5.4 Drechslera Spp ................... _................ 72
3.5.5 Exophiala Spp ...................................... 74
3.5.6 Fonsecaea spp ...................................... 76
3.5.7 Phaeococcomyces Spp ............................. 77
3.5.8 Phialophora Spp .................................... 78
3.5.9 Rhinocladiella Spp ................................ 79
3.5.10 Scedosporium Spp. . ............................... 79
3.5.11 Scytalidium Spp .................................... ~
3.5.12 Sporothrix Spp. . ................................... ~
4. Microbiology of Bacteria ...................•.................. 83-105
4.1 Shape of Bacteria ............................... " ...... '" .... S4
4.1.1 Size of Bacteria ................................... 85
4.2 The Bacterial Cell ............................................ 87
4.2.1 Cytoplasmic Membrane .......................... 87
4.2.2 Cell Wall ........................................... 88
4.2.3 Capsules ............................................ 89
4.2.4 Polysaccharide Structures ........................ 92
4.2.5 Nucleus ............................................. 92
4.2.6 Metachromatic Granules ......................... C)l
4.2.7 Fat Globules ....................................... 95
4.2.8 Motility ............................................. 95
4.2.9 Motion of Colonies ................................ 99
5. Microbiology of Viruses .............................•........ 106-167
5.1 The Nature of the Virus Particle. ... ..... .... ....... ...... 108
5.2 The Classification of Viruses .............................. 115
5.3 The Virus Host .............................................. 116
5.4 Quantification of Viruses ................................... 117
5.5 General Features of Virus Reproduction ................. 120
5.6 Early Events of Virus Multiplication ..................... 123
5.7 Viral Genetics ............................................... 128
5.8 General Overview of Bacterial Viruses .................. 130
5.9 RNA Bacteriophages ........................................ 131
(iv) CONTENTS
5.10 Single-Stranded Icosahedral
DNA Bacteriophages ....................................... 134
5.11 Single-Stranded Filamentous
5.12 Double-Stranded DNA Bacteriophages................... 139
5.13 Large Double-Stranded
5.14 Temperate Bacterial Viruses: Lysogeny .................. 147
5.15 A Transposable Phage:
Bacteriophage Mu ........................................... 156
5.16 General Overview of Animal Viruses. . . . . . . . . . . . . . . . .. . .. 160
6. General Metabolism .......................•.•..•.............. 168-178
6.1 Characterization of Metabolism.. . . . . . . . . . . . . . . . . . . . .. .. .. 168
6.2 Energy Cycles in Animate Nature ........................ 170
6.3 Energetics of Biochemical Reactions......... ............ 173
6.4 Hight-Enegry and Low-Energy Phosphates: General
Considerations ............................................... 175
6.5 Energy Transfer in Biochemical Processes ............... 177
7. Metabolism of Saccharides ................................... 179-193
7.1 Carbohydrage Catabolism in Tissues ...................... 179
7.1.1 Pentose Phosphate Cycle ....................... 180"
7.1. 2 Interrelation of the Pentose
Phosphate Cycle and Glycolysis............... 183
7.1.3 The Biological Function of
the Pentose Phosphate Cycle. . . . . . . . . . . . . . . . ... 184
7.2 Biosynthesis of Carbohydrates in Tissues. . . . . . . . . . . .. .. .. 186
7.2.1 Gluconeogenesis ................................. 186
7.2.2 Biosynthesis of Glycogen (Glycogenogenesis) 189
7.2.3 Biosynthesis of Other
Oligosaccharides and Polysaccharides ........ 190
7.3 Carbohydrate Metabolism Control in the Organism .... 191
8. Metabolism of Fats and Glycerides .......................... 194-214
8.1 Degradation of Lipids in Tissues .......................... 194
8.1.1 Intracellular Hydrolysis of Lipids ............. 194
8.1.2 Oxidation of Glycerol ........................... 195
8.1.3 Oxidation of Fatty Acids ....................... 195
CONTENTS (v)
8.2 Biosynthesis of Lipids in Tissues .......................... 200

8.2.1 Biosynthesis of Fatty Acids .................... 200
8.2.2 Biosynthesis of Triglycerides .................. 203
8.2.3 Phospholipid Biosynthesis ....................... 204
8.2.4 Biosynthesis of Ketone Bodies ................. 206
8.2.5 Biosynthesis of Cholesterol ..................... 200
8.3 Regulation of Lipid
Metabolism in the Organism ............................... 2fJ)
8.4 Pathology of Lipid Metabolism ............................ 211
8.5 Applications of Lipids and Their
Components in Pharmacotherapy .......................... 213
9. Metabolism of Nucleic Acid ••..•...••••.••.••••...••........ 215-257
9.1 Functional Roles of DNA .................................. 215
9.1.1 DNA as the Genetic Material ................. 215
9.1.2 Cellular Location of DNA ..................... 217
9.1.3 Clinical Comment. . . . . . . . . .. .. . .. . ............. 220
9.1.4 Other Conformations of DNA ................. 221
9.1.5 The "Central Dogma" .......................... 222
9.1.6 Strand Separation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 223
9.1.7 DNA Polymerases ............................... 224
9.1.8 Bacterial DNA Polymerases ................... 224
9.1.9 Stages of DNA Synthesis ....................... 226
9.1.10 Bidirectional Synthesis ......................... 727
9.2 DNA Synthesis in Animal Cells ........................... 230
9.2.1 DNA Polymerase a ............................. 230
9.2.2 DNA Polymerase b ............................. 231
9.2.3 DNA Polymerase g ............................. 231
9.2.4 Reverse Transcriptase .......................... 231
9.2.5 Nucleosome Formation ......................... 234
9.2.6 Transposable Genetic Elements ............... 235
9.3 Molecular Basis of Mutation ............................... 236
9.3.1 Mutagens ......................................... 237
9.3.2 Physical Agents .................................. 239
9.3.3 Excision Repair .................................. 239
9.3.4 Postreplication Repair .......................... 240
9.4 Chemical Carcinogenesis ................................... 241
(vi) CONTENTS
9.4.1 Initiating Agents ................................. 242
9.4.2 Promoting Agents ........................... , ... , 242
9.4.3 Oncogenes ........................................ 243
9.5 DNA Sequence Analysis ................................... 246
9.6 Recombinant DNA Technology in Medicine ............. 248
9.6.1 Restriction Endonucleases ...................... 248
9.6.2 Restriction Maps ................................ 249
9.6.3 Cloning of Recombinant DNA ................ , 249
9.6.4 Clones ............................................. 250
9.6.5 Libraries of Genomic DNA .................... 253
9.6.6 Detection of Recombinant DNA .............. 254
9.6.7 Clinical Comment .......................... , ... , 255
9.6.8 Reverse Genetics ................................ 256
9.6.9 Polymerase Chain Reaction .................... 256
9.6.10 Cloning and Sequencing
the Human Genome ............................. 257
1
Introduction
Parasitic diseases continue to cause significant morbidity and mortality
in the world, particularly in lessdeveloped tropical and subtropical
countries. In the United States, indigenous malaria was eradicated
long ago, and indigenous nematode infections such as ascariasis,
trichuriasis,· and hookworm infection have markedly decreased in both
incidence and severity. Some other infections are increasing. Giardiasis
is a frequent public health problem, with outbreaks related to water
supplies and day care centers for children. Giardia, ameba, and
Cryptosporidium infections are increasing in male homosexuals.
Pneumocystis carinii, Cryptosporidium species, Strongyloides
stercoralis, and Toxoplasma gondii are increasingly important causes
of serious infections in immunoco-mpromised hosts, especially those
with AIDS (acquired immune deficiency syndrome).
In addition to infections which are indigenous to the United States,
a wide variety of infections may be seen in U.S. citizens who have
traveled or worked in foreign countries or in foreign nationals who
are visiting or now residing in the United States. Many of these
people, such as persons infected with malaria, may be asymptomatic
for months or years before disease develops or relapses occur. Some
people are recognized as having malaria only when a recipient of
their blood develops transfusion-induced malaria or when a baby
develops congenital malaria. Other diseases such as echinococcosis
may require years before becoming clinically evident.
Efforts to eradicate parasite infections have had variable success.
Sanitary fecal disposal, improved water supplies, and improved
hygiene in food production and preparation have aided in the control
of intestinal parasites. However, much of the earlier enthusiasm for
2 MICROBIOLOGY AND BIOCHEMISTRY
the eradication of malaria has been tempered by the realization that
malaria eradication is going to be difficult because parasites are
becoming resistant to chemotherapeutic agents, mosquito vectors are
becoming resistant to common insecticides, and the use of some
insecticides may harm the environment. Human modifications of the
environment, such as the building of dams and irrigation systems,
have provided an appropriate environment for vectors such as snails
and thus allowed diseases such as schistosomiasis to flourish in areas
where these diseases had been uncommon. In addition, immunization
programs for parasite infections have developed more slowly than
was anticipated.
1.1 HOST-PARASITE RELATIONS
A knowledge of parasite life cycles is crucial in the understanding
of the ways infection is acquired and spread, the pathogenesis of
disease, and the ways in which disease might be controlled. Some
parasites which infect only humans, such as Enterobius vermicularis
(pinworm), have a narrow host specificity, whereas others such as
Trichinella spiralis infect numerous species. When oth~ animals
harbor the same parasite stage as humans, these animal species may
serve as reservoir hosts. Humans infected with a parasite stage usually
seen in other animal species are referred to as accidental hosts.
In the simplest life cycle, the parasite stage from humans is
immediately infective for other humans, as in pinworm infection or
giardiasis. In other infections such as ascariasis or trichuriasis, a
maturation period outside the body is required before the parasite is
infective. However, for many parasite infections, a second or even a
third host is required for completion of the life cycle. Hosts are defmed
as intermediate hosts if they do not contain the sexual stage and as
definitive hosts if they do contain the sexual stage. Some protozoa,
such as the amebae, flagellates and hemotlagellates, do not have a
recognized sexual stage. In the intermediate host, there may be a
massive proliferation of organisms, as occurs in -humans harboring
malaria parasites or snails harboring schistosome intermediate stages,
or there may be no proliferation, as in mosquitoes which harbor
microfilaria undergoing maturation. There may be proliferation in
definitive hosts, as in mosquitoes harboring the sexual stage of malaria
in which thousands of sporozoites are produced, or there may be no
proliferation, as in helminth infections in which one adult is developed
from each infective larva. However, in the latter, the adult helminths
do produce numerous eggs or larvae.
INTRODUCTION 3
TABLE 1.1 : INCIDENCE OF INTESTINAL PARASITES IN
322,735 FECAL SPECIMENS EXAMINED BY STATE HEALTH
DEPARTMENT LABORATORIES
Parasite No. of % of positive
examinations specimens
Protozoa
Giardia lamblia 12,947 4.0
Entamoeba histolytica 2,409 0.8
Dientamoeba fragilis 1,880 0.6
Balantidium coli 7
Isospora spp. 1
Nonpathogenic 21,120 6.5
Nematodes
Trichuris trichiura 5,481 1.7
Ascaris lumbricoides 4,630 1.4
Enterobius vermicularis 4,344 1.4
Hookworm 2,035 0.6
Strongyloides stercoralis 602 0.2
Trichostrongylus spp. 14
Trematodes
Clonorchis-Opisthorchis 205 0.06
Schistosoma mansoni 48
Fasciola hepatica
Paragonimus westermani
Cestodes
Hymenolepis nana 1,068 0.3
Taenia spp. 251 0.08
Diphyllobothrium latum 20
Hymenolepis diminuta 12
Dipylidium caninum 7
In some helminth infections, a migration through various body
tissues is essential for maturation, as in ascarasis or schistosomiasis,
whereas in other infections, the larva leaves the egg and simply matures
in the intestinal tract, as in trichuriasis and enterobiasis. Host tissues
involved vary depending upon the parasite. In severely
immunocompromised patients, sites may be involved that are not involved
in normal hosts.
Parasites of humans proliferate tremendously at certain stages,
with thousands or even millions of forms being produced for every
one that survives to perpetuate the parasite. Parasites may be quite
hardy. For example, certain stages, particularly eggs and cysts, may
survive for weeks or months in the environment.
Parasites have often developed unique ways of protection from
the defense mechanisms of the host.
The survey does not include laboratories in Guam, Puerto Rico,
or Virgin Islands. One or more parasites were found in 14.7% of
specimens. Percentages are not calculated for parasites identified less
than 100 times.
These mechanisms include the ability to change antigenic
characteristics so that although the host forms antibody, the antibody
does not react with the modified parasite, or the parasite may be
coated with host immunoglobulins, as in schistosomiasis, so that the
host does not recognize the parasite as foreign. Macrophages and
both cell-mediated and humoral immunities appear to be important
in the host response to infection. Eosinophils are particularly important
in the defense against tissue-invading helminths.
1.2 DIAGNOSIS OF PARASITIC INFECTIONS
The diagnosis of most parasitic infections is dependent upon the
laboratory. For intestinal and blood parasites, morphologic
demonstration of diagnostic stage(s) is the principal means of
diagnosis, whereas for tissue infections, immunodiagnostic teChniques
are generally more important. During the early stages before
diagnostic forms are produced (prepatent period), patients may be
symptomatic. For example, patients may have pulmonary symptoms
and eosinophilia due to ascaris larva migration at a time when eggs
are not produced. In such patients the physician may suspect parasite
infection, but the actual diagnosis must be based on a clinical
impression or immunodiagnostic tests, or diagnosis must await the
production of diagnostic stages.
In establishing a diagnosis, the clinician places a great deal of
trust in the laboratory. This trust can be misplaced if laboratory
personnel are not competent to identify or exclude parasites. The
literature clearly documents instances in which outbreaks have been
overlooked due to incompetent laboratory diagnosis or in which
inflammatory cells or other objects have been identified as parasites
and outbreaks have been diagnosed when none existed. The results
of proficiency-testing programs also suggest that laboratories have
INTRODUCTION 5
difficulty with the identification of some parasites, especially intestinal
protozoa.
Identification may be by gross examination for adult helminths
or, more commonly, by microscopic examination for protozoa,
helminth eggs, and larvae. The diagnostic forms of some parasites,
such as the eggs of Ascaris spp., are present on a regular basis.
Other forms, such as malaria parasites, Taenia eggs, or Giardia cysts,
vary from day to day.
TABLE 1.2 : PARTICIPANT PERFORMANCE
No. of Avg correct
Parasite specimens identification
(%)
Formalin-fixed
fecal specimens
Ascaris lumbricoides eggs 6 90
Hookworm 6 92
Strongyloides stercoralis larvae 4 88
Trichuris trichiura eggs 6 93
Diphyllobothrium latum eggs 6 81
Hymenolepis diminuta eggs 5 91
Taenia sp. eggs 6 87
Paragonimus westermani eggs 5 83
Giardia lamblia cysts 8 65
Entamoeba coli cysts 9 88
No parasite seen 6 92
PVA-fixed specimens
Entamoeba histolytica 5 73
Entamoeba coli 4 52
Endolimax nana 4 51
Negative for parasites 3 77
Most immunodiagnostic tests used today for parasitic infections
detect antibody. In recent years, the sensitivity and specificity of many
such tests have improved. A number of antigen detection tests have
recently been described and show promise, but none of these tests
are currently available commercially.
1.3 LABORATORY PROCEDURES
Many methods for diagnostic parasitology have been described.
'There are advantages and disadvantages to each method. Some are
particularly valuable for epidemiologic studies or for evaluations of
new therapeutic agents, whereas other methods are used primarily for
laboratory diagnosis. In this chapter we emphasize the diagnostic
procedures. From the numerous methods, we have selected those which
are widely used in this country and which are sensitive and relatively
easy to perform. These methods should prove adequate for most
laboratories. For additional procedures, laboratory manuals or
parasitology books should be consulted. When alternative methods or
methods for specific parasites are indicated, references will be given,
but the methods will not be described.
1.4 PROCEDURES FOR INTESTINAL PARASITES
Intestinal and biliary parasites are generally diagnosed by finding
diagnostic stages in feces or other intestinal material such as duodenal
or sigmoidoscopic aspirates. Studies have shown that the eggs of most
parasites are uniformly distributed in the fecal mass due to the mixing
action of the colon, although some, such as schistosome eggs, which
originate in the distal colon, may be more numerous on the surface
of formed fecal specimens. The distribution of protozoan forms is
more variable. There may be fewer protozoan trophozoites in the
first part of an evacuation than in the last because they have
deteriorated while in the lower colon.
1.4.1 Collection and Handling of Fecal Specimens
The numbers and times of collection for fecal specimens depend
somewhat on the diagnosis suspected. As a routine, because some
organisms are shed in a variable pattern, it is advisable to examine
multiple specimens before excluding parasites. The general
recommendation is to collect a specimen every second or third day,
for a total of three specimens. From a hospitalized patient, one specimen
each day for three days may be more cost effective.
A number of substances may interfere with stool examination.
Particulate materials such as barium, antacids, kaolin, and bismuth
compounds interfere with morphologic examination, and oily materials
such as mineral oil create small, refractile droplets that make
examination difficult. Antimicrobial agents, particularly broad-
spectrum antimicrobial agents, may suppress amebae. If any of these
substances have been used, specimens should not be submitted until
the substances have been cleared (generally 5 to 10 days). A fecal
specimen may appear satisfactory by gross examination when there
is still barium, etc., which can interfere with microscopic examination.
INTRODUCTION 7
Fecal specimens are best collected into widemouthed, water-tight
containers with tight-fitting lids such as waxed, pint-sized ice cream
cartons or plastic containers. Usually patients can defecate directly
into such containers. Urine should not be allowed to contaminate
specimens, as it is harmful to some parasites. If specimens are to be
collected in a bed pan, the patient should micturate into a separate
container before the specimen is collected. Toilet paper should not
be included with the specimen. Stool should not be retrieved from
toilet bowl water, as various freeliving protozoa in water might be
confused with the parasites. In addition, water is harmful to some
parasites such as schistosome eggs and amebic trophozoites. If the
patient is producing formed specimens, stool may be collected by
having the patient squat over waxed paper to defecate.
Purgation with sodium sulfate or buffered phosphosoda may be
helpful in the diagnosis of amebiasis in some patients. Purgation is
usually done after a series of fecal specimens have been negative,
and it requires the order of a physician. Prior arrangements must be
made with the laboratory, and specimens must be collected during
regular laboratory hours. The patient is given the appropriate salt
solution orally. In approximately 1 to 1.5 h, the patient will begin
to pass stool specimens, and each specimen should be promptly
transported to the laboratory for examination.
Clinical information such as the suspected diagnosis, travel history
of the patient, and clinical findings should be included on the
requisition. In addition, the time the specimen was passed and the
time it was placed in fixative should be noted. If the specimen is in
fixative, the consistency of the original specimen should be stated,
or a portion of unfixed specimen should be included with the fixed
specimen.
A laboratory may have specimens placed in fixatives in the home
or patient care area immediately after passage, may place portions
of specimen in fixatives at the time they are received in the laboratory,
or may examine the specimen unfixed. Many laboratories use a
combination of these methods depending on the location of the patient,
consistency of the specimen, time of day, and laboratory work load.
Prompt examination or fixation is particularly important for soft,
loose, or watery specimens, which are most likely to contain
protozoan trophozoites.
Formed specimens, which are likely to contain protozoan cysts
or helminth eggs or larvae, can remain satisfactory for a number of
hours at room temperature or overnight in a refrigerator. Soft and
liquid specimens should be examined or placed in fixatives promptly
(within 1 h). Specimens which cannot be examined or fixed promptly
should be either refrigerated or left at room temperature. They should
not be incubated, as incubation speeds the deterioration of the
organisms. Feces for parasite examination must not be frozen and
thawed.
The fixative system generally used is a two-vial technique with
one vial containing 5 to 10% buffered Formalin and the other vial
containing polyvinyl alcohol (PV A) fixative. A portion of the
specimen is added to the fixative in a ratio of approximately 3 parts
fixative to 1 part specimen and thoroughly mixed to ensure adequate
fixation. An alternative to Formalin is Merthiolate-iodine-formaldehyde
(MIF), which fixes and stains at the same time. If unfixed specimens
are processed in the laboratory, fecal films may be prepared and
immediately fixed in Schaudinn fixative.
1.4.2 Gross Examination of Feces
Specimens should be examined grossly to determine the consistency
(hard, formed,loose, or watery), color, and presence ofgross abnormalities
such as worms, mucus, pus, or blood. It may be profitable to examine
flecks of mucus, pus, or blood for parasites. If adult worms or portions
of tapeworms are sought, the feces may be carefully washed through
a screen. (Small worms may be difficult to see if gauze is used.) The
identification characteristics of adult worms are not discussed in this
chapter, so parasitology books should be consulted.
1.5 PROCEDURES FOR
. MICROSCOPIC EXAMINATION
The three principal microscopic examinations performed on stool
specimens are direct wet mount, wet mount after concentration, and
permanent stain. Although each examination can contribute to diagnosis,
the yield of some methods is small with certain kinds of specimens.
As a minimum, formed specimens should be examined by a concentration
procedure. Soft specimens should be examined by concentration and
permanent stain, and, if submitted fresh, by direct wet mount. Loose
and watery specimens should be examined by wet mount and permanent
stain. If specimens are received in fixative and the consistency is not
known, concentration and permanent stain should be performed. Other
examinations may be helpfuL Special procedures which may assist in
the diagnosis of specific parasites are noted below in discussions of
the parasites.
INTRODUCTION 9
1.5.1 'Calibration and Use of an Ocular Micrometer
Size is important in the differentiation of parasites and is most
accurately determined with a calibrated ocular micrometer, thus, each
laboratory performing diagnostic parasitology must have such a
micrometer.
An ocular micrometer is a disk on which is etched a scale in
units from 0 to 50 or 100. To determine the micrometer value of
each unit in a particular eyepiece and at a specific magnification,
the unit must be calibrated with a stage micrometer. A stage
micrometer has a scale 2 mm long ruled in fine intervals of 0.01
mm (10 p.m).
1.5.2 Calibration of the Ocular Micrometer
1. Insert the micrometer in the eyepiece so that the micrometer
rests on the diaphragm, with the etched scale facing the eye. In many
new microscopes, the micrometer can be dropped in and secured
with a ring retainer. (It is helpful to have an extra ocular in which
the micrometer may be left.)
2. Place the stage micrometer on the microscope stage.
TABLE 1.3: LABORATORY EXAMINATIONS FOR VARIOUS
TYPES OF FECAL SPECIMENS
Method
Type of specimen Direct wet Concen- Permanent
mount tration stain
Unpreserved
Formed + ++ ±
Soft + + ++ ++
Loose and watery ++ ± ++
Preserved
Formalin
Formed or soft + + +
Loose or liquid ++ +
PVA fixative
Formed ±
Soft, loose, or liquid - ++
+ +, Essential for basic examination; +, recommended for basic examination;
±, optional for basic examination.
3. Focus on the etched scale. Since the micrometer must be
calibrated for each objective, begin with the lowest magnification
(e.g., x 10).
4. Align the two scales so that the zero points are superimposed.
5. Find a point far down the scales at which a line of the stage
micrometer coincides with a line of the ocular micrometer. Count
the number of ocular units and the number of stage units from zero
to these coinciding lines.
6. Multiply the number of stage micrometer units by 1,000 to
convert millimeters to micrometers.
7. Divide the. product of step 6 by the number of ocular units to
determine the 'value of an ocular unit.
Repeat the calibration for each objective. Keep a record of the
unit value for each objective for each microscope used. Calibration
must be done separately for each microscope and must be repeated
if an ocular or objective is changed.
Use of the micrometer. Insert the eyepiece containing the
calibrated ocular micrometer in the microscope. Count the number
of ocular units which equal the structure to be measured. Multiply
the number by the micrometer value of the ocular unit for the
objective being used. If an ocular micrometer is properly used,
parasites which are similar in appearance but different in size can be
readily differentiated.
1.5.3 Direct Wet Mount
The direct wet mount made from unconcentrated fresh feces is
most useful for the detection of the motile trophozoites of intestinal
protozoa and the motile larvae of Strongyloides spp. It is also useful
for the detection of protozoan cysts and helminth eggs. For fixed
feces, the direct wet mount may allow the detection of parasites which
do not concentrate well. This method is also useful for the
examination of specific portions of feces, such as flecks of blood or
mucus.
Direct wet mounts are prepared by placing a small drop of 0.85%
saline toward one end of a glass slide (2 by 3 in. [ca. 5 by 7.5 cm])
and a small drop of appropriate iodine solution (see below) toward
the other end. With an applicator stick, a small portion of specimen
(1 to 2 mg) is thoroughly mixed in each diluent, and a no. I cover
slip (22 mm) is added. The density of fecal material should be such
that newspaper print can be read with difficulty through the smear.
The material should not overflow the edges of the cover slip. Grit
or debris may prevent the cover slip from seating and may be
INTRODUCTION 11
removed with a comer of the cover slip or an applicator stick. Mounts

may be sealed with Vaspar (50% petroleum jelly, 50% paraffin) which
is melted on a hot plate (not over an open flame). A cotton applicator
or small brush is used to apply small drops of Vaspar to opposite
comers to attach the cover slip and then to seal it with even strokes.
The amount of Vaspar on top of the cover slzip should be minimal.
Sealing slows drying and allows oil immersion magnification to be
used. Alternatively, drying can be slowed by placing wet gauze or
paper toweling in a petri dish, laying portions of applicator sticks or
glass rods on the moist material, laying the slide on the .sticks or
rOds, and replacing the lid of the dish.
The iodine solution should be that of Dobell and O'Connor (1 %)
or a 1:5 dilution of Lugol iodine. Iodine solution, if too weak, will
not stain organisms properly, and if too strong, it will cause clumping
of fecal material. Stock iodine solution should be stored in a tightly
stoppered brown bottle away from sunlight. Keep the iodine and saline
solutions in small dropper bottles, and replace (don't replenish) the
solutions weekly. Iodine solution keeps longer if it is refrigerated.
Iodine stain solution can be quality controlled by the observation of
appropriate staining in positive clinical specimens or Formalin-fixed
specimens kept for that purpose.
For the examination of wet mounts, the light of the microscope
must be properly adjusted. To achieve optimal resolution, the
condenser should be centered and focused for Kohler illumination
(racked up). To achieve contrast of the objects in the field, light
intensity is diminished with the iris diaphragm of the condenser rather
than by lowering the condenser.
The entire saline wet mount cover slip should be systematically
scanned with x 100 to X 200 magnification. Suspicious objects are
confirmed at higher magnification. In addition, the preparation should
be scanned at higher power (X 400 to x 500) for a couple of passes
across the cover slip to look for protozoan cysts which might be
missed with lower power. Screening a slide should take an
experienced microscopist about 10 min. If debris is covering a
suspicious object, the debris may be removed by pressing or tapping
on the cover slip with an applicator stick. This pressure may also
help in reorienting an egg, as when one is looking for an operculum.
The saline wet mount is best for the detection of helminth eggs and
larvae, and it is especially good for protozoan cysts, which appear
refractile. The principal usefulness of the iodine mount is to study
the morphology of protozoan cysts, as this stain shows nuclear detail
and glycogen masses (but does not stain chromatoid material). If
suspicious objects are seen, they can be examined under oil immersion
( xl, (00). If definite or possible protozoan cysts or trophozoites
are detected which cannot be identified in wet mounts, permanent
stains are required.
A solution of buffered methylene blue (pH 3.6) may be used as
a vital stain for the examination of fresh specimens for protozoa. The
wet mount is prepared as described above, with buffered methylene
blue substituted as diluent and 5 to 10 min allowed for the dye to
become incorporated in the organisms before examination. Organisms
become overstained in 20 to 30 min.
1.5.4 Concentration Procedures
Concentration procedures are used to separate parasites from fecal
detritus. These procedures are based on differences in the specific
gravity of parasite forms and fecal material. In sedimen-tation, the
parasite forms are heavier than the solution and are found in the
sediment, whereas in flotation, solutions of high specific gravity are
used, and parasite forms float to the surface. An initial washing step
removes some of the soluble or fmely particulate material, and straining
removes larger portions of debris. A wide variety of sedimentation
and flotation methods have been described. The Formalin-ethyl acetate
modification of the Formalin-ether sedimentation technique and a zinc
sulfate flotation technique are widely used and are the only methods
described in this chapter. Both methods require that centrifugation be
performed in centrifuges with free-swinging carriers. Squeeze bottles
for Formalin, saline, or water simplify the processing of large numbers
of specimens.
1.5.4.1 Formalin-ethyl acetate centrifugal sedimentation
The original procedure from which the Formalinethyl acetate
centrifugal sedimentation technique was adapted was the Formalin-
ether concentration method of Ritchie. The Formalin-ethyl acetate
procedure avoids problems with the flammability and storage of ether.
This procedure can be performed on specimens which have been fixed
in Formalin for a time or on specimens with Formalin added during
the processing. The procedure can also be performed on material
fixed in MIF.
The procedure with Formalin-preserved specimens is as follows.
1. Thoroughly mix the formalinized specimens.
INTRODUCTION 13
2. Depending on the density of the specimen, strain a sufficient
quantity through gauze into a 15-ml conical centrifuge tube to give
the desired amount of sediment. (Wet gauze in a 4-oz [ca. 120-ml]
conical paper cup with the tip cut off can be used for straining.)
3. Add tap water or saline, mix the solution thoroughly, and
centrifuge it at 650 x g for 1 min. The amount of the resulting
sediment should be about 1 ml. The amount of sediment may be
adjusted by the addition of more feces and centrifugation again or
by the addition of water, suspension again, the removal of an
appropriate amount of material, and then recentrifugation.
4. Decant the supernatant, and wash it again with tap water, if
desired.
5. To the sediment, add 10% Formalin to the 9-ml mark, and
mix the solution thoroughly.
6. Add 4 ml of ethyl acetate, stopper the tube, and shake the
tube vigorously in an inverted position for 30 s. Remove the stopper
with care.
7. Centrifuge the solution at 450 to 500 x g for 1 min. Four
layers should result: ethyl acetate, plug of debris, Formalin, and
sediment.
8. Free the plug of debris from the sides of the tube by ringing
the tube with an applicator stick, and carefully pour the top three
layers into a discard container. With the tube still tipped, use a swab
to remove debris from the sides of the tube. This step is very
important, for lipid droplets which reach the sediment make
examination difficult.
9. Mix the remaining sediment with the small amount of fluid
that drains back down from the sides of the tube (or add a drop of
saline or Formalin). If mounts are to be prepared later, a small
amount of Formalin may be added to the sediment and the tube may
be stoppered.
10. Prepare wet mounts as described above, and examine them.
The procedure for Formalin-ethyl acetate centrifugal sedimen-
tation with fresh specimens is as follows.
1. Comminute a portion of stool about 1.5 cm in diameter in 10
ml of saline or water.
2. Strain about 10 ml of the fecal suspension into a 15-ml conical
centrifuge tube.
3. Centrifuge the suspension at 650 x g for 2 min. This step
should provide about 1 ml of sediment. If not, adjust the amount of
sediment as described above.
4. Wash the sediment again if desired.
5. To the sediment, add 10% buffered Formalin lo the 9-ml
mark, mix thoroughly, and allow the mixture to stand for 5 min or
longer.
6. Proceed as for step 6 of the procedure for fixed specimens.
(Note that either saline or water can be used. Tap water will
lyse Blastocystis hominis. If schistosomiasis is suspected, the specimen
should be preserved in Formalin before concentration to prevent
hatching.)
1.5.4.2 Zinc sUlfate centrifugal flotation
The zinc sulfate concentration method originally described by
Faust et al. may be performed on unfixed or Formalinfixed specimens,
although the specific gravity of the zinc sulfate solution required
differs. The disadvantages of the zinc sulfate concentration are: (i)
dense schistosome eggs do not concentrate well; (ii) opercula often
pop, and thus operculate eggs may be missed; and (iii) larvae and
cysts may collapse. The modified procedure with Formalin-fixed feces
slows the collapse of larvae and cysts and largely prevents the popping
or opercula. The advantages are that it leaves a rather clean
background, has less grit than the sedimentation procedure, and is
better for the concentration of some parasites, such as Giardia cysts.
The procedure for Formalin-preserved specimens is as follows.
The specific gravity of the zinc suflate must be 1.20. Centrifugation
must be performed in round-bottomed tubes such as 16- by l00-mm
disposable tubes.
1. The Formalin-preserved fecal material is mixed, strained
through one layer of cheesecloth into a conical paper cup, poured
into the tube to a level about 1 cm from the top, and then centrifuged.
2. The tubes are centrifuged for 3 min at about 650 x g. There
should be 1 to 1.5 cm of sediment.
3. Decant the supernatant from each tube, and drain the last
drop against a clean section of paper towel.
4. To the packed sediment of each tube, add zinc sulfate to within
1 cm of the rim.
5. Insert two applicator sticks, and thoroughly mix the packed
sediment.
6. Immediately centrifuge the suspension at 500 rpm for 1.5 min.
INTRODUCTION 15
7. Very carefully transfer the tubes to a rack of the proper size,
so that the tubes remain vertical. Do not disturb the surface ftlms,
which now contain the parasites. Allow the tubes to stand for 1 min
to compensate for any movement. The countertop must be vibration
free.
8. With a loop which is bent at a right angle, transfer to a slide
(2 by 3 in.) two loops of surface material beside I drop of saline
and two loops beside I drop of iodine. With the heel of the loop,
mix first the saline and then the iodine with the surface material.
Cover each mixture with a 22-mm no. I cover slip. The slide should
be made within 20 min.
9. To retard drying, place each prepared slide on a bent glass
rod or portions of applicator sticks in a petri dish containing a damp
paper towel. Petri dishes may be placed in the refrigerator if
examination will be delayed. Alternatively, cover slips may be sealed
with Vaspar.
The procedure with fresh specimens is as follows. /
1. Comminute a fecal specimen about 1 cm in diameter in a
tube (16 by 100 mm) half filled with tap water. Add additional water
to within 1 to 2 cm of the top.
2. Centrifuge the tube at 650 x g for 1 min.
3 .. Discard the supernatant, and add a zinc sulfate solution of
specific gravity 1.18 to within 1 cm of the rim.
4. Proceed as from step 5 above.
1.5.4.3 Sheather sugar ./lotation
She ather sugar flotation is recommended for the concentration
of Cryptosporidium cysts. Although these oocysts will concentrate
when the Formalin-ethyl acetate or zinc sulfate technique is used,
they are more readily detected with the Sheather sugar flotation, for
they stand out sharply from the background in this solution of high
specific gravity. This procedure may be performed on unfixed or
Formalin-fixed feces. The procedure for Sheather sugar flotation is
outlined below.
l. (a) Fonned stool. Place approximately 0.5 g of stool in a
tube (16 by 100 mm) about half full of Sheather sugar solution. Mix
the solution thoroughly, and then add more sugar solution to within
1 cm of the rim.
(b) Watery stool. Centrifuge the fecal specimen and mix 0.5 to
1 ml of sediment with Sheather solution as described above.
2. Centrifuge the solution at 400 x g for 5 to 10 min.
3. Remove the top portion of the sample with a wire loop bent
at a right angle. Place severalloopfuls on a glass slide (2 by 3 in.).
Cover the specimen with a 22-mm cover slip, and examine the slide
with a x 40 objective. Oocysts are found just beneath the cover slip
and are refractile. Saline or iodine is not used in the pr~paration of
these mounts.
1.5.4.4 Baermann concentration
The Baermann concentration technique has greater sensitivity for
the detection of strongyloides larvae than do the standard concentration
techniques described above. This technique is useful clinically for
the diagnosis and monitoring of therapy of strongyloides infections,
and it is useful epidemiologically for the examination of soil for the
larvae of nematode parasites.
A funnel with a clamped rubber tube on the stem is placed in a
ring stand. A circular mesh screen is placed across the funnel
approximately one-third from the top, a portion of coarse fabric such
as muslin is placed on the screen, and feces is added. Tap water at
37°C is added so that the water just touches the feces. Let the
specimen stand I h, remove 2 ml of fluid from the stem, and
centrifuge the sample at 300 x g for 3 min. Prepare a wet mount
of sediment, and examine it for larvae.
1.5.4.5 Hatching technique for viable schistosome eggs
Place a large amount of feces (5 to 10 g) in a large flask (1 to
2 liters), and add water while mixing to break up the feces to a
fine suspension. Bring the water level to 2 to 5 em from the top of
the flask. Cover the sides of the tlask with foil or other material to
shield all but the top of the liquid from light. Allow the tlask to
stand at room temperature for several hours. With a hand lens,
examine the material at the top of the tlask neck for swimming
miracidia. Remove the miracidia with a Pasteur pipette for
examination with a x 10 objective. It is not possible to determine
the species of schistosome from the miracidia.
1.5.4.6 Other concentration procedures
Concentration procedures have been described for feces preserved
in MIF, sodium acetate-Formalin, or PYA fixative. MIF- or sodium
acetate-Formalin-fixed feces may be used in place of Formalin-fixed
feces in the Formalin-ethyl acetate concentration procedure. Some
workers feel that organisms do not concentrate as well from material
INTRODUCTION 17
fixed in PV A fixative or from material which has been in MIF for
extended periods.
If large amounts of specimen are to be concentrated, as when
specimens of C)ggs are prepared for teaching, gravity sedimentation is
usualiy used. The feces is thoroughly mixed in liquid (water, saline,
or 10% Formalin) and allowed to settle in a sedimentation jar or funnel
for several hours ot overnight. Supernatant fluid is discarded, and the
sediment is again suspended and allowed to settle. This procedure can
be continued if desired until the supern-atant is clear.
1.5.5 Permanent Stains
Permanent stains of fecal smears are most needed for the detection
and identification of protozoan trophozoites, but they are also used
for the identification of cysts. Wet mounts of fresh feces, even with
stains such as methylene blue, are not as sensitive for trophozoites
and therefore do not substitute for permanent stains. It is sometimes
difficult to identify cysts which are detected in wet mounts; thus,
for each specimen, regardless of consistency, it may be worthwhile
to fix a portion in PV A fixative or to prepare two fecal films fixed
in Schaudinn fixative so that permanent stains can be performed if
needed. Permanent stains also provide a permanent record and are
easily referred to consultants if there are questions on identification.
A number of staining procedures have been described. Some
stains, such as chlorazol black, require fresh specimens and are not
widely used. A variety of stains for fecal smears preserved by
Schaudinn or PV A fixative have been described, including various
hematoxylin stains. The stain most widely used in the United States
is the Wheatley trichrome stain, which is the only permanent stain
described in this chapter. The trichrome staining procedure uses
reagents with a relatively long shelf life and is easy to perform. Note
that there are differences in staining times depending on whether the
specimen is fixed in Schaudinn or PV A fixative, as penetration is
slower in the latter.
Preparation of smears. (i) Unpreserved specimens with
Schaudinn fixative.
1. To prepare thin, uniform smears, place a drop of saline on a
glass slide (l by 3 in. [ca. 2.5 by 7.5 cm]). With an applicator
stick, transfer a small, representative portion of the specimen to the
drop of saline, and mix the two. Spread the solution into a film by
rolling the applicator stick along the surface. Remove any lumps.
Before watery specimens are smeared, apply an adhesive such
as serum or albumin to the slide. Liquid specimens may be
centrifuged, and the sediment may be used for smear preparation.
2. Place fresh smears immediately into Schaudinn ftxative. Do
not allow the smears to dry at any time before they are stained.
Smears should ftx for at least I h at room temperature or for 5 min
at 50°C; however, they can be left in ftxative for several days. After
ftxation, slides may be kept in 70% alcohol indeftnitely before they
are stained.
(ii) Unpreserved specimens with PV A fixative.
1. On a slide (l by 3 in.), thoroughly mix I drop of unftxed
specimen ~ith I drop of PYA ftxative.
2. Spread the specimen as described below.
3. Allow the smear to dry, preferably overnight, before it is
stained.
(iii) PVA fixative-preserved specimens.
1. Preserve I part specimen in 3 parts PV A fixative. Mix
thoroughly. Fix for at least I h. Specimens keep indefinitely.
2. Add I drop of PV A-fixed specimen to a slide.
(a) If there is little sediment, remove a portion of the sediment
with a Pasteur pipette.
(b) If there is abundant sediment, mix the specimen thoroughly,
and add I drop of specimen to a slide with applicator sticks or a
Pasteur pipette.
3. Spread the material over the center third of the slide by rolling
the specimen with an applicator stick. Remove any lumps. The film
should extend to both the top and bottom edges of the slide, as this
helps prevent peeling.
4. Allow the slide to dry overnight at room temperature or 35°C.
In an urgent situation, the slide can be dried for 4 h at 35°C and
then stained.
1.5.5.1 Trichrome staining procedure
Table elsewhere outlines the steps in the trichrome staining
procedure.
Permanently stained slides may be mounted with a cover slip or
may be air dried and examined after oil is added. Slides should be
examined at a magniftcation of x 400 to X 500 or greater after they
are scanned under lower power to fmd optimal areas. A x 50 oil
immersion objective is particularly helpful, as it allows the easy use
,
INTRODUCTION 19
of aX 100 oil immersion objective for the detailed examination of
organisms while allowing more rapid screening with a x50 objective.
Oculars of x 5 or x 6 can provide the same result. A x20 dry
objective may also assist in screening.
Permanently stained slides should be kept for 2 years.
1.5.5.2 Stain reactions
In an ideal stain, the cytoplasm of cysts and trophozoites is blue-
green tinged with purple. Entamoeba coli cyst cytoplasm is often
more purple than that of other species. Nuclear chromatin, chromatoid
bodies, erythrocytes, and bacteria stain red or purplish red. Other
ingested particles such as yeasts often stain green. Parasite eggs and
larvae usually stain red. Inflammatory cells and tissue cells stain in
a fashion similar to that of protozoa. Color reactions may vary from
the above.
Incompletely fixed cysts may stain predominantly red, and
organisms which have degenerated before fixation often stain pale
green. Poor fixation due to an inadequate mixing of the specimen in
fixative may result in both of these" appearances. In some specimens,
degeneration has occurred before the specimen is placed in fixative,
either in the patient before the specimen was evacuated or because
of delay in fixing the specimen.
1.5.5.3 Troubleshooting the trichrome stain
Except for problems with delayed or inadequate fixation as noted
above, problems with the trichrome stain are usually related to
reagents other than the stain. If crystalline material is apparent after
the specimen is stained, the crystals are probably mercuric chloride
in the fixative which was not adequately removed because the iodine
in the alcohol-iodine solution was too weak or because the slide was
in this solution too short a time. If crystals are present after treatment
with proper-strength iodine-alcohol, they are present in the specimen,
which is thus unsatisfactory, and another specimen should be
requested.
If the stain appears washed out, it is likely that the slide was
destained too much. This washed-out appearance can be either because
the specimen was left too long in the acid-alcohol destain or because
the alcohol wash after the acid-alcohol destain had become acidic as
a result of transfer by previous slides.
The trichrome may become diluted by carry-over alcohol if more
than 10 slides per day are stained in one Coplin jar. To restore the
stain, the lid may be left off for several hours to allow alcohol to
evaporate, and then the volume is replaced with new stain.
Control slides should be used to monitor the staining. Specimens
containing protozoa are best for controls; however, feces containing
inflammatory cells or added buffy-coat leukocytes also are satisfactory.
1.5.5.4 Restaining
Should the stain be unsatisfactory, the slide can be destained by
placing it in xylene to remove the cover slip or immersion oil and
then placing it in 50 % alcohol for 10 min to hydrate the slide. Destain
the slide in 10% acetic acid in water for several hours, and then
wash it thoroughly first in water and then in 50 and 70% alcohols.
Place the slide in stain for 8 min, and then complete the stain
procedures. It is helpful to e.Jiminate or shorten the destain step.
1.5.5.5 Acid-fast stain for Cryptosporidium sp.
Acid-fast staining for Crypiosporidium. sp. has recently become
important because this parasite is now recognized as a cause of severe
diarrhea in immunodeficient patients such as those with AIDS, and
it can cause transient diarrhea in immunocompetent individuals. The
modified acid-fast stain recommended is similar to that used to stain
Nocardia spp. in that it uses milder acid decolorization. A variety
of acid-fast and fluorochrome staining procedures have been described
for Cryptosporidium spp., and all the procedures appear to work.
The following procedure is useful for staining Nocardia species
as well as Cryptosporidium species. This procedure may be used on
fresh or Formalinfixed material or on material from concentration
procedures. If the specimen is liquid, centrifuge it, and use the
sediment to prepare a smear.
1. Pick a portion of material with an applicator stick, mix the
material in a drop of saline, spread it on a glass slide (1 by 3 in.),
and allow it to dry.
2. Fix the dried film in absolute methanol for 1 min, and air
dry the slide.
3. Flood the slide with Kinyon carbol-fuchsin, and stain the smear
for 5 min.
4. Wash the slide with 50% ethyl alcohol in water, and
immediately rinse it with water.
5. Destain the smear with 1 % sulfuric acid for 2 min or until
no color runs from the slide.
6. Wash the slide with water.
INTRODUCTION 21
7; Counterstain the smear with Loeffler methylene blue for 1
min.
8. Rinse the slide with water, dry it, and examine the smear
with oil inunersion.
The results are that Cryptosporidium oocysts stain bright red,
and background materials stain blue or pale red.
1.5.6 Egg Counts
Egg-counting methods are used in clinical studies to assess the
intensity of infections (especially infections by intestinal nematodes)
and the efficacy of therapeutic agents, and these methods are
commonly used for epidemiologic studies. Methods used for scientific
studies, such as Kato thick smear or Stoll egg counting, require
greater accuracy than methods used for patient care. The simplest,
most practical method is to use a standard fecal suspension which
contains approximately 2 mg of feces mixed in a drop of saline and
covered with a cover slip. The entire cover slip is examined at a
magnification of 100 x, field by field, and the number of eggs is
counted. For research work, the density of the smear can be
standardized with a light meter, but this standardization is not essential
for patient care. The number of eggs per cover slip provides a rough
index of the severity of the infection.
1.5.7 Duodenal Material
The examination of duodenal fluid or duodenal biopsy material
may be useful for the diagnosis of giardiasis, strongyloidiasis, or
other upper intestinal parasite infections in patients in whom parasites
cannot be detected in the feces. In addition, duodenal fluid
occasionally can be useful in showing whether helminth eggs are
originating in the biliary or intestinal tract. Duodenal material may
be obtained by passing a tube through the nose and stomach into the
upper small intestine and then aspirating enteric fluid. As an
alternative, a string test may be used. A weighted gelatin capsule
attached to a string is swallowed, and the proximal end of the string
is taped to the face of the patient. Over a period of several hours,
helped with small sips of water, the string reaches the upper small
intestine. After 4 to 5 h, the string is retrieved, and the material on
the bilestained portion is stripped from the string and examined for
parasites with direct wet mounts or with permanent stains when wet-
mount fmdings are questionable. Aspirated duodenal fluid is examined
in a similar fashion. The material for permanent stains can be fixed
in Schaudinn or PV A fixative, although the latter may adhere better
to the slide. If question able organisms are seen in the direct wet
mount, the coverslip can be removed, the material can be mixed
with a drop of PV A fixative, and a film can be made for later
permanent staining.
A duodenal biopsy can be used to demonstrate Giardia organisms.
A biopsy is usually obtained by a swallowed biopsy capsule. In
searching for Giardia spp., it is generally preferable to make both
impression smears and sections of biopsy tissue. Giardia spp. are
usually present in mucus or attached to epithelium rather than in
tissue. Biopsies occasionally can confirm a diagnosis of
strongyloidiasis or cryptosporidiosis.
1.5.8 Sigmoidoscopic Material
Materials obtained by sigmoidoscopy may be helpful in the diagnosis
or monitoring of amebiases, schistosomiasis, or cryptosporidiosis.
Patients suspected of having amebiasis may have ulcerations of the
l:olon which can be visualized by sigmoidoscopy or colonoscopy.
Scrapings or aspirates of material from ulcers can be examined by
direct wet mounts and permanent stain as described above. The rmding
of typical, erythrophagocytic, motile trophozoites in direct wet mounts
or in permanently stained preparations allows a diagnosis of amebiasis.
Material is best aspirated with a pipette or scraped with an instrument.
Swabs should not be used, as the parasites may be killed or trapped
by swab material.
Biopsy material for amebiasis should be processed for surgical
pathology and then examined for ulcers containing amebae. The
periodic acid-Schiff stain counterstained with hematoxylin is
particularly helpful because amebae stain more positively with periodic
acid-Schiff stain than do inflammatory cells, and amebae show typical
amebic nuclei. Of course, there are no amebic cysts in tissue.
Biopsy material for schistosomiasis is better examined in teased
preparations than in sections, as the entire thickness can be examined
at once, and the viability of eggs can be determined by observation
of the movement of the larvae within the eggs.
In cryptosporidiosis, biopsy material shows organisms at the
luminal surface of the epithelial cells, but the organisms are small,
and the study of structural detail requires electron microscopy.
1.5.9 Abscess Material
Abscesses suspected of being caused by Entamoeba histolytica
may be aspirated, and the material may be submitted to the laboratory.
INTRODUCTION 23
The last material aspirated is most likely to contain amebae. Material
may be examined microscopically in wet mounts and permanent stains,
and in addition, it can be cultured for amebae if bacteria are also
added to the culture as described below. Abscess material is often
thick and difficult to examine. It may be treated with streptokinase
and streptodonase enzymes to liquefy the specimen.
1. Reconstitute streptokinase and streptodonase per the instructions
of the manufacturer.
2. Add 1 part enzyme solution to 5 parts aspirated material.
3. Incubate the mixture at 35 to 37°C for I h. Shake the mixture
at intervals.
4. Centrifuge the mixture at 300 to 400 x g for 5 min.
5. The sediment may be used for microscopic examinations for
amebae (wet mounts and permanent stains) and for the culture of
amebae.
1.5.10 Cellophane Tape
Cellophane tape is used for finding the eggs of Enterobius
vermicularis or Taenia species from the perianal area. The tape used
must be clear cellophane and not slightly cloudy or opaque.
Alternatively, a Vaspar swab may be used. Specimens from more
than 1 day may be required to diagnose light infections.
1.5.11 Examination of Cellophane Tape
1. If the specimen is difficult to examine, raise the tape from
the front of the glass slide, add a drop of toluene to the slide, and
replace the tape smoothly with an applicator stick. (Remember.
Enterobius spp. and Taenia solium eggs are infective!)
2. Examine the entire tape, including the edges, with x 100
magnification (x 10 objective).
3. Confirm suspicious objects with high dry objectives (x40 to
x50).
1.5.12 Culture for Amoebae
Cultures for amoebae have improved detection in some studies,
but they are not widely used. Although Giardia spp. have been
cultured in research laboratories, cultures are not useful for diagnosis.
A variety of culture media for amebae have heen described, and
some may be purchased from commercial meUlum manufacturen.
The method described here uses the modified charcoal agar slant
diphasic medium described by McQuay.
1. Place 3 ml of sterile 0.5% phosphate-buffered saline on a
charcoal agar slant.
2. Add approximately 30 mg of sterile rice starch to the tube.
3. Warm the medium to 35°C before it is inoculated.
4. (a) Inoculate the medium with fecal specimen (approximately
0.5 ml of liquid specimen or a 0.5-cm sphere of formed specimen)
which is mixed with the saline overlay.
(b) If abscess material is cultured, bacteria must be inoculated
into the culture in addition to the inoculation with 0.5 ml of specimen.
A heavy inoculum with Clostridium perjringens or Escherichia coli
is satisfactory.
5. Incubate the culture at 35°C.
6. At 24 and 48 h, remove I drop of liquid from the lowest
point of the overlay, and prepare a wet mount.
7. Examine the wet mount for amebae.
8. Permanent stains can be prepared by the fixation of sediment
in PV A fixative, with the subsequent preparation of smears and
staining.
1.5.13 Larval Maturation
Larval maturation studies, sometimes referred to as cultures, can
be performed on fecal specimens applied to wet filter paper. Nematode
larvae such as Strongyloides spp. or hookworm mature to the
filariform stages in the culture container and migrate from feces into
water, where they are detected microscopically. The procedure can
be performed in a petri dish with a square of filter paper or in a
large test tube with a strip of filter paper.
1. Smear approximately 0.5 g of feces on the filter paper.
2. (a) For the tube ·method, insert the filter paper strip into the
tube so that the bottom of the strip is in 3 ml of water. The feces-
smeared portion of the strip need not be immersed in the water.
(b) For the petri dish method, place feces on one half of a piece
of filter paper. Lay the feces-bearing end of the filter paper on a
glass rod or a portion of an applicator stick in the petri dish. Add
approximately 3 ml or sufficient water so that the feces-free end of
the filter paper is in the liquid.
3. Leave the tube or dish at room temperature in the dark. Add
water as needed to ensure that the filter paper is in contact with the
water.
4. Examine the liquid for larvae either by direct microscopic
INTRODUCTION 25
examination with an inverted microscope or by examination of a wet
mount of sediment from the liquid. With the petri dish method, the
surface of the feces also may be examined with a dissecting
microscope.
5. Examine the specimen on days 3, 5, and 7. Strongyloides
filariform larvae are found on days 2 and 3, and hookworm larvae
are found on days 5 through 7. Larvae are identified by their
morphological characteristics.
1.5.14 Adult Worms
Adult worms, or objects suspected to be adult worms, may be
submitted to the laboratory. The laboratory must determine if these
are helminths and, if so, if they are parasites. Identification
characteristics are described in standard references. Tapeworm
proglottids, particularly those of the Taenia species, are difficult to
differentiate grossly unless they are cleared so that the internal
structure can be seen and the number of lateral uterine branches can
be counted. One procedure for clearing the proglottids is outlined
below.
1.5.14.1 Clearing Taenia proglottids and other helminths
Proglottids are first relaxed by placing them in warm saline (56°C)
for I h and then clearing them in carbolxylene while they are kept flat.
They may be kept flat in a number of ways. One way is to press the
proglottid between two glass slides held together with membrane clips
or string. Clearing takes from several hours to overnight.
The proglottid is examined under a dissecting microscope or with
a hand lens, and the uterine branching is observed. Glycerine and
beechwood creosote can also be used with good results. Cleared
proglottids may be mounted or stained if desired.
Small nematodes may also be cleared in carbolxylene or beech-
wood creosote and mounted in permount or balsam. This method is
particularly good for hookworm adults.
1.6 BLOOD AND TISSUE PARASITES
Blood and tissue parasites whose diagnostic forms circulate in
the peripheral blood are generally diagnosed by the demonstration
of parasites in Giemsastained thick or thin films of blood. Special
concentration techniques may be helpful for the diagnosis of some
diseases such as filarial or trypanosornal infection. Other tissue
parasites which do not circulate in the blood may be diagnosed by
the detection of parasites in skin snips, lesion scrapings, body fluids,
or biopsy material or by the detection of antibody or antigen in serum
or other body fluids.
1. 7 COLLECTION AND HANDLING OF
BLOOD SPECIMENS
The timing of the collection of blood specimens depends on the
parasite disease suspected. For example, for certain filarial infections,
specimens are best obtained between 10:00 p.m. and midnight, whereas
for other infections, specimens are best obtained during the day. In
malaria, the numbers and stages of parasites in the peripheral blood
vary with different parts of the cycle.
Blood films are best made from blood which is not anticoagulated,
such as that obtained from finger stick or ear lobe puncture.
Anticoagulants may interfere with parasite morphology and staining.
Care should be taken that the alcohol disinfectant is allowed to dry
before the area is punctured, or there may be fixation of erythrocytes,
which will interfere with the preparation and staining of thick films.
Both thick and thin films can be prepared from blood obtained by
venipuncture, although it is best if the blood remaining in the needle
of the venipuncture device is used, because it is anticoagulant free.
Thick and thin films can be prepared from blood that is
anticoagulated, but the staining characteristics are not as good. EDTA-
anticoagulated blood is better for staining than citrate- or heparin-
anticoagulated blood.
Both thick and thin blood films are useful. Thick films are more
sensitive because the same amount of blood can be examined in a
thick film in 5 min as can be examined in a thin film in 30 min.
However, thin films allow the study of the effects of parasites on
erythrocytes and provide better parasite morphology.
Thick and thin films may be prepared on separate slides or on
the same slide, with the thick film at one end and the thin film at
the other end.
The thick film is prepared by spreading 1 drop or puddling
several small drops of blood into an area approximately 1.5 cm in
diameter. A properly prepared thick film should be thin enough so
that newspaper print can barely be read through it. If the film is too
thick, it will fragment and peel, and if the film is too thin, the
increased sensitivity will be lost. Thick films should be allowed to
dry overnight and should be stained within 3 days. They must not
be heated, and they should be protected from dust. If the erythrocytes
are fixed, they will not dehemoglobinize. If prompt examination is
INTRODUCTION 27
required, prepare a slightly thinner thick film, dry it for I h, and
stain it.
The thin film is prepared in the same manner as a film for a
differential leukocyte count. A small drop of blood is placed on one
end of a microscope slide. A second slide held at an acute angle of
30 to 45 0 is backed into the drop of blood, which spreads along the
junction of the slides. The spreader slide is then pushed along the
slide, and it pulls the drop of blood along behind the angled edge of
glass. A properly prepared thin film should have a significant area
near the end which is only one erythrocyte thick and in which the
erythrocytes show good morphology. The angle and speed of the
spreader slide and the size of the drop of blood will influence the
thickness and size of the film.
Slides with only a thin film can be fixed by being immersed in
absolute methyl or ethyl alcohol for I min and allowed to air dry. If
the thick film is on' one end of the slide and the thin film is on the
other end, the thin film is fixed by a brief flooding or by immersion
in alcohol and allowed to air dry, while the thick film is protected
from alcohol or alcohol fumes. In a well-ventilated area, the slide
may be dried vertically with the thick film up or horizontally after
the thick film is covered with a dry paper towel.
Thick and thin films are best stained with Giemsa stain, as it
provides the most detailed and intense staining of parasites. Wright
stain can be used for thin films but not for thick films, as it contains
alcohol, which will fix the erythrocytes. Wright stain does not stain
parasites as well as Giemsa stain. The staining procedure is outlined
below.
1. 7 .1 Tissue
Biopsy or necropsy tissue may be examined by histology sections
or impression smears.
To prepare impression smears, tissue should be blotted to remove
as much blood or other fluid as possible and then pressed against
glass slides (1 by 3 in.) to make a series of impressions. Tissue
should stick to the slide slightly and leave an irregular film on the
slide. Similar impressions may be made on multiple slices from the
same portion of tissue. Portions of biopsy tissue with different gross
appearances can be used with the impressions from each portion
placed in a longitudinal row. Impressions must be close together,
preferably with slight overlapping to make slide scanning easier.
Impressions from small fragments may be placed in a small area (1
cm in diameter). After being dried, the area with impressions is
circled with a diamond marker to facilitate the location and scanning
of the material. Fixatives and stains appropriate for the parasites
suspected are used. If amebiasis is suspected, impression smears must
be fixed promptly in Schaudinn fixative and not allowed to air dry.
For most other parasites, the slides are allowed to dry before fixation
in methyl alcohol. Giemsa is the usual stain, but other stains such as
Gram-Weigert or hematoxylin may be used depending on the parasite
suspected.
1.7.2 Aspirates of Bone Marrow or Spleen
Aspirates of bone marrow or spleen may be useful in the
diagnosis of infections such as leishmaniasis, trypanosomiasis, and
occasionally malaria. In such instances, Giemsa stains of alcohol-
fixed bone marrow films are most useful. Splenic aspiration is r~ely
performed in the United States because it is dangerous.
1.7.3 Fluids
Fluids such as tissue aspirates, cyst fluid, bronchial washings,
cerebrospinal fluid, pleural fluid, and peritoneal fluid can be examined
directly, or they can be centrifuged and the sediment examined by
wet mounts or stains (or both), depending on the parasite suspected,
as described above for abscesses or tissue.
1.7.4 Skin Snips
Skin snips may be useful in the diagnosis of microfilarial
infections such as onchocerciasis in which the parasites circulate in
the skin and not the blood. A small (2-mm) skin snip is taken with
a needle and a knife. The needle point is stuck into the skin, and
the skin is raised. With a sharp knife or razor blade, the skin is
excised just below the needle. Alternatively, a scleral punch may be
used. The skin snip is then placed in a small volume (0.2 ml) of
saline in a tube or a microtiter well, teased, and allowed to stand
for 30 min or more. The microfilariae migrate from the tissue into
the saline, which is then examined microscopically to demonstrate
the wiggling microfilariae.
1. 7.5 Concentration Procedures for Blood
A number of procedures have been described for the
concentration of blood specimens. Most of these procedures have
been developed to diagnose filarial infections.
The three most widely used methods are membrane filter, saponin
lysis, and Knotts concentration. Procedures for the first two methods
INTRODUCTION 29
will be given here, as these methods are the most sensitive. Membrane
filter techniques use 5- or 3-lLm filters. Both filters give satisfactory
results, but the procedures with the Nuclepore filters do not require
the lysis of erythrocytes. Parasites on filters are often not as suitable
for morphologic study as are those in thick films.
1.7.6 Membrane Filter Concentration for Filariae
1. Collect approximately 7 ml of blood in EDTA.
2. With a syringe and firm pressure, pass 5 to 7 ml of blood
through a 5-lLm Nuclepore filter held in a Swinney adapter.
3. Wash the membrane several times with a small amount of
distilled water or physiologic saline.
4. The moist filter may be e}t'cUtlined directly or fixed and stained
in the usual fashion for a thin blood film.
1. 7.7 Saponin Lysis Concentration for Filariae
The saponin lysis method can be performed on either EDT'\- or
citrate-anticoagulated blood. Saponin solution to lyse erythrocytes is
available in most laboratories for use with automated hematology
instruments.
1. Centrifuge up to 10 ml of blood at 150 x g for 10 min.
2. Remove and discard the plasma.
3. Mix the packed erythrocytes with 50 ml of 0.5% saponin
solution in 0.85% saline.
4. Mix the solution at intervals for 15 min.
5. Centrifuge the solution at 650 x g for 10 min.
6. Decant and discard the supernatant (there should be about 1
ml of sediment).
7. Spread several drops of sediment on a glass slide (1 by 3
in.), and examine two such uncovered wet mounts for motile
microfilariae. Allow the wet mounts to dry before they are fixed
and stained.
8. Prepare four or five similar wet mounts and examine them as
described above. To. each slide, immediately add 2 drops of 1 % acetic
acid solution and mix it well (microfilariae will be killed and
straightened). Allow the slide to air dry.
9. Dip the dried slides in buffered methylene bluephosphate
solution.
10. Rinse the slides in distilled water, and let them air dry.
11. Stain the mounts for 10 min in a 1:20 dilution of Giemsa
stain in buffered water.
12. Examine the slides microscopically.
1.8 STAINING PROCEDURES
1.8.1 Giemsa Stain Procedure
The procedures for staining thick. and thin fIlms differ. Staining
is usually done in a Coplin jar. The ,&tain must be made fresh each
day.
Stain slides with only a thin fIlm as follows.
1. Fix and dry the blood film as described above.
2. Prepare a 1:40 dilution of stock Giemsa stain in neutral buffered
water, pH 7.0 to 7.2 (generally, 2 ml of Giemsa stock plus 38 ml of
buffered water with 0.01 % Triton X-l00).
3. Stain the film for approximately 60 min (the time, which will
vary slightly with different lots of stock Giemsa stain, can be
determined by the staining of leukocytes and erythrocytes).
4. Wash the slide brietly by dipping it in buffered water.
5. Air dry the slide in a vertical position.
Note that, alternatively, a 1:20 dilution for 20 to 30 min may be
used.
Stain slides with only a thick fIlm as follows.
.,1. Do not fix the slide .
2. Prepare a 1:50 dilution of stock Giemsa stain in neutral buffered
water (pH 7.0 to 7.2).
3. Stain the film for approximately 50 min (the optimal time may
vary with different lots of stain).
4. Wash the slide by placing it in buffered water for 3 to 4 min.
5. Air dry the slide in a vertical position.
For combination thick and thin fIlms, the procedure is as follows.
1. Fix the thin film but not the thick film as described above.
2. Stain the film in a 1:50 dilution of Giemsa stain in neutral
buffered water (pH 7.0 to 7.2) for approximately 50 min.
3. Rinse the thin film brietly by dipping it in buffered water.
Wash the thick film by immersing it in buffered water for 3 to 5 min.
4. Dry the slide in a vertical position with the thick film down.
1.8.2 Gram-Weigert Stain Procedure
The Gram-Weigert stain is used to stain the cyst walls of P.
carinii cysts. It also stains fungi and many bacteria. Impression
INTRODUCTION 31
smears, sediment smears, or sections are fixed in methanol and air
dried. For sections, when reagents are added, flood the slide gently
from the end opposite the section, and rinse the slide carefully so
that the tissue is not washed from the slide.
The stain procedure is as follows.
1. Stain the slide with eosin Y for 5 min.
2. Wash the slide with water.
3. Stain the slide with crystal violet for 5 min.
4. Rinse the crystal violet from the slide with Gram iodine
solution.
5. Leave the iodine solution on the slide for 5 min.
6. Rinse the slide with water.
7. Blot the smears carefully (do not blot the sections).
8. Wipe the reverse of the slide.
9. Air dry the slide completely.
10. Decolorize the smear in aniline-xylene, agitating the slide
gently until no purple runs from it (the use of a second Coplin jar
of aniline-xylene after the majority of blue stain has been removed
aids the visual assessment of decolorization).
11. Rinse the slide in xylene.
12. Air dry the slide, add immersion oil to it, and examine it.
P. carinii cysts and fungi stain dark blue and somewhat
irregularly. Cell nuclei may stain blue if they are inadequately
decolorized, but they are not as dark as P. carinii cysts.
1.8.3 Culture Procedures for Blood and Tissue Parasites
Culture procedures have been developed for a number of blood
and tissue parasites, but these procedures are used primarily in
research. The culturing of Leishmania spp. and Trypanosoma cruz;
may be helpful for diagnosis, and the procedures are easy to use.
Biopsy or blood specimens may be cultured for Leishmania spp.
or T. cruzi with Novy-MacNeal-Nicolie (NNN) medium. Biopsy
specimens are ground in a small amount of saline. Biopsies from
skin lesions or other tissues which may contain bacteria may have
penicillin (0.1 ml of 1,000 U/ml) added to the medium with the
inoculum. The inoculum is 1 drop of ground tissue or blood. Incubate
the culture at room temperature (22°C), and at days 3 and 7, examine
a direct mount of liquid from the bottom of the slant at x400
magnification. These cultured organisms are potentially infective for
humans.
1.9 URINE
Urine specimens usually are examined for the eggs of Schistosoma
haemotobium or the trophozoites of Trichomonas vaginalis, although
occasionally the larvae of Strongyloides stercoralis may be found in
patients with hyperinfection syndrome. Urine is the usual specimen
for the diagnosis of Trichomonas infection in males. See below
(Vaginal Material) for culture method. Urine is centrifuged, and the
sediment is examined microscopically.
1.10 SPUTUM
Sputum may be examined to diagnose Paragonimus infection or
hyperinfection due to Strongyloides stercoralis. Occasionally an amebic
abscess or hydatid cyst may rupture, and amebic trophozoites or
hydatid sand, respectively, may be found in sputum. Entamoeba
histolytica must be differentiated from Entamoeba gingivalis, which
may be found in the oral cavity of over 30% of people. Occasionally,
the migrating larvae of ascarids, strongyloides, or hookworm can be
found. Sputum may be examined directly by wet mount or treated
with a mucolytic agent such as Nacetyl-cysteine and then concentrated
by simple centrifugation, with subsequent examination of the sediment.
1.11 VAGINAL MATERIAL
T. vagina lis frequently infects the vagina, and Enterobius
vermicularis adults or eggs occasionally may be found. Direct wet
mounts of vaginal material for typical, tumbling T. vagina lis
organisms are widely used and generally allow the diagnosis of
symptomatic infection, but wet mounts are not as sensitive as culture
methods.
Vaginal material is best submitted as liquid in a tube, although
swabs submitted in a small amount of saline may be used. A drop
of the material is covered with a cover slip and examined with
reduced light. To culture, 1 or 2 drops of urine sediment or vaginal
exudate are inoculated into tubes of warmed, modified Diamond
medium. If vaginal swabs are submitted, the swab is immersed in
the medium and pressed against the side of the tube to express
material. Tubes are incubated at 35°C, and drops of culture are
examined by wet mount at 48 and 72 h for motile trophozoites.
1.12 REFERRAL OF MATERIALS
Few laboratories perform complete parasitological examination,
whereas many perform limited studies, and some perform none. Referral
laboratories may provide services not available in the individual
INTRODUCTION 33
laboratory and can provide consultation on specimens with questionable
laboratory findings. Referral laboratories with a special interest and
competence in parasitology may be found in major cities, university
medical centers, and state public health laboratories. The major national
resource is the Centers for Disease Control in Atlanta, Ga. Specimens
for the Centers must be sent via the state health laboratory, and
appropriate clinical information must be provided. Of course, the
recommendations of the specific referral laboratory should supersede'
these guidelines.
1.13 SAFETY
The parasitology laboratory has infection hazards for personJil.el.
Blood, feces, and other body materials as well as parasite cultures
may be infective. Eggs of Ascaris spp. can survive and embryonate
even in Formalin, and Cryptosporidium oocysts are hardy. In fresh
fecal specimens, the cysts of Entamoeba histolytica and Giardia spp ..
the oocysts of Cryptosporidium spp., the eggs of Enterobius
vennicularis. Taenia solium, and Hymenolepis nana, and the lanae
of Strongyloides stercoralis may be infective. In addition, feces may
contain other infectious agents such as hepatitis A, rotavir~s,
Salmonella spp .. Shigella spp., and Campylobacter spp. Blood and
tissue specimens can be infectious for trypanosomes, Leishmania sp~.,
malaria, and Babesia spp., as well as for non-A, non-B hepatids,
hepatitis B, and possibly AIDS.
TABLE 1.4 : HANDLING OF SPECIMENS FOR REFERRA~
Specimen Handling
Feces, for
Helminths Fix in 10% buffered Formalin.
Protozoa Fix a portion in 10% buffered Formalin and
either fix a portion in PVA fixative or
prepare three Schaudinn-fixed fecal films.
Cryptosporidium spp. Fix a portion in 10% buffered Formalin.
Material from suspected

amebic abscess Place the last material aspirated in a sterile
tube and send it on ice for culture (do not
freeze). Prepare Schaudinn-fixed fecal
films, or fix a portion in PVA fixative.
Obtain serum for serology.
Duodenal aspirate Centrifuge, and remove the supernatant.
Prepare two films from sediment. Fix in
Schaudinn or PYA fixative. Preserve the
remainder of sediment in 10% Formalin.
Urine, for
Trichomoniasis Centrifuge. Cover the sediment with sterile
saline and send it on ice (not frozen) for
direct mounts and culture.
Schistosomiasis Centrifuge entire midday urine. Add an
equal volume of 10% buffered Formalin to
the sediment.
Sputum, for
Nematode larvae or
Paragonimus eggs Break up mechanically or digest I part
sputum plus 5 parts 3% NaOH for 1 h.
Centrifuge, and preserve the sediment in an
equal volume of 10% buffered Formalin.
Amebae Prepare films fixed in Schaudinn fixative,
or fix a portion in PYA fixative.
Blood
Malaria and babesiasis Send unstained and, if available, Giemsa-
stained thick and thin films. Fix thin film
(but not thick) in alcohol before it is sent.
Filariasis Send 5 ml of citrate- or EDTA-anticoa-
gulated blood on ice (not frozen).
Unfixed thick films may be sent in addition.
Send serum for serologic tests.
Trypanosomiasis Send 5 ml of anticoagulated blood as for
filariasis (above).
Send fixed thin films.
Cerebrospinal fluid
Trypanosomes, Send on ice (not frozen).
toxoplasma, leishmania, Send in a sterile container without
trichinella refrigeration.
Free-living amebae
Sigmoidoscopic material Fix films in Schaudinn fixative or mix
material with PYA fixative.
Tissue For impression smears when E. histolytica
INTRODUCTION 35
is suspected, fix in Schaudinn or PVA
fixative. When toxoplasma, leishmania,
Pneumcoystis spp., or Trypanosoma cruzi
is suspected, prepare multiple impression
smears and fix in methyl alcohol.
For surgical pathology, fix the tissue in
buffered Formalin.
Whole worms or
proglottids Wash debris from the specimen and send it
in saline. If there are mUltiple worms or
proglottids, some may be fixed in Formalin.
~eagents such as mercury-containing fixatives may be toxic, and
solvents such as ether may be flammable. These materials must be
handled and discarded properly.
1.14 QUALITY ASSURANCE
The parasitology laboratory must have an up-todate procedure
manual and appropriate reference materials which might include color
atlases or 35-mm slide collections permanently stained glass slides, wet
fecal material containing parasites, and one or more standard reference
books on laboratory methods or general medical parasitology. The
persons performing parasitic examinations must be competent in the
identification of parasites which might be found in patients from whom
they receive specimens. Methods should allow the ready use of outside
consultants, if there is a question of diagnosis. Personnel may maintain
proficiency through participation in formal courses or workshops, review
of self-study sets, and periodic review of known positive materials.
Participation in external survey programs is particularly valuable, as
the performance of the laboratory in the identification of unknown
specimens can be compared with the performance of other laboratories.
If a laboratory is unable to do accurate parasitology because of
either the types of procedures offered or the quality of personnel
available, it should arrange to have specimens appropriately prepared
and submitted to a reference laboratory.
tI
Origin of Microbiology
Before the dawn of civilization in the Mesopotamian regions and
farther east some 7000 to 8000 years ago there was little exact
knowledge of either the causes or nature of natural phenomena.
However, scholarly thinkers and their works were not wholly lacking.
By the time writing and written history had been "invented" 5000 to
6000 years ago 'in Sumeria, Egypt, Syria and adjacent regions, many
keen and ambitious minds in the ancient priesthoods, secular upper
classes and royal families had learned of the medicinal and poisonous
properties of certain plants and of the venoms of certain snakes and
insects. They knew how to exploit nature for political and other
purposes. For thousands of years after the beginnings of civilization
magic, incantation, abracadabra and witchcraft passed for science and
usually also for religion. Even as recently as the Middle Ages (c.
500-1400 AD.) and later in the European Renaissance (c. 1400-1700
A.D.) astrology (aided by imaginative charlatans, with weird grimaces
and impressive passes) passed for astronomy; alchemy (strongly
flavoured with wizardry) masqueraded as chemistry; the most
outrageous quackery was accepted, even by royalty, as medicine.
As always, however, honest, imaginative and inquisitive men here
and there were still capable of analytical and creative thought and
the proposing of working hypotheses to be tested experimentally. They
were sometimes reviled, persecuted and tortured for their supposed
dealings with "The Evil One." Century after century these pioneer
scientists (seekers after experimentally demonstrable truth) began to
establish a system of knowledge based on accurate, purposeful
36
ORIGIN OF MICROBIOLOGY 37
observation; logical inference; imaginative hypothesis; and ingenious
experiments designed to establish indisputable fact or destroy fallacy.
Because of great difficulties in travel and communications, ancient
scholars shared little of one another's learning. As the centuries passed,
exploration began and travel became more common, populations
increased, and vast interminglings of peoples occurred because of
wars and trade. Scientific information thus began to spread from
country to country and, more recently, from continent to continent.
Instead of a few great scholars who were thought (even by themselves!)
to know everything, men began to realize that there were boundless
deserts and plains and illimitable dark forests of ignorance only awaiting
the axe and plow of the devoted researcher to yield rich crops of
wonderful, golden knowledge. Men also realized the awesome truth
that knowledge is power-to create or to destroy utterly. Eventually
scientific thought, experimentation and communications became
permissible and even respectable. They also became incalculably
profitable, and frightening.
Scientists interested in the mysteries of life collected, over the
centuries, a considerable mass of reasonably accurate information
about such living things as could be seen with the naked eye, and
even with "magnifying glasses" (magnifications of about 10
diameters). By 350 B.C. Aristotle and his students had drawn up a
systematic, though limited and (as we now know) often erroneous
classification of hundreds of plants and animals. Accumulating
knowledge of living organisms slowly became arranged into a more
or less orderly system and the study of life was eventually dignified
with a given name: biology (Gr. bios = life; logos = study or
description). Most of biology was at first largely descriptive of
outward form and macroscopic (Gr. makros = large; skopion = to
see) anatomy. These descriptions became the basis of classifications
and taxonomy-major preoccup-ations of most early botanists and
zoologists. Until the seven-teenth and eighteenth centuries chemistry
and physics were almost completely separate fields of study and little
used in biology. Life and living substance were commonly thought
of as mysterious and beyond physical and chemical analysis.
2.1 BEGINNINGS OF MICROSCOPY
Until about 1660 A.D. all knowledge of the form, structure and
life processes of plants and animals was narrowly restricted to what
could be seen with the naked (or very feebly assisted) human eye.
Microorganisms were merely "fabulous monsters." Visual limitations
38 MICRnBIOLOGY AND BIOCHEMISTRY
of the pitiably restricted eye of man had always stood, like an
impenetrable curtain, between man and the fantastic and glittering
cosmos of the microscopic world.
Unaided human vision fails to see objects less than about 100 p,
(0.004 or 11254 inch) in diameter or to perceive as separate objects
TABLE 2.1 SOME LINEAR MEASURES COMMONLY USED
IN MICROBIOLOGY
1 inch = 2.54 cm.
1 cm. 10 mm. 1 mm.:::: 1000 p.
1 p. = 0.001 mm. = 0.00003937 or 1125,400 inch
= 1000 mp.
I mp' 0.001 p. = 10.0 Angstrom (A)
1 A = .0001 p. = 0.0000001 mm. ;:; 1/254,000,000 inch
(i.e., resolve) particles separated by distances less than about 100 p..
Microscopic linear units are shown in Table 1. 1. The development
of practical, relatively high-power microscopy about the middle of
the seventeenth century was like turning on a SOD-watt lamp in a
pitch-dark curiosity shop. It gave men the power to see a universe
of objects, living and inanimate, so minute that their very existence
had never before even been suspected.
2.2 THE FIRST MICROSCOPES
By the end of the thirteenth century simple lenses (magnifying
glasses) had already been used in various ways for many years. Such
lenses, however, did not magnify very highly. About 1590, a Dutch
spectacle maker, Zacharias J anssen, used a second lens to magnify
the image produced by a primary lens. This is the basic principle of
the compound microscope used by every microbiologist today. Galileo
invented an improved compound microscope in 1610. Robert Hooke
(1635-1703) made and used a compound microscope in the 1660's
and described his fascinating explorlttions of the newly discovered
universe of the microscopic in his classic "Micrographia" (1665),
published at request of the Royal Society in London. Although
Hooke's highest magnifications were possibly enough to reveal
bacteria, he apparently made no observations of them. probably
because he studied mainly opaque objects in the dry state by reflected
light, conditions that, as will be explained, are not optimal for
observation of microorganisms. However, his pictures of "white
mould" (probably a Mucor species) are very informative and accurate.
ORlGIN OF MICROBIOLOGY 39
f'iiure 2.1 : Hooke's compound microscope: drawn by himself.

A contemporary of Hooke, and the man mainly responsible for
revealing the hitherto unknown and unseen world of microorganisms,
did not use a compound microscope. He was the Dutch investigator,
Antonj van Leeuwenhoek (1632-1723), a linen merchant by trade
and a man of public and commercial affairs in the city of Delft. He
was nO! a trained scientist but was self-educated, and amused himself
by means of his skill and craftsmanship in glass blowing and fine
metal work. He lived in relatively easy circumstances with leisure
time for his avocation of making minute, simple but powerful lenses.
With these he delighted in examining a great variety of objects: saliva,
pepper decoctions, cork, the leaves of plants, circulating blood in
the tail of a salamander , ~minal nuid, urine, cow dung. scrapings
from the teeth and &0, on. ln' many of these be saw living creatures ,
some of which we now know were protowa and bacteria but all of
which he called "animalcules."
In spite of the fact that his microscopes were not compound he
obtained remarkable results with them.
he showed rare ingenuity and expert craftsmanship in the
grinding and mounting of his simple lenses, a skill which he zealously
kept to himself; and in spite of the requests of his learned friends,
he refused to disclose the secret of his success.
I..eeuwennoek's instruments are not true microscopes at all in
Figure 2.2 : Drawing of "white mould".

the sense in which we ' think of microscopes, but rather simple
magnifying glasses generally consisting of a small, single, biconvex
lens. The object, and not the lens, was moved into focus by means
of screws.
Figure 2.3 : Antonj VllJI Leeuwenhoek .. A fanciful delineation based on a famous portrait.
The picture shows accurately the size and shape of the first microscopes. the manner
in which they were used. and the simple laboratory apparatus of the "Father of
Bacteriology. "
To adjust the lens to the object was so long and tedious a task
that it is not surprising that Leeuwenhoek used an individual lens
for each object.... The magnification varied and at best did not exceed
two hundred to three hundred diameters .... The size of objects which
Leeuwenhock examined was determined by comparison. For this
purpose he used at various times a grain of sand, the seed of millet
or mustard, the eye of a louse, a vinegar eel, and still later hair or
blood corpuscles. In this way he secured fairly accurate measurements
of a great variety of objects . . . . he was forced to admit that the
sand grain was more than one million times the size of one of the
animalcules.
Leeuwenhoek was so interested in the things he observed that,
like Hooke, he wrote minutely detailed reports about them to the
Royal Society in London, beginning in 1674. He was later elected a
fellow of the Royal Society. Some of his observations are at once
quaint and epochmaking. For example, after examining material which
he scraped from between his teeth, he said:
Though my teeth are kept usually very clean, nevertheless when
I view them in a Magnifying Glass, I fmd growing between them a
little white matter as thick as wetted flour; in this substam.:e, though
I could not perceive any motion, I judged there might probably be
living Creatures.
Figure 2.4 : One of Leeuwenhoek's microscopes: front, back and side views. The
tiny spherical or hemispherical lens is held in the slightly raised structure in the upper
part of the metal plate. The object to be examined was mounted at the tip of the
sharp-pointed mounting pin. Focusing was accomplished by means of the three·
thumbscrews to which the mounting pin is attached. These are approximately actual
size.
I therefore took some of this flour and mix it either with pure
rain water wherein were no animals; or else with some of my spittle
(having no Air bubbles to cause a motion in it) and then to my
great surprise perceived that the aforesaid matter contained very small
living animals, which moved themselves very extravagantly. The
42 MICROBIOLOGY ANO:BIOCHEMISTRY
biggest-sort had the .shape of A. Theirnidtron was stron.g -arid nimble;
and they darted themselves through the' water or spittle~ 'as a: JaCK ot
Pike· does' through the water. These' were 'generally not many; 'In
number.· The .second sort had the' shape of B. These sjnm 'abOut like
a top, or took 'a' course sometimes on one side; as is 'shown' at C
arid D: They were more in number than' the first. In the third s~rt I
could n6t well distinguish the Figure, for sometimes It seem'd to' be
an oval, and other times a circle. These were so small they'seem'a
no bigger than E and therewithal so swift, 'that I can compare them
to nothing better than a swarm of Flies or Gnats, flying and tUrning
among one another in a small space. -
cPdA<=>
'."
.',; ,
Figure 2.5 : Leeuwenh;~;~ drawings bacteria. Heii; may be seen cocci, bacilli
and (probably) a spirochet~. The motion of one of the bactli is clearly indicated. Today
such observations are conbnonplace. But l,eeuwenhoek itas seeing them for the first
time in the history of the human race! iwas as momtfuous a discovery as that of
Columbus -a new world! " . ,.!. ... '
,. : Note, that, unlike Hooke, Leeuwenhoek made many of his

observations' by light transmitted through the' object' and that the
ri'lic:too'rganisms were Slispended in vaiiolls f\uids,:nOl ipiInobiliied
oi-'l:)therwise altered by drying. ' ,
2.'3:':, MICROORGANISMS AND':~·' ., ',-c., ..
,:,'<:THE' ORIGIN OF LIFE" ", - ,",.' ( "

>,:i 1'lie"aricients Iffiew nothing of ~ctQOrgani~rits, of-~vQ1Qt[~n,: :6r
of!:ijie 'fut:t 'that only 'living things could beget liyini( tiirog~':' r'tiey'
befieveci, that creatures like frogs, mice, bees 'and 'other ariiiDais -
sprang'
'ORIGIN OF MICROBIOLOGY 43
fully formed from fertile mud, decaying carcasses, warm rain or fog
. and the like. Van Helmont (1577-1644) devised a method for
manufacturing mice. He recommended putting some wheat grains with
soiled linen and cheese into an appropriate receptacle and leaving it
undisturbed for a time in an attic or stable. Mice would then appear.
This observation may still be experimentally confirmed but the
conclusions drawn from it differ today.
Belief in spontaneous generation lived on for years, as it !lad
for centuries. For example, an elderly lady of the writer's early
acquaintance complained bitterly that she had been cheated by a
merchant who sold her a woolen coat which was of such a quality
that it turned entirely into moths when left undisturbed in a closed
for SOme months!
. In the earlier years, in the absence of exact knowledge of
microorganisms· or chemistry, there had arisen much skepticism and
bitter feeling over the question of the origin of life. One "scientist"
who still held to the ancient ideas says of the views of another who
doubted,
So may we doubt whether, in cheese and timber, worms are
generated, or if beetles and wasps in cow dung, or if butterflies,
locusts, shell-fish, snails, eels, and such life be procreated of putrefied
matter which is to receive the forms of that creature to which it is
by formative power disposed. To question this is to question reason,
sense and experience. If he doubts this let him go to Egypt and there
he will fmd the fields swarming with mice begot of the mud of Nylus,
to the great calamity of the inhabitants.
There Was a great deal of such acrid discussion by wordy savants
of the-times, who tried to settle everything by argument. Experimen-
tation was regarded as rather undignified and even smacking of relations
with the devil.
Francesco Redi (1626-1679). The experimental method was,
however, being invoked here and there .. For example, it had always
been· supposed that the maggots in decaying meat were derived
spontaneously from transformations of the putrid meat itself.
Francesco Redi, a physician of Arezzo, questioned this hypothesis.
He placed meat and fish in jars covered with very fine gauze and·
saw flies approach the jars and crawl on the gauze. He saw the eggs
of the flies caught on the gauze and observed that the meat then
putrefied without maggot formation. Maggots developed only when
the flies' eggs were deposited on the meat itself. Obviously the meat
itself did not turn into maggots. Redi's work was not widely noted,
however, and it was not until much later that another series of
experiments was made.
Louis Joblot (1645-1723). After Leeuwenhoek's disc-overy of
microor~anisms it was thought by many who belie-ved in the
Aristotelian doctrine concerning spontaneous generation of life that
animal or vegetable matter contained a "vital or vegetative force,"
capable. of converting such matter into new and different forms of
life. A popular notion was that geese and lambs could grow from
certain kinds of trees. Leeuwenhoek's animalcules were hailed by
many as proof of this. In 1710, Louis loblot observed that hay, when
infused in water and allowed to stand for some days, gave rise to
countless animalcules, or infusoria (bacteria and protozoa). The hay
was thought by some to change into animalcules; anyone today can
observe the development of these for himself. loblot, however, boiled
hay infusion and divided it into two portions, placing one in a
carefully baked (sterilized) and closed vessel, which he heated
thoroughly and kept closed. The other portion was not heated and
was kept in an open vessel. The infusion in the open vessel teemed
with microorganisms in a few days. In the closed vessel no life
appeared as long as it remained closed, thus showing that the infusion
alone, once freed of life by heat, was incapable of generating new
life. spontaneously.
John Needham (1713-1781). Similar experiments carried out by
an English scientist, John Needham, gave conflicting results. Life
developed in Needham's heated closed vessels as well as in the open
unheated ones. He therefore believed in spontaneous generation. We
shall see later that this result was due to insufficient heating which
failed to kill heat-resistant forms of bacteria called spores. But nothing
was known about spores at that time.
Lazzaro Spallanzani (1729-1799). Spallanzani, an Italian
naturalist, published the results of a whole series of the same type
of experiments which disagreed with those of Needham. He showed
that if heating was prolonged sufficiently and the vessels kept closed
to exclude dust and air, no animalcules developed in hay infusions
or in any other kind of organic matter, such as urine and beef broth.
Needham, in reply, said that the prolonged heating destroyed the
"vegetative force" of the organic matter which, he said, was necessary
for the spontaneous generation of life. Spallanzani answered
Needham's objections by showing that the heated infusions in the
closed flasks could still develop animalcules when exposed to air (i.e.,
when microorganisms were introduced with dust).
In 1775 Lavoisier discovered oxygen and the relation between
air and life: This renewed the controversy about spontaneous
generation, the objection to Spallanzani's results being that it was
the exclusion of air (oxygen) from the flasks which prevented the
development of ,life.
Schulze and Schwann. New experiments were perfo-rmed in
which unheated air was admitted freely to the previously heated
infusions of meat or hay, but only after passing through sulfuric acid
or potassium hydroxide solutions (Schulze, 1836) or through very
hot glass tubes (Schwann, 1836), the idea being that the air itself
introduced the germs of life into the infusions. When the infusions
exposed to air so treated failed to develop any life, it was claimed
by others that this was not due to a destruction of any germs of life
in the air by the sulfuric acid or hot glass, but that the "life-giving"
power of the air had been destroyed by these methods, thus preventing
spontaneous generation. ,
Schroder and von Dusch. This objection was oyercome by
Schroder and von Dusch (1854-1861) who performed similar
experiments in which the air Was not heated or passed through acid
or alkali but merely filtered through cotton wool. This method
prevented the appearance of animalcules in the heated broth or
infusions until the vessels were opened. It was therefore apparent
that the method of treatment of the air had nothing to do with the
development of animalcules and that these did not develop
spontaneously, but that there were particles of living matter floating
on dust in the air which not only were killed by heat, acids and
alkalis, but which could be caught and withheld by the cotton wool
alone. The presence of the microorganisms in the cotton wool was
later proved by Pasteur. The experiments of Schroder and von Dusch
were the origin of our present-day use' of cotton plugs for
bacteriological culture tubes and flasks.
In spite of these demonstrations, long and bitter controv-ersies
still raged. Schroder and von Dusch were not convinced by their
own ' experiments and admitted the possibility that spontaneous
generation might occur under natural condi-tions.
Louis Pasteur (1822-1895). Pasteur, one of the most famous
French scientists, was born in Dole, December 27, 1822. Son of a
moderately prosperous tanner who had fought for and been decorated
;(') ) ~t)J:'~(;;i )nl ·i~) ~../i,_)!}i;}
, 46 MICROBI<)L()GY AND BIOCHEMISTRY
"-'r''''-'''-~' -!,: .·~:·~;t.;·t~:1 t:.,.A"J;·.;L.~ j:;fi\;-)} '.i~j-;H{~~~'l:"': ~
o? the'" battlefiel~ .bY" N.~Fle~?~!. ~Mf~pr~,,~~ ~ 1!~fll.1~~-<!4Q!l ifor
hIS father's soldler~y . acco~phsfurit:~ts : l n~ f~a!eL~~ pt9~~9 ~ many
! ofbi§;'beSfscientitk aChievementS bY:
Ilis: p~m9ilc ·~. ~ pi~ .p'<?'yq~
"he W~'~;~itf~re~t' ~t#d~nt:~~i I,~~~~~ami!~,~p~~~~~q,~~~Q!~,
'devdtirlg':?~~. ~~~~e~ '~o ~ ·> ~:~dx,,;~r,~~~~~~,;·:. ~t. ~qYcrf~dJ~e
'-te1l'ition-Shtp .of the st~tebls~omenc forms of ~P-~.tf ;IHY.~f!J~ r~qd
revealed a whole new series of poss~biI!t~es , ~11. R1i:rsif~ ..$.~rN.stry.
,_: ; ':, ;t_C1 i -:,{' ...' ; '.~ '~. ~ '~-¥' '_~'. ,;/"!,-- .IL~~~;(\) j). ,)n .J ..' t .. . ...
~2 ..4 ~ "QI~~A~ES~' OF. ;~S, ;;:jL,J.

'!~ iT 'I',. ','f"ii:~';: :L:::'i
t '.'); 'P'¥';~m:, i p:ow~y:et,' :w~! ~Qt ! one 'to gloat 'over.' such ,successes :1arld
rest qp)~\~'lla~e~'. ' H,e S9.llgQtJor"other.fields -of. ; mVeSlf.Jga~brfj His
, cti()i.ce ,'~~.',guiq~d; ~argely.;[ l)y, patrioti~ ;motiNes (::An <f:iriglisbJrian lh~d
:~-~~~~~·~oJ~:.~.;·;.: ~!"~''';'\':! -,!:: 0_idf 'j/ri ': \ ?Li!'.~~ '.,n.~ L. ~i1fVti!~;:
;, ."; :~9PJ~- ,.ar,e astonisht9 -lll/ France-'wl the '- slile ~of i Fte'n6-J{wm~s
~lwuld WLhaY~k become -,more extend¢d' Dere' {m Ehgfand)"sllice t1(e
t
CO.~r~~!ll ·Dea,tie~ ': ITbe ,:~asoh li'S L>$impl@l, ebo~". /IAi ; fiiSt We
~~g~lj~.Jlw~.med ·those ,[French» 'Witl~Si;) iOt" we,ckdOn ')h~l(f' ) die;; !;'ad
experience that there was ·too much loss occasieried' by tlie'rais'el:i§ies
~~'pllf,w:gl:~ r~Wc)l ~ey,·are subject. .! v;;{~ tid' 'Hll ,;h.mb?
l"l!..; / Ln~, ·~~, :)01rl· .. ~
t~·... t;'lh '_.j:; r.')j{l'.:'" ll1i
. . :JrlI innu . .~ni.~:}UIL;

;j " (1 ! j ' i!1' '. . ~!: i'!: tl,~r: i
Ir '}';I noLc. .1 ..-L .:d

,!' .:/ ~i"'!flh' li.;:>.'.l .;, 1 "")j:-;', ..:·r·i ... r:{ 'Ji! :~c
~~r.c: 2~; ;Pasteur, -in his; J3bo'ratoiiyl' m :'!p3$leUr' ;f~~xa1tJi'niiig'~ s~(iMR cif J~h?J
CfP:? fr~Il?Cf a ,; f~~i9 ; animal.,: The L mateFial ' ra n ,the ! bottblll ';Of ·tJ\~ j~r.i i's
sodium liydroxide (drying agent), :.:~:,' if!," ,Ii;): ,~,:;:i;,~ :r;:::;',:' i!;i i'i
-' ' qe~~:-v~)_a~ tl1~t. t~. ~~,;~; ffi~S1?-J3ttJ~J;r pe~~ ~'~i1i~ce.

~~i 'Pa~tep';~)~}f~~to~~_ t~~ ~~. rnm,cre.;
aG~~c<r~st.W>PVi}l)~IP- llhat
r~;~~~Tt:, l~'i?fd~f.! to 4? i ~p .h.~ , Fpe ~> ,~~: ~Wq~i J;lf ,l?PC;f'i~'"11)fjictufe
and of the ·cause of" ~ow~fi , ~~':§~fl~~~d~q!W~e~ ~d~f;;~.r:-~
wines ." ~s ,a ~e~ul~o~, th~S(!;i stHdi~QRf_~!,j~e~\dl.h l~fr:;{s~~ing
ciJ~~f~~~ns.:J 1i)~'> ~a~. ? at ~.~ . .~Hae$ff~ 9f;s Mfi~ltri9~..~ ~J~Uff9" f!~&94:1
;' . ,I ~~ir\~ ~9.1(;~e .Ois~<~( ~~fts ,p~u,¥.¥! ,by' :Qr~(iJc:~t&'1 ,9f
riiiC'fdst:hp.c'~egetationS £yeastS,- mOlds, bacteria], of which the germs
'ORIGiN OF'MicROBIOLOdv 47
would de~~i~p: -~h~n'icer{ai~ dr'cum~t~nc~~ 'of' temp:r~~~re, -~f
atmospheric variations, of exposure to air, would favour )heir
evolution or their introduction into wines? ... I have indeed re~ched
this result that the alterations ("diseases"1 of wines ate co-eXistent
witIi 'the presence arid multipliCation of microscopic' vegetatlons. '
Pasteur had; found that acid wines, "ropy" wines, bitter wines,
sour beer and so on were caused by the growth in them bf undesirable
contaminating, organisms which produced, these so-called diseases.
The solution of the problem, as later proved 'by Pasteur, lay in
preventing the' growth of foreign orgartisms, '''wild'' yeasts and
bacteria, which caused the 'uridesirable conditions. After' considera15le
experimentation along these liIles he discovered that the wine; did
not spoil in transit if it were held for some '~nutes at a temperatUre
between 50 0 and 6(j°C.' He said, l have '. ascertained that wine was
'neverilltered by th'at preliminary operation (healing), and as nothing
'prevents it afterwards from undergoing . '. . improvement with. age-
it is evident that this process [heating] offers every advantage.
Ris ~l>eriDiems\ were ~'Stlccessftll'Utat !-a 'Practical :test; oftlie
efficacy of his methods was :made, He wrote. to a friend:
.. , . experiments on the heating of wines will be made by the
M~ter of the Navy. Great quantities of heated and of non-heated
wine are to be sent to Gabpn so as to test the process; at, present
our ,cplonial crews h;lVe to ,drink mere vipegar.
~asteur . laid, down· three great principles:
1. Every alteration. either of beer or of wine, depends -on the
devel9pm~llt in. it of 'Jl}icrool1ganisms which are ferments, or ."diseases"
of the beer or wine;' ,.'
.. 2",These "germs or ferments" are btoughtby the'air, by the
ingredients or by the apparatuS used in breweries .
. . 3. Whenever beer 'or, wiriecontains nO living microorg-'anistris 'it
remains unchanged.
2.5 :'PASTEURIZATION"
, - "- ,',
, : IIqbe same "Y~y~, wille$ could be pn:;l!ervedbybeat~g irom

varipus c~ses of alteration, bQtt}.ed beer c~>ulq es«ape the develppment
ofdiseas~ ferlnen~ by being brought to a te~rature of 50· to 55 0
t;,: The 'app1lcation of this pr,ocess soon gave tise to,the neW word.
')Jasteuri?-eiJ" beer, whi~ti"be~e cUI;fent in technicallangQage_, Today"
Pilsteuiization of ,millc(hea~ing at 63°C., for 3Q minutes) .isroutine·.
The heating kilis pathogemc (disease-producrng)' microorganisms.
2.6 PASTEUR ON SPECIFICITY OF DISEASE
Pasteur foresaw the consequences of his studies, and wrote in
his book on beer:
When we see beer and wine subjected to deep alterations because
they have given refuge to micro-organisms invisibly introduced and
now swarming within them, it is impossible not to be pursued by
the thought that similar facts may, must, take place in animals and
in man.
It was obvious from Pasteur's studies that each special kind of
fermentation or disease of beer or wine was the result of the growth
and activity in it of a special, distinct form of yeast or other
microorganism, depending on the type of fermentation or disease
under investigation. This furthered an idea, already old, of the
specificity of biological action, and supported the view that animal
and human diseases also, like different sorts of putrefaction and
fermentation, were each caused by a single, specific type of
microorganism.
2.7 PASTEUR OF SPONTANEOUS GENERATION
After Pasteur's views of the nature of fermentation had been
made public he became involved in the bitter quarrel over the
apparently mysterious appearance of "germs" in fermentable or
putrescible liquids like wine, beer, urine and broth. Pasteur carried
out many ingenious experiments to answer the various objections and
fallacies of previous workers and to show that the animalcules in
spoiled beer and wine were merely descendants of microorganisms
that had gained access to the fluids from dust in the air and that, by
their growth and metabolism, caused fermentation and putrefaction.
First, he redemonstrated that living creatures float in the air attached
to particles of dust. Then he showed, as Schulze and Schwann had
done, that when they could be excluded from vaTious substances such
as sterilized broth and urine, these substances did not ferment or
putrefy. By using flasks with long open necks having several vertical
bends in them, he showed that although unheated, untreated and
unfiltered air communicated freely with the interior, the dust was
caught by gravity in the bends of the neck, and no life appeared in
the infusions. Not until the flask was tilted so that the fluid came
into contact with this dust and was allowed to run back into the
flask, or until the neck of the flask was broken off close to the body,
did growth occur in the fluids. Some of Pasteur's flasks which were
sterile in 1864 have been preserved and are still sterile (if they have
not been destroyed by wars) after over a century!
2.8 MODERN STYLE
In 1864 Pasteur received a prize from the French Academy of
Science for his studies that conclusively disproved the Aristotelian
doctrine of spontaneous generation. Since that time, studies of organic
chemistry have produced a mass of evidence to revive the doctrine
of spontaneous generation in a modem form called chemical evolution.
There is now good reason to believe that life evolved during many
millions of years from combinations of a few elements (chiefly C,
H, 0, N, S and P) by a series of reactions that were compatible
with natural laws (second law of thermodynamics or law of entropy)
and were actually inevitable under what are called "primitive earth"
or prebiotic conditions. These are conditions that are thought to have
existed during the later stages of the formation of the earth over
4,000,000,000 years ago, eons before the Cambrian period that began
as recently as 600,000,000 years ago-practically yesterday,
geologically speaking.
2.9 CHEMICAL EVOLUTION
Prior to 1828 the formation of organic compounds was believed
to be absolutely restricted to living organisms (hence organic). In
1828 Friedrich Wohler, a German chemist, produced urea,
0=C=(NH2)2' an organic substance common in the wastes of many
animals, by the simple process of evaporating an aqueous solution
of ammonium cyanate, 0=C=N(NH4), an inorganic substance. The
old-time distin-ction between organic and inorganic evaporated with
the water from Wohler's solution; Wohler had demonstrated that
"spontaneous generation" of organic substances from inorg-anic ones
could occur. Everyone was naturally excited with the implications
for the spontaneous generation of life. If urea could be made to appear
"spontaneously," what about other organic substances? Could living
substance, or anything like it, be made to appear?
Without recounting the research studies dealing with or
formaldehyde under the influence of ultraviolet and gamma rays.
These sugars are, as will be detailed later, parts of the most important
and absolutely essential materials, ribonucleic and deoxyribonucleic
acids (RNA and DNA, respectively), of all living cells and also of
viruses. Certain purines and pyrimidines, other essential parts of DNA
and RNA, are also reported to have appeared under experimental
50 MICROBIOL~)(,Y AND BIOCHEMIST.RY
Geologic EVOLUlJONARY 'SUCCESSION Of liFE' IQRM$

&os and Epochs , (It. f.w r.pr...ntati•• types)
(Duration of Eras "MOdem" M a n ' '

, in~)
20,OOO(?) yean _____ " . Q.
ta "Space Ag." ~ .' ~
Yeana:,..Epochs ~'_'
o~ ..,
'~S-:AtI*" - ' - '500,000 -

~
I ,000,00() " ,..
"'-* 11,000,000
(fin' "_"?I
MioceM 25,000,000 ~
oao- 040,000,000
'fJlI
~ 70,000,000
Meoozoic &0
(155.000,0001 (
135,000,000
180,000,000 ~ ,( ~ -:: , -,; .' ~
Tria" 225,000.000 '
. I ;
PciI_oic Era " II
(375,000,000) , ' II'
~ ~
nV
270,000.000 .I
~
e'
Carboniferous 310,000,000
Dftonian 375,000.000--...:,' \ ~yll. ~i@
Silurian 0425,000,000 •• • ~ II I'
Ordo¥iclan
Cambrian
0475,000,000
6OO.ooo,OOO(?) ___
~
~ II
I'~
r
"":c=:i:
(m binlan) I
Entirely l,ooo.ooo.ooorn) ~~ I
Speculative 2,OOO,OOO,OOO(??) I ~I I
rifP' Ijfj, O~
Oldest dated' 3,500,000.000
racks
m,ooo,ooo,ooo,m years • Origin of? •
to "Creation" "Lif." •
Figure 2.7 : Geologic time scale (left) showing ancient origin of bacteria and other
microorganisms in pre·Cambrian times (entirely s~culative) at foot of evolutionary
scale. Pictures I, 2, protozoa; 3, 4, bacteria; 5, spi;ochete (protozoa.like bacterium);
6, marine worms; 7, mold·like bacteria; 8, aquatic fungus (Saprolegnia); 9, trilobite
(fossil marine arthropod); 10, 13, bacteria·like algae (Cyanophyceae, desmids); 11,
higher algae; 12, fossil fish; 14, cotylosaur (fossil reptile); 15, Psilopsida, first vascular
land plants (Silurian); 16, Triceratops, 'a dinosaur; 17, cycad tree (Jurassic); 18, fossil
man·like ape; 19, hybrid (1968) daiSies; 20, man in space. *Oldest rocks dated by
modern measurements of radioactivity :
prebiotic conditions: adenine in solutions of amn10nium cyanide, uracil
in mixtures of CH4 , H2 and HP, and soon.
Thus we see actually demonstrated the "spontaneous" formation
of the organic molecules like amino acids, simple sugars, purines
and pyrimidines and a number of others that are the "building stones"
of the enormously complex macromolecules (fats, proteins,
polysaccharides, nucleic acids, corijugates such as apoenzymes,
lipoproteins, respiratory metalloproteins and so on) that make up living
cells.
Figure 2.8 : Reconstruction of a Middle Cambrian sea floor about 600,000,000 years
ago. The fauna includes siliceous sponges (the upright cones), jellyfish, and two genera
of trilobites (Paradoxides, the large form, and Ellipsocephalus, the small form). BacteTla
had probably already been in existence for millions of years.
Unfortunately (or fortunately!) human attempts, even by the most
advanced methods, to combine these "building stones" into complex
macromolecules as they occur in living cells have failed. Some sugars,
some enzyme-like complexes and some genelike structures have been
made synthetically in minute quantities at great cost. The last steps,
the formation of the type of integrated colloidal complexes that are
found in living cells, are still far in the future. (It would be so
profitable if we could cheaply synthesize cane sugar or beef!)
3
Microbiology of Fungi
3.1 CHARACTERIZATION
Dematiaceous fungi are characterized by the development of a
brown-to-olive-to-black color in the cell walls of their vegetative cells,
conidia, or both. This cell coloring results in colonies that are olive
to black. These ubiquitous and cosmopolitan opportunistic pathogens
are normally associated with soil and plants, but occasionally they may
cause ,infections in humans and animals. In medical mycology,
dematiaceous fungi often are thought of as being exclusively
hyphomycetes. This idea is in error because some ascomycetes,
basidiomycetes, coelomycetes, and zygomycetes may be dematiaceous.
Mycotic infections caused by dematiaceous fungi include
chromoblastomycosis, mycetoma, phaeohyphomycosis, and
sporotrichosis. In this chapter, only chromoblastomycosis,
phaeohyphomycosis, and sporotrichosis will be considered.
Sporotrichosis is treated here because the etiologic agent is
dematiaceous in culture, even though the yeast form in tissue is hyaline.
Deciding whether a particular dematiaceous fungus is involved
in the disease process can at times be difficult, since these fungi
occasionally are recovered from clinical specimens as contarni-nants.
Documentation of a dematiaceous fungus as the etiologic agent of a
mycotic infection necessitates sound evidence that the infection is
compatible with a mycosis, that the suspected etiologic agent is seen
in clinical specimens, that the morphology of the fungus in the clinical
specimens is compatible with the suspected etiologic agent, and that
the recovered fungus is properly identified. The repeated recovery
of a suspected etiologic agent, especially from more than one type
of clinical specimen, is highly significant. The recovery of a fungus
52
MICROBIOLOGY OF FUNGI 53
from body sites that normally are sterile is important.
3.2 COLLECTION AND
STORAGE OF SPECIMENS
Clinical specimens must be collected aseptically and then promptly
transported to the clinical laboratory in a properly labeled sterile
container. Specimens collected on swabs or transported to the clinical
laboratory in a transport medium are unacceptable for mycological
study. An adequate quantity of clinical material is necessary if the
information obtained in the laboratory is to be meaningful.
The most frequently submitted specimens for the recovery of
dematiaceous fungi include aspirates, biopsy material, scrapings, and
tissue specimens. Specimens other than skin scrapings must be
protected from dehydration at all times. This protection can be
accomplished by ensuring that a few drops of sterile saline or distilled
water is added to the specimens at the time of their collection. Biopsy
and tissue specimens can be kept moist by placing them between
two pieces of sterile gauze moistened with sterile saline or distilled
w~ter. Clinical specimens should never be placed on cotton pads,
since cotton fibers can be confused with hyphae in the direct
microscopic examination. In addition, it is often impossible to recover
all of the. clinical specimen from among the cotton fibers. Direct
microscopic examination of the specimens and subsequent plating must
be done promptly.
3.3 DIRECT EXAMINATION
Clinical specimens obtained for the recovery of dematiaceous
fungi usually do not require extensive processing. If aspirated'
specimens contain a substantial amount of purulent material, this can
be dissolved with N-acetyl-L-cysteine without sodium hydroxide.
Tissue specimens and biopsy material should be homogenized in a
tissue homogenizer after highly suspicious areas consisting of necrotic,
purulent, or caseous material are selectively, examined microscopically
and inoculated onto isolation media.
Specimens are typically examined microscopically in 10% KOH.
The clearing process can be accelerated by gently heating the KOH
preparation. The dematiaceous nature of fungal elements in clinical
specimens should be determined only by bright-field microscopy.
Phase-contrast microscopy is an excellent method for examining
specimens, but it does not always permit the demonstration of the
dematiaceous nature of these fungi. In some instances, the dark color
of these fungi can be seen in tissue sections stained with hematoxylin
and eosin. However, if a dematiaceous fungus is suspected, an
unstained tissue section should be examined microscopically by bright-
field microscopy. A drop of immersion oil can be placed directly
onto a paraffin section mounted on a microscope slide, and then the
section is examined microscopically.
The etiologic agents of chromoblastomycosis may be filamen-
tous at the surface of the skin. In the deeper subcutaneous tissues,
they occur as muriform cells (sclerotic bodies). Muriform cells are
typically chestnut brown and variable in size, with thick cross walls
arranged in a muriform manner. The cells result from vegetative
growth without the elongation seen in hyphae. The presence of
muriform cells in clinical specimens is diagnostic of
chromoblastomycosis. However, the various etiologic agents of this
mycosis cannot be identified solely on the basis of their morphology
in tissue.
Phaeohyphomycosis is characterized by the presence in tissue of
dematiaceous yeastlike cells, hyphae, or both. The hyphae may be
regular and uniform in diameter or irregular in shape with many
swollen. cells, and they can be either short or very long. The name
phaeohyphomycosis is not meant to be restricted to hyphomycetes,
but it encompasses all dark hyphae causing disease in tissue, regardless
of the taxonomic classification of the etiologic agent. As with
chromoblastomycosis, the etiologic agents of phaeohyph-omycosis
c.umot be identified in clinical specimens. These fungi must be grown
on laboratory culture medium before they can be identified.
Figure 3.1 : Alternaria alternata. Taken by phase-contrast microscopy after 2 weeks on

potato dextrose agar. Bar equals 10 /Lm.
Most mycologists consider it fruitless to directly examine clinical
specimens for the yeast form of Sporothrix schenck;;. The number of
yeast cells in the specimen is typically very limited, and their small
size and shape are not distinctive. The yeast form of S. schenck;; is
not easily seen in sections of tissue stained with hematoxylin and
eosin.
Figure 3.2 : Aureobasidium pullulans. Taken by phase-contrast microscopy after 2

weeks on potato dextrose agar. Bar equals IO /Lm.
The fungus usually can be seen in tissue sections when the
sections first are treated with diastase and then stained by the Gomori
or periodic acid-Schiff technique. Fluorescent-antibody-specific
conjugates are ideal, although they are available only at certain
reference laboratories at the present time. Even though the yeast form
may be difficult to see in specimens, there is generally no difficulty
in recovering this fungus by the cultivation of clinical materials on
media that are routinely used for the isolation of fungi.
Figure 3.3 : Cladosporium carrionii. Taken by phas~:-dlltJ:ast microscopy after 2 weeks

on potato dextrose agar. Bar equals 10 /Lm. • .
3.4 CULTURE AND ISOLATION
Dematiaceous fungi are easily isolated on most routine media.
Some of them are sensitive to cycloheximide, and for that reason,
Sabouraud dextrose agar (2 % glucose) should be used in conjunction
with a medium containing cycloheximide. Most dematiaceous fungi
grow well at 30°C; some species grow poorly or not at all at 37°C.
The majority of the pathogenic dematiaceous fungi usually are visible
on isolation media within a week. However, cultures should not be
discarded as negative until 6 weeks. Once a dematiaceous fungus is
isolated, it must be determined whether or not the isolate is a pure
culture. If the culture is not pure, then the fungus must be purified
by the isolation of hyphal tips or individual germinating conidia. This
Figure 3.4 : Cladosporium bantianum. Taken by phase-contrast microscopy after 2

weeks on potato dextrose agar. Bar equals 10 "m.
purification is extremely important because many of the opportunistic
dematiaceous pathogens are polymorphic; that is, they can produce
several different kinds of conidia in the 'same culture. For an accurate
identification, it must be known whether the various types of conidia
present in a culture were formed by one fungus or by several fungi.
The colony characteristics and microscopic morphology used for
the identification of dematiaceous fungi are based upon cultures that
are approximately 2 weeks old and have been grown at 25 to 30°C on
a medium such as potato dextrose agar or cornmeal agar. These media
usually stimulate the formation of conidia. If a suspected pathogen
cultivated as described above does not produce conidia, exposure to
a naked daylight-type bulb for several days in a 12-hlight, 12-h-dark
cycle while the pathogen is growing on a medium such as 2 % water
agar, sterile wooden sticks, potato dextrose agar, cornmeal agar, or
filter paper may stimulate it to form conidia. The culture also can be
lyophilized and then regrown, a procedure which often stimulates the
development of conidia. Exposure to UV light often stimulates the
development of conidia and other kinds of structures.
Figure 3.5 : Curvularia lunata. Taken by phase-contrast microscopy after 2 weeks on

potato dextrose agar. Bar equals 10 !LID.
It has been suggested that pathogenic and nonpathogenic Cladosp-
orium isolates can be distinguished from each other by their inability
or ability, respectively, to hydrolyze casein, gelatin, or Loeffler serum
medium. Although this test may be of some value for designating a
Cladosporium species as a saprophyte, it cannot be used in place of
morphological studies.
When isolates are believed to be S. schenckii, they should be
subcultured on an enriched medium such as blood agar to determine
whether they are dimorphic; that is, whether they grow vegetatively
as hyphae at 25°C and as yeast cells at 35°C. The conversion from
the mold form to the yeast form is enhanced by incubation of the
inoculated medium in a candle jar.
When temperature studies are conducted, it is important to
concurrently incubate an additional tube of medium inoculated with the
fungus at 25°C to ensure viability of the inoculum. For an isolate to
be considered dimorphic, only a few cells of its typical tissue form need
VI
00
TABLE 3.1: DIAGNOSTIC FEATURES FOR SOME MEDICALLY IMPORTANT DEMATIACEOUS FUNGI
Genus Diagnostic characteristics Comments Selected references
Alternaria Conidiophores dark, septate. simple or Recognized by the distinctive muriform, 3,4,6, ll,
branched. Conidia muriform, obclavate, obclavate conidia with a beak. 12,19
with a beak, darkly pigmented. in simple
or branched acropetal chains.
Aureobasidium Conidiophores hyaline to chestnut brown, Differentiated from Hormonema spp. by the 10-12,19
undifferentiated from hyphae. Conidia production of conidia in a synchronous
borne laterally, hyaline, one celled, oflen manner. Differentiated from ~
producing secondary blastoconidia. Phaeococcomyces spp. by the lack of ?)
~
-5
Large, dark, one- or two-celled, thick- dematiaceous yeast cells.
walled arthroconidia commonly present. t:x:I
0
Cladosporium Conidiophores dark, erect, often septate. C. bantianum grows at 42 to 43°C and 5, 19,21
Conidia one to several celled in some forms long, sparsely branching chains of Cl
species, with dark hila, occurring in conidia from hyphalike conidiophores, -<
fragile, branched acropetal chains. with conidia ca. 6.4 I!m long. C. carrionii >
Z
Conidia at base of chains usually shield grows up to 36°C and forms short, 0
shaped. branching chains of conidia from distinct
conidiophores, with conidia ca. 5 I!m
long.
-g
t:x:I
:I:
trI
Curvularia Conidiophores dark, erect, geniculate due
to sympodial development. Conidia
Differentiated from Drechslera spp. by
possessing conidia which have an
11,12, 19
Table 3. J Contd.
-
3:
~
~
Table 3.1 Contd. ~
Genus Diagnostic characteristic~
multiseptate, usually curved, with
central cell larger and darker than end
Comments
enlarged and darker central cell and
Selected references
narrow septa. cells and thickness of septa and outer

~
o
cell wall approximately the same. 5
Drechslera Conidiophores dark, erect geniculate due Differentiated from Curvularia spp. by 11, 12, 19 Q
to sympodial development. Conidia possessing conidia that are oblong to o'T1
multiseptate, cylindrical to oblong, dark, cylindrical with thickened septal walls,
with septal walls thickened. 2lz
Exophiala Conidiophores hyaline to subhyaline,
hyphalike or distinct. Conidiogenous
cells annellides that are cylindrical to
E. werneckii has annellides reduced to
yeast cells, one to two celled, the latter
predominant, tapering towards the end
10,17-19,23
-
o
lageniform. Conidia one to several celled bearing annellations. E. jeanselmei has

(one species), hyaline to pale brown, cylindrical-to-lageniform annellides
accumulating in balls at the apices of the produced from conidiophores, with some
annellides. Phaeococcomyces annellides intercalary. Growth up to ca.
synanamorph often present. 3TC. E.jeanselmei IS differentiated from
W dermatitidls by lack of the ability to
grow at 40°C and by the development of
annellides instead ofphialides.
Fonsecaea Conidiophores pale brown, usually erect F pedrosoi is differentiated from F 19
swollen apically due to sympodial compacta by the formation of conidia
development. Conidia one celled, pale that are more elongate and in loose
Table 3.1 Contd.
VI
IQ
Table 3.1 Contd. ~
brown; primary conidia function as conidial heads. sympodial conidiogenous cells to
produce secondary conidia. Tertiary
conidia may be formed in the same
manner. Rhinocladiella, Cladosporium, or
Phialophora synanamorphs often are
present.
Phaeococcomyces Conidiophores and hyphae absent. Yeast Often will produce synanamorphs when 8,10, 19 ~
cells one celled, pale brown to black; grown on either cornmeal agar or potato n
pseudo hyphae may be formed. May
occur as a synanamorph associated with
dextrose agar.
~
t::!1
species of Exophiala, Phialophora, o
Phialophora
Wangiella, and other genera.
Conidiophores absent or present, pale P. verrucosa produces flask-shaped 11-13, 19
5
Cl
brown. Conidiogenous cells phialides phialides with cup-shaped, dark, often >-<:
with distinct collarettes. Conidia one deep collarettes. P. parasitica produces >
celled, hyaline to pale brown, phialides of variable length, some ~
accumulating as balls at the apices of the isolates forming extremely long t::!1
phialides. phialides; phial ides often swollen near o
n
base, with prominent encrustations on the ::t:
tI1
-;3
cell wall. Conidia elliptical to cylindrical,
~
often curved. P. repens produces intercalary CIl
Table 3.1 Contd.
>-<:
Table 3.1 Contd. ....("')a;::
Genus Diagnostic characteristics Comments Selected references ~
0
phiCllides without basal septa or phialides
cylindrical to slightly lageniform with a
....0t:tI
delicate collarette. P. richardsiae produces
phial ides of variable size and shape. some
5
Cl
phial ides long with flaring, flattened -<
collarettes. Conidia of two shapes: globose 0
'Tl
conidia from phial ides with flattened 'Tl
collarettes and cylindrical conidia that are c:::
often curved from other phialides. Z
Rhinocladiella Conidiophores pale brown, erect, usually Conidia occur along a rachis. 11,12.19,26
....
Cl
with distinct scars, and sympodial in

development. Conidia one celled,
fusiform to obovate, pale brown, with a
dark basal scar.
Scedosporium Conidiophores hyaline, short or long. Several species of ascomycetes besides 19.22
Predominant conidiogenous cells Pseudallescheria boydii may produce a S.
annellides with slight swelling just apiospermum anamorph.
below the apex. Conidia one celled,
obovate. truncate, subhyaline to light
black. single or in balls.
Scytalidium Hyp!lae produce one- to two-ce\led May occur as synanamorphs with 19,27
arthroconidia, pale brown to brown, Aureobasidium spp.
subglobose to ellipsoidal. H. toruloidea
Table 3.1 Contd 0'1
.-
Table 3.1 Contd.
synanamorph may be produced by some
isolates.
Sporothrix Conidiophores erect, hyaline, and S. schenckii is a dimorphic fungus capable 9,19
sympodial in development. They may be of growing as a yeast form at 37°C and as
apically swollen or geniculate and gently a maid at 25°C. Black aleurioconidia are
tapering. Conidia of two types: one often no longer produced after repeated
celled, hyaline, arising on denticles from subculture.
sympodial conidiophores, and one celled,
thick walled, black, arising laterally
from the hyphae in some isolates.
Wangiella Conidiophores hyphalike, subhyaline to W dermatitidis is differentiated from E. 16, 19,24

pale brown. Conidiogenous cells jeanselmei and similar fungi by the
phialides without distinct collarettes, production of phial ides and the ability to
intercalary or laCeral from hyphae. grow at 40'C.
Phial ides cylindrical with rounded
apices. Conidia one celled, subglobose,
pale brown, occurring as balls that slip
down the conidiogenous cells. Annellidic
and apical sympodial development also
may occur. Phaeococcomyces
synanamorph often present.
to be present; the entire colony does not have to be converted to its
corresponding tissue form.
Figure 3.6 : Drechslera spicifera. Taken by phase-contrast microscopy after 2 weeks

on potato dextrose agar. Bar equals 10 /Lm.
Figure 3.7 : Helm inthosporiurn solani. Taken by phase-contrast microscopy after 2

weeks on potato dextrose agar. Bar equals 10 /Lm.
3.5 IDENTIFICATION
The identification of dematiaceous fungi ultimately rests upon
their microscopic morphology and, to a lesser extent, upon their gross
colonial morphology. The importance of conidium development in
defining the numerous genera of dematiaceous fungi makes it essential
to determine how a particular fungus forms its conidia. For this reason,
slide culture preparations with potato dextrose agar or cornmeal agar
are ideal for identification purposes.
Figure 3.8 : Exophiala jeanselmei. Taken by phase-contrast microscopy after 2 weeks

on potato dextrose agar. Bar equals 10 I'm.
Figure 3.9 : Exophiala moniliae. Taken by phase-contrast microscopy after ;2 weeks

Our new understanding of conidium development has resulted in
the redefinition of many genera of medically important fungi. Terms
such as spore and conidium (plural, conidia) are no longer used
interchangeably. Many mycologists consider spores to be propagules
that arise either from meiosis (ascospores, basidio-spores, oospores,
or zygospores) or by mitosis within a sporangium (sporangiospores).
All other asexual, nonmotile propagules are considered conidia. Conidia
usually occur on specialized hyphae or hyphal branches called
conidiophores. The actual cells that give rise to the conidia are referred
to as conidiogenous cells. The distinction between the various kinds
of conidiogenous cells is important for the identification of species of
dematiaceous fungi. Phialides usually are flask shaped to cylindrical
and have an apex that neither increases in length nor changes in
diameter as the phialoconidia are formed. A cup-shaped structure called
a collarette may be present at the apex of the phialide. In contrast to
phialides, the apices of annellides increase in length, become narrower,
and have apical rings called annellations. The annellations result when
the annelloconidia separate from the apex of the annellide.
Some fungi produce conidia by a blowing-out process. Such
conidia are called blastoconidia. They may occur individually or in
chains. The term acropetal is used when the conidia at the apex of the
chain are the youngest. The term basipetal refers to the condition of
a chain of conidia when the youngest conidium is at the base of the
chain. A number of the medically important dematia-ceous fungi
produce conidiophores that are sympodial. In this type of development,
a conidium is formed at the apex of the conidiophore. The conidiophore
then increases in length by the formation of a new growing point just
below and to one side of the conidium. At the apex of this new
growth, a second conidium develops. The entire process is repeated,
which often results in a conidiophore that has the appearance of a
series of bent knees, which is said to be geniculate.
The term anamorph is used to characterize an asexual reproductive
structure or form produced by fungi. Occasionally, some fungi seen
in the clinical laboratory produce more than one asexual form, or
anamorph. An example of this polymorphic nature is Fonsecaea
pedrosoi, which may form a sympodiaJ anamorph (RhinocladieUa
form), a phial ide anamorph (Phialoph-ora form), and an anamorph
consisting of branched chains of blastoconidia (Cladosporium form).
When a single fungus produces more than one anamorph, the term
synanamorph can be used to designate any of these concurrently
existing forms. The Scytalidium form is often associated with the
pycnidial fungus Hendersonula toruloidae. This form can be referred
to as a synanamorph associated with H. toruloidae. The Rhinocladiella,
Phialophora, and Cladosporium forms are all synanamorphs of F.
pedrosoi.
Figure 3.10 : Exophiala spinifera. Taken by phase-contrast microscopy after 2 weeks

A number of medically important fungi have the ability to
produce sexual forms. The sexual form of a fungus is referred to as
a teleomorph. Pseudallescheria boydii is characterized by the
formation of cleistothecia; hence, it is a teleomorph. P. boyd;; also
Figure 3.11 : Exophiala werneckli. Taken by phase-contrast microscopy after 2 weeks

may produce two anamorphs, that is, Scedosporium and Graphium
forms. The term holomorph is used to encompass the whole fungus.
In this example, the whole fungus consists of the Pseudallescheria,
Scedosporium, and Graphium fom1S. Because fungi are classified by
sexual structures, the name used for the teleomorph also is used for
the whole fungus. Problems occasionally arise with anamorph-
teleomorph connections because a teleomorph may have more than
one anamorph and a single anamorph may be produced by several
different teleomorphs. For example, Scedosporium apiospermum is
one anamorph that is produced by more than one Pseudallescheria
species.
The black yeasts at times are extremely difficult and frustrating
to identify. Black yeasts typically represent one growth form or
anamorph of polymorphic fungi. The genus Phaeococcomyces was
established to accommodate isolates that consisted of black, budding
yeasts with occasional short elements of pseudohyphae, or toruloid
hyphae. The assumption that a black yeast, regardless of whether
the colony is initially dematiaceous or not, should be identified as
Aureobasidium pullulans is incorrect. One of the most frequently
isolated black yeasts in the clinical laboratory is the Phaeococcomyces
synanamorph of Exophiala jeanselmei. When fresh isolates of this
fungus are transferred from Sabouraud dextrose agar to potato
dextrose agar or cornmeal agar, the typical conidiogenous cells and
conidia of E. jeanselmei rapidly become evident. Dematiaceous,
yeastlike co lonies may be formed by such fungi as A. pullulans, E.
jeanselmei, and many other species as well.
Figure 3.12 : Fonsecaea IJedrosoi. Taken by phase-contrast microscopy after 2 weeks

on potato dextrose agar. Bar equals 10 Wm.
Figure 3.13 : Fonsecaea compacta. Taken by phase-contrast microscopy after 2 weeks

Sterile isolates represent a second group of medically important
fungi that are especially difficult to identify. They commonly are
referred to as members of the form-order Mycelia Sterilia. These
fungi have been shown to cause phaeohyphomycosis and mycetoma.
When sterile fungi are isolated, they should be exposed to near-UV
radiation from a black light (310 to 410 nm) for several days in a
12-h-light, 12-h-dark cycle, incubated at both low and high
temperatures, and subcultured onto media such as 2% water agar,
hay infusion agar, soil extract agar, cereal agar, potato dextrose agar,
cornmeal agar, and sterile, moist, wooden applicator sticks or filter
paper. These media may help stimulate the production of conidia or
fruiting bodies. The cultures should be kept for several weeks before
they are discarded. With time, some of these fungi may develop
structures that produce spores or conidia, such as ascocarps, pycnidia,
or synnemata, either in the agar or at the colony surface.
Alternaria spp.
Members of the genus Alternaria occasionally are implicated as
agents of phaeohyphomycosis. These fungi have been associated with
infections involving bone, cutaneous tissue, ears, eyes, and the urinary
tract. An ALternaria sp. and Alternaria aLtemata (synonym, A. tenuis)
are the only well-documented human pathogens in this genus. The
Alternaria anamorph of Pleospora infectoria has been reported to be
a pathogen of humans, but this report has not been convincingly
documented.
MICROBIOLOGY OF FUNGI . 69
Alternaria colonies are rapid growing, cottony, and gray to black.
The erect conidiophores are dematiaceous, simple or branched, and
usually solitary, but they occasionally occur in small groups. The
conidia of Alternaria spp. develop at the apex of the conidiophore
in branching chains, with the youngest conidium at the apex of each
chain. The conidia are dematiaceous, muriform, smooth or rough,
tapering toward the distal end, and. typically with a short cylindrical
beak at their apices.
Alternaria isolates are difficult to identify beyond the generic
level. If an isolate is recovered that must be identified to species, it
should be sent to a specialist.
Figure 3.14 : Phaeococcomyces exophialae. Taken by phasecontrast microscopy after

2 weeks on potato dextrose agar- Bar equals 10 /LID.
Figure 3.15 : Phialophora parasitIca. Taken by phase-contrast microscopy after 2 weeks

3.5.1 Aureobasidium Spp.
Aureobasidium pullulans has been implicated as an agent of
phaeohyphomycosis in humans and other animals. This hyphom-ycete
is capable of causing opportunistic infections and has been reported
from skin, nail, subcutaneous, and deeper tissues.
Colonies of A. pullulans are smooth, moist, and yellow, white,
cream, light pink, or light brown, fmally becoming black due to the
development of arthroconidia. The conidiogenous cells are
undifferentiated from the vegetative hyphae and may be intercalary,
terminal, or arising as short lateral branches from the hyphae. The
conidia are hyaline, one celled, smooth, ellipsoidal, and variable in
shape and size. The conidia develop in a synchronous manner from
the conidiogenous cells. Blastoconidia commonly are produced from
the conidia that arise from the undifferentiated hyphal cells. A
Scytalidium anamorph consisting of dematiaceous arthroconidia
typically is present.
Hormonema species occasionally are confused with Aureoba-sidium
spp. In the genus Hormonema, the conidia arise in a basipetal succession
from either hyaline or dematiaceous hyphalike conidiogenous cells. In
contrast, A. pullulans produces its conidia in a synchronous manner.
Because of the confusion which has surrounded these two genera,
some of the reported cases of infection ascribed to A. pullulans may
have been caused by misidentified isolates of Hormonema spp. This
speculation is based upon the fact that several authors have illustrated
Hormonema spp. under the name Aureobasidium.
3.5.2 Cladosporium Spp.
Cladosporium bantianum (synonym, c. trichoides) and C. carrionii
are the most important pathogenic members of the genus Cladosporium.
C. bantianum is the most frequently reported etiologic agent of cerebral
phaeohyphomycosis, whereas C. carrionii occasionally is recovered
from patients with chromoblas-tomycosis. C. cladosporioides is of
some interest since it was the etiobgic agent of a pulmonary fungus
ball in one patient. This species has been unconvincingly implicated
as a pathogen in eye and nail infections. Occasionally, other
Cladosporium spp. are reported from cutaneous, eye, and nail infections.
Because pathogenesis of C. bantianum other than cerebral involvement
has not been well defined, it would be appropriate to handle this
organism in a safety cabinet.
Cladosporium isolates are rapid growing, velvety or cottony, and
usually some shade of olive gray to olive brown or black. From the
mycelium, erect, tall, dematiaceous conidiophores arise. At the apex
of the branching conidiophore, acropetally branching chains consisting
of one- to several-celled, smooth or rough, dematiaceous blastoconidia
form; the conidia have a dark hilum (basal scar). The conidia at the
bottom of the chains tend to have the appearance of and are
commonly referred to as shield cells.
Figure 3.16 : PhIalophora repens. Taken by phase-contrast mIcroscopy after 2 weeks

C. bantianum and C. carrionii are morphologically similar. C.
carrionii can be distinguished from C. bantianum by its slower growth
rate, shorter conidia (2 to 3 by 4 to 5 JLm versus 2 to 2.5 by 4 to 7
JLm, with some being 3 by 15 to 20 JLm), dermotropic nature in
contrast to the neurotropic nature of C. bantianum, and maximum
growth temperature of 35 to 36°C compared with 42 to 43°C for
C. bantianum. Both of thef\e species may form long chains of
blastoconidia. C. carrionii can hydrolyze casein and starch, whereas
C. bantianum apparently does not have this ability.
3.5.3 Curvularia Spp.
Curvularia geniculata, C. lunata, C. pallescens, C. senegalensis.
and C. verruculosa have been implicated in a number of opportunistic
infections. Members of this genus have caused endocarditis, eye
infections, mycetoma, and pulmonary phaeohyphomycosis.
Curvularia colonies are rapid growing, woolly, and gray to
grayish black or brown. The conidiophores are dernatiaceous, solitary
or in groups, simple or branched, septate, and typically geniculate.
The conidia are two to several celled, usually curved, dark with pale
ends, solitary, and typically with a dark hilum. The conidia develop
from a sympodial conidiophore. Works by Ellis should be consulted
if a Curvularia isolate must be identified to species.
3.5.4 Drechslera Spp.
Several Drechslera species have caused opportunistic infections
in humans, including meningitis and cutaneous, eye" nasal, and
pulmonary infections. The presently recognized pathogenic members
of this genus include an unidentified Drechslera sp., Drechslera
hawaiiensis, D. longirostrata, D. rostrata, and D. spici!fera. Alcorn
has suggested that some of these species should be classified in the
genera Bipolaris and Exserohilum. Additional study is necessary before
this issue can be adequately resolved.
Drechslera species form rapid-growing, woolly, gray-to-black
colonies. The conidiophores are dernatiaceous, solitary or in groups,
simple or branched, septate, and geniculate. The dematiaceous,
oblong-tocylindrical conidia are multicelled and develop from a
sympodial conidiophore.
Figure 3.17 : Phialophora richardsiae. Taken by phase-contrast Illicroscopy after 2

weeks on potato dextrose agar. Bar equals 10 "m.
Some medical microbiologists have confused Helminthosp-orium
spp. with Drechslera spp. The conidiophores of Helmintho-sporium
spp. are straight, and they stop-lengthening when the terminal conidium
is formed. The conidia develop along the conidiophore; hence, the
conidiophore is not sympodial. Helminth-osporium spp. are rarely, if
ever, isolated in the clinical laboratory, and members of this genus
have not caused phaeohy-phomycosis in humans. If it is necessary to
identify Drechslera isolates, either the works of Ellis or a specialist
in this genus should be consulted.
Figure 3.18 : Phialophora verrucosa. Taken by phase-contrast microscopy after 2 weeks

Figure 3.19 : Lecythophora mutabilis. Taken by phase-contrast microscopy after 2

3.5.5 Exophiala Spp.
Exophiala jeanselmei, previously known as Phialophora
jeanselmei or Phialophora gougerotii is a relatively common etiologic
agent of mycotic subcutaneous abscesses. This demati-aceous
hyphomycete may cause either mycetoma or phaeohypho-mycosis.
E. moniliae and E. spiniJera also have been reported as agents of
phaeohyphomycosis, in which they caused subcutaneous cysts. The
last member of this genus known to be pathogenic for humans is E.
werneckii (synonym, Cladosporium werneckii), which causes
superficial phaeohyphomycosis (synonym, tinea nigra).
Figure 3.20 : Lecytbophora mutabilis. Note the presence of chlamydoconidia. Taken

by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10
I'm.
The colonial morphology of the members of the genus Exophiala
is varied. The colonies are slow to rapid growing, often moist and
yeastlike at first, becoming woolly with age, and gray to black. Some
isolates of E. wemeckii and the Phaeococcomyces synanamorph of
E. jeanselmei may remain black and yeastlike. Conidiophores are
dematiaceous, simple, or hyphalike. The conidiogenous cells are
annellides. In E. jeanselmei, the annellides are lageniform to
cylindrical, tapering to a narrow apex; in E. moniliae, they are
inflated to elliptical, tapering to a very long, narrow apex; in E.
spini lera, they are lageniform to cylindrical, tapering to a narrow;
i
apex, and they arise from distinct spinelike conidiophores; and in E.
werneckii, either they are hyphalike or they consist of two-celled yeast
cells that are clavate.
The conidia are one-celled in most species and accumulate in
balls at the apices of the annellides. With careful study utilizing the
oil immersion objective, annellations (rings) usually can be seen at
the apices of the annellides.
Figure 3.21': Lecythophora hoffmannll Taken by phase-contrast rrucroscopy after 2

E. jeanselmei was incorrectly believed by some to belong to the
genus Phialophora. However, when it was discovered that the
conidiogenous cells of E. jeanselmei were annellides and not phialides,
the fungus was transferred to the genus Exophiala. An identical
situation occurred when it was discovered that the conidiogenous cells
of E. spini fera were annellides and not phialides. E. werneckii
produces one- to two-celled conidia from annellides that exist as either
yeast cells or intercalary conidio-genous cells incorporated within the
hyphae.
The fungUs originally described as Sporotrichum gougerotii was
considered a morphological variant of Sporothrix schenckii. The name
S. gougerotii is best considered a nomen dubium.fungi that are
currently identified as either S. gou~erotii or P":',i,?~gerotii are
typically misidentified isolates of E. jeanselmei. At one time, these
two supposedly different fungi were erroneously distinguished from
each other by their tissue morphology. In the sense of some
contemporary medical mycologists, P. gougerotii is a misapplied name
for isolates of E. jeanselmei that do not form granules in tissue.
3.5.6 Fonsecaea spp.
The genus Fonsecaea contains two species, Fonsecaea compacta
(incorrectly spelled compactum by some) and F. pedrosoi. Both of
these species are agents of chromoblastomycosis.
Figure 3.22 ; RhinocIadiella aquaspersa. Taken by phase-contrast microscopy after 2

weeks on potato dextrose agar. Bar equals 10 pm.
Fonsecaea colonies are slow growing, velvety to woolly, and
olive to black. Fonsecaea isolates are extremely polymorphic. They
are charac"terized by the development of one-celled primary conidia
that form on erect, dark, sympodial conidiophores. The primary
conidia in turn become conidiogenous cells and form secondary one-
celled conidia. This form of development has been incorrectly called
Acrotheca-like by some. It is actually more similar to the form seen
in the genus Rhinocladiella. Some of the conidia occur as branching
chains of blastoconidia, identical to those found in the genus
Cladosporium. Fonsecaea spp. may produce phialides with the
collarettes bearing the balls of one-celled conidia that are typical of
the genus Phialophora.
F. pedrosoi and F. compacta are morphologically distinct. F.
pedrosoi is differentiated from F. compacta by its elongate conidia
Figure 3.23 : Scedosporium apIOspermum. Taken by phase-contrast microscopy after

2 weeks on potato dextrose agar. Bar equals 10 I'm.
that occur in loose heads, in contrast to the rounded conidia in compact
heads produced by F. compacta. As a result of their polymorphic
nature, F. pedrosoi and F. compacta have been placed inappropriately
in the genera Phialophora and Rhinocladielia by some mycologists.
3.5.7 Phaeococcomyces Spp.
Phaeococcomyces is a genus that contains black yeasts. Black
yeasts are often synanamorphs associated with several of the medically
important polymorphic dematiaceous hyphomycetes such as E.
jeanselmei and Wangielia dermatitidis. The genus Phaeococcomyces,
which was originally named Phaeococcus, contains four species that
are distinguished from each other primarily on morphological criteria.
Phaeococcomyces exophialae forms slimy, mucoid, slow-growing,
smooth colonies that are grayish black. Budding yeast cells which
are at first subhyaline are abundant. With age, some of the cells
become darker, with thickened cell walls. Some pseudohyphae usually
are present. Hyphal development may become dominant in some
isolates of this species with subsequent subculture.
Black yeasts occasionally are isolated in the clinical laboratory.
They are recognized by their black, mucoid, yeastlike colonies. When
grown on cornmeal agar or potato dextrose agar, many black yeasts
will rapidly produce the hyphae and conidiogenous cells typical of
genera such as Exophiala and Wangielia. Based upon conidiogenesis,
other genera of black yeasts probably will be needed in the future to
accommodate this group of fungi.
Figure 3.24 : Scytalidium Iignacola. Taken by phase-<:ontrast microscopy after 2 weeks

3.5.8 Phialophora Spp.
Members of the genus Phialophora are well-recognized etiologic
agents of phaeohyphomycosis and chromoblastomycosis. In addition
to cutaneous and subcutaneous tissue invasion, some species have
caused endocarditis and mycotic keratitis. The pathogenic species of
Phialophora include Phialophora bubakii, P. parasitica, P. repens,
P. richardsiae, and P. verrucosa.
Phialophora colonies are rapid growing, cottony to woolly, and
usually some shade of olive gray. When conidiophores are present,
they usually are short. The conidiogenous cells are hyaline to
dematiaceous phialides that are cylindrical to flask shaped. At the
apices of the phial ides , distinct collarettes are present. The conidia
are one celled and usually hyaline, and they occur in balls that may
occasionally slip down along the phialides in some species. Some
species commonly produce intercalary phialides; a yeast form may
occur in some isolates.
P. mutabilis and P. hojjmannii have been considered species of
the genus Phialophora for a number of years. Gams and McGinnis
recently have reclassified these species in the genus Lecythophora.
This reclassification was necessary because these fungi produce
intercalary phialides with short, lateral, cylindrical necks, which bear
the conidia in balls at their apices. Collarettes are present at the tips
of the phialides.
3.5.9 Rhinocladiella Spp.
Rhinocladiella aquaspersa, previously known as Acrotheca
aquaspersa, is a rare etiologic agent of chromoblastomycosis. Human
cases of chromoblastomycosis caused by R. aquaspersa have occurred
in Brazil and Mexico.
Colonies of R. aquaspersa are rapid growing, velvety, slightly
elevated, and olive black. The conidiophores are sympodial, usually
darker than the vegetative hyphae, unbranched, and erect. Conidia
are one celled, rarely two celled, fusiform, elliptical or obovate,
smooth, light brown, and with a dark basal scar. Annellides like
those of Exophiala spp. and phialides like those of Wangiella spp.
may be present.
Figure. 3.25 : ScytahdlUm hyahnum. Taken by phase-contrast Il1Icroscopy after 2 weeks

on potato dextrose agar Bar equals 10 j!m.
3.5.10 Scedosporium Spp.
Scedosporium apiospermunl, previously known as MOlloSporiuni
apiospermunl, is an anamorph of Pseudallescheria boydii, a fungus
once classified as Petriellidium boydii and Allescheria boydii. The
fungus may cause mycetoma, as well as infections involving the lungs
and brain, where the fungus grows in the form of hyphae that look
like those produced by Aspergillus spp. S. apiospermum rapidly
produces colonies that are cottony and smoky gray to dark brown.
One-celled conidia may occur singly along the hyphae or in clusters
at the apices of annellides. The conidia are obovate, truncate, and
subhyaline to light black.
S. apiospermum occasionally has a Graphium synanamorph
present. Several members of the genera Pseudallescheria and Petri ella
may produce a S. apiospermum anamorph. Therefore, without having
the teleomorph present, it is not possible to determine if an isolate of
S. apiospermum was produced by Pseu dallescheria boydii.
3.5.11 Scytalidium Spp.
Members of the genus Scytalidium have been well documented
as opportunistic fungal pathogens of nail, skin, and subcutaneous
tissue. The Scytalidium synanamorph associated with the pycnidial
fungus H. toruloidea, as well as Scytalidium lignicola and S.
hyalinum, all have caused disease. S. hyalinum, because of its hyaline
nature, would best be classified in a genus other than Scytalidium.
Scytalidium spp. produce rapid-growing colonies that are at first
white, becoming dark gray with age. In S. lignicola, arthroconidia
of two types are formed. In the first type, the arthroconidia are
cylindrical, one celled, and hyaline. In the second type, they are
thick walled, yellowish brown, and one or two celled. The Scytalidium
synanamorph of H. toruloidea forms arthroconidia that are brown,
cylindrical at first, becoming rounded, barrel shaped or subglobose,
and one or two celled.
The monograph on Malbranchae spp. by Sigler and Carmichael
should be consulted for the identification of Scytalidium species and
similar hyphomycetes that produce arthroconidia.
3.5.12 Sporothrix Spp.
S. schenckii, a dimorphic fungus, is considered the only
pathogenic member of the genus Sporothrix. Recently, a new species,
Sporothrix cyanescens, was added to the genus. Some of the isolates
upon which the species description was based were isolated from
patients with mycosis of human skin. Whether or not S. cyanescens
is another etiologic agent of sporotrichosis remains to be proven.
Colonies of S. schenckii are rapid growing and at first moist,
flat, and yeastlike, later developing aerial hyphae. They are initially
white, becoming brown to black with age. The conidia are of two
kinds in most fresh isolates. Hyaline, one-celled conidia develop
solitarily upon denticles along the hyphae; laterally from sympodial,
slender, tapering, erect conidiophores; and tenninally in clusters at
the apices of swollen conidiophores. The second type of conidia are
one celled, thick walled, and black. These conidia develop along the
hyphae. At 37°C on enriched media, the mold form of S. schenckii
converts to a yeast form.
Figure 3.26 : Sporothrix schenckii. Taken by phase-contrast microscopy after 2 weeks

on potato dextrose agar. Bar equals 10 /tm.
The etiologic agent of sporotrichosis originally was described as
S. schenckii. Later, the fungus erroneously was transferred to the
genus Sporotrichum. Members of the genus Sporotrichum are
characterized by the formation of large hyphae with clamp connections
and large, one-celled, thick-walled, golden conidia. They are neither
dimorphic nor pathogenic for humans and other animals.
Wangiella sp.
W. dermatitidis is an agent of phaeohyphomycosis that typically
causes infections involving cutaneous and subcutaneous tissue. The
fungus most frequently has been seen in patients living in Japan.
The colonies of W. dermatitidis are moist and at first yeastlike,
developing some aerial hyphae with age. They are olive to black.
Distinct conidiophores are absent. The conidiogenous cells are
phialides which do not have collarettes. Some conidiogenous cells
appear to possess a group of slightly raised, truncate denticles at
their apices that occur as a result of sympodial development. Rare
annellides may be produced by isolates of this fungus. The one-celled,
lightto-dark, smooth phialoconidia form in balls at the apices of the
phialides and then slide down their sides. The phial ides develop from
conidiophores that are indistinguishable from the hyphae. Most isolates
produce an abundant yeast form and large amounts of toruloid hyphae.
Figure 3.27 : Wangiella dermatitidis. Taken by phase-contrast microscopy

after 2 weeks on potato dextrose agar. Bar equals 10 JLm.
The genus Wangiella was established to accommodate date the
fungus known as either Hormiscium dermatitidis or Phialophora
dermatitidis. The new genus Wangiella was necessary because the
phial ides without coUarettes that are typical of W. dermatitidis could
not be accommodated in any known genus. W. dermatitidis can be
recognized by its ability to grow at 40°C, whereas similar
dematiaceous hyphomycetes do not grow at that temperature.
4
Microbiology
of Bacteria
Bacteria belong to the class of organisms known as the Schizomycetes
(schizo, fission, and mycetes, fungi). The organisms are single-celled
and reproduce normally by transverse or binary fission.
The class Schizomycetes is divided into ten orders. The largest
order is the Eubacteriales; it includes most of the common bacterial
species.
Bacteria are typically unicellular plants, the cells being usually
small, sometimes ultrarnicroscopic. They are frequently motile. By
means of modem techniques, a true nucleus has been demonstrated in
bacterial cells. Individual cells may be spherical or straight, curved
or spiral rods. Cells may occur in regular or irregular masses, or
even in cysts. Where they remain attached to each other after cell
division, l~ey may form chains or even definite trichomes. The latter
may show some differentiation into holdfast cells and into motile or
nonmotile reproductive cells. Some grow as branching mycelial threads
whose diameter is not greater than that of ordinary bacterial cells,
i.e., about Ill. Some species produce pigments. The true purple and
green bacteria possess photosynthetic pigments much like or related
to the true chlorophylls of higher p~ants. The phycocyanin found in
blue-green algae does not occur in the Schizomycetes. Multiplication
is typically by cell division. Endospores are formed by some species
of Eubacteriales. Sporocysts are found in Myxobacteriales. Bacteria
are free-living, saprophytic, parasitic, or even pathogenic. The latter
types cause diseases of either plants or animals.
Filament Formation. Cells that reproduce and divide in a normal
manner may be induced to grow in filaments by changing the
83
conditions of the medium. According to Webb, "the division of the
bacterial cell follows a complex sequence, which in many respects,
. resembles that occurring in the cellular reproduction of higher forms.
It is now known, for example, that bacterial cell division entails
division of the nuclear element, division of the cytoplasm, secretion
of new cell wall material, and the separation of the daughter cells.
Some or all of the events of this sequence are readily thrown
out of balance, or even completely inhibited. Thus bacteria,
particularly the rod-shaped organisms, may be induced to elongate
into filaments by various treatments which apparently inhibit cell
division but which do not inhibit growth. Such an effect is produced
by various chemical substances, by sub-bacteriostatic concentration
of certain antibacterial agents, as, for example, methyl violet,
sulfonamides, m-cresol, penicillin, irradiation, and higher temperatures
of incubation.
These changes in morphology induced by chemical substances
are usually temporary, since reversion to normal form occurs promptly
when the filamentous bacteria are subcultured in the absence of the
inhibitory agents. Irradiation, on the other band, may give rise to a
temporary or permanent induction of filamentous cells.
From ob~.!rvations such as these the concept has arisen that
bacterial growth, in the sense of an irreversible increase in cell
substance or volume. and cell division may be considered to some
extent as separate anu independent processes; at least, in so far as
growth may occur either with or without the operation of the cell
division mechanism."
Variation in the magnesium (Mg) content of the medium may
exert a marked effect on cell division of some bacteria. In a Mg-
deficient medium, Gram-positive rods grow in the form of long
filaments. Such filaments revert to normal forms when transferred
to the same medium supplemented with suitable concentrations of
Mg. Filament formation is enhanced by the addition of zinc and
cobalt. Inhibition of cell division occurs also in media supplemented
with an excess of Mg.
Deibel et aI. produced filamentous Lactobacillus leichmannii in
the absence of vitamin Bn' Reversion to the normal cell form occurred
on the addition of either vitamin B12 to a medium lacking the growth
factor or of an excess of the desoxyriboside thymidine.
4.1 SHAPE OF BACTERIA
Bacteria exhibit three fundamental shapes: (1) spherical, (2) rod,
-MICROBIOLOGY OF BACTERIA 85
and (3) spiral or curved rod. All bacteria exhibit pleomorphism in
more or less degree under normal or other conditions, but a bacterial
species is still generally associated with a definite cell form when
grown on a standard medium under controlled conditions.
The spherical bacteria (singular, coccus; plural, cocci) divide in
one, two, or three planes, producing pairs or chains, clusters, or
packets of cells. Some are apparently perfect spheres; others are
slightly elongated or ellipsoidal in shape.
The streptococci divide in only one plane. They grow normally
in pairs or chains. Depending upon the species, the distal ends of
each pair may be lancet-shaped, or flattened at the adjacent sides to
resemble a coffee bean.
The staphylococci divide in two planes, producing pairs, tetrads,
or clusters of bacteria, the latter resembling bunches of grapes.
The sarcinae divide in three planes, producing regular packets.
These are cubicle masses with one layer of bacteria atop another.
The rod forms also show considerable variation. A rod is usually
considered to be a cylinder with the ends more or less rounded.
Some rod forms are definitely ellipsoidal in shape. The ends of rods
also show considerable variation. Some species are markedly rounded;
others exhibit flat ends perpendicular to the sides. Gradations between
these two forms may be seen.
Rods may show marked variation in their length/width ratio.
Some rods are very long in comparison to their width; others are so
short they may be confused with the spherical forms.
The shape of an organism may also vary depending upon certain
environmental factors, such as temperature of incubation, age of the
culture, concentration of the substrate, and composition of the
medium. Bacteria usually exhibit their characteristic morphology in
young cultures and on media possessing favorable conditions for
growth.
Young cells are, in general, larger than old organisms of the
same species. As a culture ages, the cells become progressively larger
until a maximum is reached, after which the reverse effect occurs.
Bacterial variations resulting from changes in age are only temporary;
the original forms reappear when the organisms are transferred to
fresh medium.
4.1.1 Size of Bacteria
Bacteria vary greatly in size according to the species. Some are
so small they approach the limit of visibility when viewed with the
light microsco~. Others are so large they are almost visible with
the normal eye. However, the sizes of the majority of bacteria occupy
a range intermediate between these two extremes. Regardless of size,
none can be clearly seen without the aid of a microscope.
A spherical form is measured by its diameter; a rod or spiral
form by its length and width. Calculation of the length of a spiral
organism by this method gives only the apparent length, not the true
length. The true length may be computed by actually measuring the
length of each turn of the spiral. Mathematical expressions have been
formulated for making such computations.
The method employed for fixing and staining bacteria may make
a difference in their size. The bacterial cell shrinks considerably
during drying and fixing. This will vary somewhat depending upon
the type of medium employed for their cultivation. Shrinkage
generally averages about one-third of the length of the cel! as
compared to an unstained hanging-drop preparation. Young cells of
Bacillus megaterium may shrink from 15 to 25 per cent when
transferred from nutrient broth to the same medium containing sodium
chloride in 2 M concentration.
Measurements show some variation depending upon the staining
solution used and the method of application. In dried and fixed
SD'pars, the cell wall and slime layer do not stain with weakly staining
dyes such as methylene blue but do stain with the intensely staining
pararosaniline, new fuchsin, crystal violet, and methyl violet. The
great majority of bacteria have been measured in fixed and stained
preparations. In some instances dried, negatively stained smears have
been used. Therefore, the method employed should be specified when
measurements of bacteria are reported; otherwise the results will be
of doubtful value.
The unit for measuring bacteria is the micron. It is expressed
by the symbol p.. It is 0.001 mm. or 0.0001 cm. A millimicron is
0.001 p. or 0.000001 mm. It is expressed by the symbol m. p
Some bacteria measure as large as 80 p. in length; others as small

as 0.2 p.. However, the majority of the commonly encountered
bacteria, including the disease producers, measure about 0.5 p. in
diameter for the spherical cells and 0.5 by 2 to 3 p. for the rod
forms. Bacteria producing spores are generally larger than the non-
spore-producing species. The sizes of some common species in dried
and stained smears are as follows: Escherichia coli, 0.5 by 1 to 3
MICROBIOLOGY OF BACTERIA 87
p.; Proteus vulgaris, 0.5 to 1 by 1 to 3 p.; Salmonella typhosa, 0.6
to 0.7 by 2 to 3 p.; Streptococcus lactis, 0.5 to 1 p. in diameter;
S. pyogenes, 0.6 to 1 u in diameter; Staphylococcus aureus, 0.8 to
1 u in diameter; Lactobacillus acidophilus, 0.6 to 0.9 by 1.5 to 6
p.; Bacillus subtilis rods, 0.7 to 0.8 by 2 to 3 p., spores, 0.6 to 0.9
by 1 to 1.5 p.; B. megaterium rods, 0.9 to 2.2 by 1 to 5 p., spores,
1 ·to 1.2 by 1.5 to 2 p.; B. anthracis rods, 1 to 1.3 by 3 to 10 p..
spores, 0.8 to 1 by 1.3 to l.5 p..
The most commonly employed method for measuring bacteria is
by means of an ocular micrometer. Measurements may also be made
by using a camera-Iucida attachment and drawing oculars, or by
projecting the real image on a screen and measuring the bacteria.
The same factors that cause variations in the shape of bacteria
also affect their size. With few exceptions, young cells are much
larger than old or mature forms. Cells of B. subtilis from a 4-hr.
culture measure five to seven times longer than cells from a 24-hr.
culture. Variations in width are less pronounced. The organism
Corynebacterium diphtheriae is a notable exception to the rule of
decreasing cell size with age.
Variations in cell size with age are due to a variety of factors.
The major causes appear to be changes in the environment with the
accumulation of waste products. An increase in the osmotic pressure
of the medium will also cause a decrease in cell size and may very
well be the most important factor.
4.2 THE BACTERIAL CELL
Bacteria do not show the same morphological picture. Differences
in structure exist between species. It is generally agreed that a
bacterial cell consists of a compound membrane enclosing cytoplasm
and nuclear material and often containing various granules, fat
globules, and one or more vacuoles. In addition, some species contain
resistant bodies known as spores, and some have one or more organs
of locomotion called flagella.
The term protoplasm is used to indicate the thick viscous
semifluid or almost jelly-like colorless, transparent material which
makes up the essential substance of both the cell body and the nucleus,
including the cytoplasmic membrane but not the cell wall. It contains
a high percentage of water and holds fine granules in suspension.
4.2.1 CytopIasmic Membrane
This membrane appears in young cells as an interfacial fluid film,
becoming thicker and denser as surface-active material accumulates.
It is finally converted into a firm structure. The membrane is believed
to be composed mainly of lipide and protein. Polysaccharide has not
been demonstrated as a component.
The membrane is acid in reaction because of its content of ribon-
ucleic acid. It stains deeply with basic and neutral dyes over a wide
range of pH. The membrane stains Gram-positive in Gram-positive;:
bacteria and acid-fast in acid-fast organisms. It is a semipermeable
membrane and is principally responsible for the Gram and acid-fast
reactions. When a cell is plasmolyzed by immersion in a hypertonic
solution, this membrane is drawn in with the cytoplasmic constituents.
The thickness of the membrane varies even in a single cell. Measur-
ements on a strain of Bacillus cereus at various stages of development
ranged from 5 to 10 mp. in thickness.
4.2.2 Cell Wall
The cell wall is a more rigid structure and is responsible for the
form of the bacterial body. It behaves as a selectively permeable
membrane and apparently plays a fundamental role in the life activities
of the cell.
The cell wall has a low affinity for dyes, which means that it is
probably not stained in some of the usual staining procedures. It is
lightly stained by certain basic dyes such as basic fuchsin and the
methyl violets. Where deep staining of the wall is desired, the use
of a mordant, such as tannic acid, is necessary. The mordant not
only increases the affmity of the cell for dye, but it may increase
the thickness of the wall.
The cell wall accounts for an average of about 20 per cent of the
dry weight of bacteria and represents .the major structural component.
In thickness, it ranges from 10 to 23 mp., depending upon the species.
According to Saltori, chemical analyses of cell walls have revealed
differences in Gram-positive and Gram-negative bacteria. Cell walls
of Gram-positive bacteria are lacking in aromatic and certain sulfur-
containing amino acids, arginine, and proline. On the other hand, cell
walls of Gram-negative bacteria show the presence of aromatic and
sulfur-containing amino acids, arginine, and proline.
Gram-negative cell walls are generally richer in lipides than
Grampositive bacteria.
Cell walls of a number of Gram-positive and negative bacteria
contain the amino acid diaminopimelic acid.
Polysaccharides have been detected in both Gram-positive and
Gramnegative bacteria. The polysaccharide is determined as reducing
substances after acid hydrolysis. Some polysaccharides yielded only
one reducing sugar; others yielded two or more sugars.
Gram-positive organisms gave rhamnose, galactose, and glucose;
glucose only; rhamnose only; arabinose, galactose, and mannose.
Gram-negative bacteria yielded galactose and glucose; galactose,
glucose, mannose, and rhamnose.
In addition, all organisms studied contained an amino sugar or
hexosamine. Generally, the walls of Gram-positive bacteria are richer
in hexosamine than the Gram-negative forms.
Work pointed to the existence in Gram-positive bacteria of a
common basal structure containing the following constituents: a
hexosamine component comprising glucosamine and muramic acid
and sometimes also galactosMIlind; a peptide component made up of
alanine, glutamic acid, and either diaminopimelic acid or lysine with
sometimes also glycine, aspartic acid, or serine; and usually a
polysaccharide containing not more than four different sugar residues.
Other substances may also be attached to the walls, as for example
the protein antigens.
Smithies, Gibbons, and Bayley reported a relatively hig}} nitrogen
content in the walls of several halophilic bacteria which indicated
that the cell material was predominantly protein. They contained only
small amounts of lipides. The cell walls were lipoprotein.
Barkulis and Jones found that approximately one-third of streptoc-
occal cell walls was made up of rhamnose and hexosamine. The
remaining two-thirds was protein in nature.
For more information see Cummins and Harris (1956), Graziosi
and Tecce (1957), Hayashi and Barkulis (1959), Kakutani (1957),
Salton (1956), Strange (1959), Tanaka (1957), Tomcsik and Grace
(1955), Trucco and Pardee (1958), Yoshida et al. (1956).
4.2.3 Capsules
Extracellular material of a "Hmy or gelatinous nature is formed
by many bacteria, especially those producing mucoid growths. This
material may remain firmly adherent as a discrete covering layer on
each cell, or it may part freely from the cells. In the former case it
is known as a capsule; in the latter, as free slime or gum.
Capsules and slime are believed to be distinct from the morphol-
ogical and biochemical point of view. The capsule is a part of the
cell, the slime a secretion. According to Klieneberger-Nobel, capsules
are of definite shape, of more or less definite density throughout,
and of defmite outline, whereas slime envelopes are amorphous and
can be drawn out into manifold structures, are most concentrated in
the vicinity of the bacterial cell~, and decrease in density with
increasing distance from the cell.
Broth cultures of capsule-producing organisms are usually stringy
in texture, and agar colonies exhibit a very moist, glistening surface
which is described as mucoid. Capsule formation is dependent upon
the composition of the medium but especially the variant phase of
the organism. Some disease-producing organisms form large capsules
in culture media rich in animal fluids. Others produce prominent
dapsules when cultures are incubated at low temperatures (4 to
20°C.).
Chemical analyses of capsular material from a number of bacteria
show wide differences in composition. For this reason it is impossible
to make statements which apply to all bacteria. In some organisms
the capsular material appears to be a glycoprotein; in others, a protein-
polysaccharide complex; in still others, a polysaccharide framework
with the spaces filled in by a larger amount of glutamyl polypeptide.
Capsular material is difficult to distinguish from those gums which
flow away from the cells as they are formed. Organisms producing
gums do so when grown in sugar solutions. Some organisms produce
gums only in the presence of a specific sugar; others produce gums
in the presence of anyone of several sugars. In the absence of sugar,
usually very little, if any, gum is formed. Organisms producing gums
of this type are the cause of considerable losses in the sugar industry.
The increased viscosity produced by the gum interferes with the
filtration of the sugar solution.
The species commonly encountered in sugar-cane juice is
Leuconostoc esenteroides. The cells are surrounded by a thick, .
gelatinous, colorless polysaccharide consisting of dextran (glucose
poiymer).
The formation of gums is of common occurrence by soil bacteria.
From 5 to 16 per cent of such forms have been shown to be capable
of synthesizing gums from sugars.
When the cell wall is damaged, the protoplasm usually
disintegrates. However, methods are available for removing the cell
membranes without destroying the vital nature of protoplasm. The
term protoplast is used to indicate living protoplasm exclusive of the
cell membranes.
Action of Lysozyme. Some bacteria are rapidly lysed or dissolved
by the action of lysozyme. Weibull reported that lysozyme possessed
a specific depolymerizing action on- the cell wall and that this appeared
to be the only portion of the cell that was affected by such treatment.
As a result of the destruction of the wall, the -protoplasts were
liberated.
Protoplasmic structures are not very stable. Consequently, if
protective agents were not employed, destruction of the walls was
accompanied by a rapid lysis of the protoplasts, followed by the
liberation of most of the cell protein and nucleic acid in soluble form.
This could be prevented by employment of the enzyme in a 0.2 M
solution of sucrose or can~ sugar. After digestion of the cell walls,
the living protoplasts rounded up into spheres.
Spiegelman, Aronson, and Fitz-James found that digestion of
protoplasts of Bacillus megaterium led to the liberation of nuclear
bodies of the protoplasts. Such bodies were collected by centrifugation
for 5 min. at 10,000 x g.
Properties of Protoplasts. The difficulty in handling and studying
protoplasts is their extreme fragility and sensitivity to osmotic shock,
shaking, centrifugation, and aeration. Removal of the cell wall does
not change the structure and capabilities of the protoplasm.
Permeability, respiration, and spore formation appear to be the same
for protoplasts and intact cells. Also both can support the development·
of bacteriophages. Under special conditions the protoplasts grow and
probably divide like intact cells. However, there is no evidence that
protoplasts form colonies.) The metabolism of protoplasts and intact
cells appear to be very similar but probably not identical.
Filterability of Protoplasts. Sinkovics reported the spontaneous
occurrence of units in aged cultures of Escherichia coli which
conformed to the description of artificially induced bacterial
protoplasts. The disintegration of cells from aged cultures was
preceded by swelling of the cell and rupture of the rigid cell wall.
Centrifugation of the culture gave a supernate which, after filtration,
contained units capable of regeneration when placed in fresh medium.
The smallest units capable of regeneration measured about 350 mls
in diameter. The units underwent fusion before cell-wall formation
occurred.
The results supported the assumption that aged E. coli cultures
could survive in the form of units having no cell walls and which,
under adequate conditions, regenerated into vegetative forms.
4.2.4 Polysaccharide Structures
P'Olysaccharides occur (1) in cell walls, (2) extracellularly in
capsules and gums, and (3) inside 'Of bacterial cells. The first tW'O
have already been discussed.
Penningt'On (1949) revealed the presence 'Of polysaccharides by
treating bacteria with sodium metaperiodate f'Oll'Owed by staining with
sulfitedec'OI'Orized basic fuchsin. In Bacillus cereus the polysaccharide
was c'Oncentrated in the cyt'Oplasmic membrane as well as in the cell
wall.
Selective staining 'Of P'Olysaccharide in the cell is said t'O depend
uP'On the 'Oxidizing acti'On 'Of periodate 'On such chemical c'Onfigurati'Ons
as a, 13 glyc'Ols and a-hydroxyket'Ones. P'Olyaldehydes generated by
this selective 'Oxidation react with sulfite-dec'OI'Orized fuchsin.
P'Olysaccharide areas in the cell are c'OI'Ored red by the stain.
4.2.5 Nucleus
The questi'On 'Of the presence 'Of a well-defined nucleus in bacteria
has been the subject 'Of investigati'Ons by bacteri'OI'Ogists almost from
the beginning 'Of bacteri'OI'Ogy.
a <l)
b <Jl)
c ClJ) ~e'
d ~IU)
e<UJIV
f'~lIrTi)
Figure 4.1 : Escherichia coli. Diagram of successive division stages of the chromatinic
bodies from the beginning of the lag phase, after transfer to a fresh nutrient medium,
to the first division of the growing organism. c-c' and c-f are alternative modes of
development, c-f being that most commonly followed.
Some 'Of the earlier cyt'OI'Ogists maintained that bacteria were very
primitive 'Organisms dev'Oid 'Of nuclei and c'Onsisting simply 'Of
cyt'Oplasm, granules, and vacu'Oles. This view was based 'On their
failure t'O 'Observe a nucleus in a bacterial cell. Others held the view
that the nuclear material was present in a diffuse f'Orm thr'Ough'Out the
cyt'Oplasm. Still 'Others believed that the wh'Ole cell sh'Ould be regarded
as a "naked nucleus," corresponding to the nucleus of higher
organisms. The naked nucleus is regarded as a primitive form of
living matter. Since bacteria have the structural and physiological
attributes of true cells, this concept cannot apply to these organisms.
Much of the confusion was caused by the inadequacy of the
staining procedures. By means of the HCI-Giemsa staining technique
of Piekarski, many observations have been reported demonstrating
the presence of chromatinic structures in bacteria.
Robinow prepared wet smears of Escherichia coli. Slides were
fixed in osmic acid vapor, dried, and immersed in normal HCl for
about 9 min. at 53 to 55°C., then washed and stained in 1:20 Giemsa
solution for 10 to 60 min., depending on the staining properties of
the specimen.
The chromatinic structures in E. coli from old cultures were too
small to be resolved accurately. After transfer to fresh medium the
chromatinic structures increased in size and gave rise to short, often
dumbbell-shaped rods or chromosomes, which multiplied by splitting
leQgthwise in a plane more or less parallel to the short axis of the
cell. A single cell of E. coli contained one chromatinic body or one
or two pairs of these representing primary and secondary division
products.
The Smith technique consisted of fixing the smear in osmium
tetroxide vapor, immersion in HCI, mordanting in dilute
formaldehyde, and staining with aqueous basic fuchsin. The method
was said to possess certain advantages over the procedure of Robinow.
Another cause of confusion in the recognition of nuclear structures
in bacteria was the lack of appreciation for the ages of the cultures.
At certain times nuclear structures cannot be seen. In general, most
of the early observations of tidy, intelligible "nuclei" were made on
preparations from very young cultures, whereas haphazardly scattered,
unintelligible granules of chromatin were persistently observed in
preparations made from cultures of the same bacteria beyond the
logarithmic growth phase.
Recognition of the fact that the configuration of nuclear material
in bacteria might change with age removed one of the chief causes
of confusion. As Dobell said (1911), "My own belief is that the
nucleus in bacteria may display not one but many forms during the
whole life cycle. Many of the nuclear structures which have been
shown to exist in these organisms shOUld, I think, be regarded as
temporary states rather than aspermanent conditions. The different
'.
results which have been reached by different workers when working,
apparently, upon the same species, may to some extent find an
explanation in this circumstance.»
The chromatin bodies, according to Robinow, appear to have
the following properties:
They are simple structures of relatively low density, not markedly
basophilic but reacting positively in the Feulgen test. Normally they
lie separately in the cytoplasm, and all those in one bacterium are
homologous. Changes in the balance of ions in the cytoplasm may
cause the aggregation of several chromatin bodies into a single
continuous structure. This effect is reversible. Growth and division
of chromatin bodies are attended by changes of form only, not by
visible changes of texture. In the simplest type of body, that which
in profile looks like a bar or dumbbell, division begins at one end
and causes the successive appearance of V-, U-, and H-shaped phases.
The chromatin structures of certain bacteria are netlike or spongelike,
and their mode of division is not easily imagined.
Direct division of the chromatin bodies of E. coli has been
demonstrated by Mason and Powelson in a series of remarkable
phasecontrast photomicrographs. These observations were made on
living bacteria. The nuclear areas in the dividing cells appeared to
be as clearly defined as the areas in fixed, hydrolyzed, and stained
cells.
Vacuoles. Vacuoles have been identified in young bacteria. They
are cavities in the protoplasm and contain a fluid known as cell sap.
As the cells approach maturity, some of the water-soluble reserve
food materials manufactured by the cell dissolve in the vacuoles.
Insoluble constituents precipitate out as cytoplasmic inclusion bodies.
4.2.6 Metachromatic Granules
The best-known inclusion bodies in bacterial cells are known as
volutin or metachromatic granules. The granules are small in young
cells and become larger with the age of the culture. They are believed
to originate in the cytoplasm of young cells and to localize in the
vacuoles of mature forms. The granules show a "Strong afrmity for
basic dyes, indicating that they are acid in character. They are usually
considered to be a reserve source of food.
Grula and Hartsell found the granules to be composed of
metaphosphate or another form of inorganic phosphate, some fat,
and possibly small amounts of protein. Their presence and size in
cells were related to the phosphate concentration of the growth
medium in the presence of an energy source and specific divalent
ions (Mn and Zn). Older cells possessed larger granules. Their
basophilic nature did not depend on either ribonucleic acid (RNA)
or desoxyribonucleic acid.
On the other hand, Widra found metachromatic granules to
contain protein-bound lipide, RNA, and polyphosphates.
4.2.7 Fat Globules
Bacteria are capable of storing fat in the form of globules. Fat
globules may be demonstrated in 24-hr. cultures and usually reach a
maximum in about 48 hr.
Some cells may contain only one large globule; others may show
the presence of a number of small, scattered globules.
Figure 4.2 : Composite representation of a metachromatic granule.

It is generally believed that fat is stored as reserve food material.
Globules usually cannot be demonstrated in young, vigorously growing
cells. As cells age and slow down in activity, fat globules appear in
the cytoplasm and may be recognized by appropriate staining.
4.2.8 Motility
Bacterial motion is generally associated with the presence of
organs of locomotion known as flagella (singular, flagellum). They
were first observed in stained preparations by Cohn. The presence
of flagella does not mean necessarily that the organisms are always
motile, but it indicates a potential power to move.
Independent bacterial motion is a true movement of translation
and must be distinguished from the quivering or back-and-forth motion
exhibited by very small particles suspended in a liquid. This latter
type of motion is called Brownian movement and is caused by the
bombardment of the bacteria by the molecules of the suspending fluid.
4.2.8.1 Properties of Flagella
Flagella are very delicate organs and easily detached from the
cell. In the stained condition they are long, slender, undulating organs.
They are directed backward to the direction of motion at an angle
of about 45 0 • Reversal of direction occurs by swinging the flagella
through an angle of about 90°. Turning movements take place, by
swinging the flagella forward on one side only. They propel the
organism by a spiral or corkscrew motion.
The thickness of flagella varies from species to species. In Proteus
vuLgaris they measure about 12 mJL. This figure is considerably below
the shortest wave length of visible light and explains why flagella
cannot be seen in hanging-drop preparations or in smears stained by
the usual simple procedures. When special staining methods are
employed, sufficient dye becomes deposited on the flagella to make
their diameters greater than the wave length of visible light. They
may then be seen under a light microscope.
4.2.8.2 Chemistry of Flagella
Flagella and bacterial bodies differ in composition. Flagella break
up on boiling or when exposed to pH values below 4 or above 11.
Their composition is largely protein, having a molecular weight of
about 41,000. Weibull found the flagella of P. vuLgaris to be
composed of 98 per cent protein, traces of carbohydrate and fat,
and no phosphorus. The protein contained only 14 known amino
acids. It is an incomplete protein, lacking in some of the essential
amino acids.
It is well established that the H antigens of bacteria are associated
with the flagella and the 0 antigens with the bodies. Purified flagella
are agglutinated by H antiserum but not by 0 antiserum; 0 antigens
are not agglutinated by H antiserum. This is another indication that
flagella and bacterial bodies differ in composition.
4.2.8.3 Origin of Flagella
Some believe flagella originate from the cell wall; others believe
they traverse the cell wall into the protoplasm.
Flagella differ chemically both from the cell wall and the
protoplasm.
Two observations have been made relative to the site of origin
of flagella.
Electron micrographs by van Iterson and others show the flagella
penetrating the faint outer zones and extending into the cytoplasm.
If the outer zone is the cell wall, then the flagella have their origin
in the cytoplasm.
Weibull showed that removal of the cell wall of some bacteria
by means of lysozyme produces a spherical protoplast which still
retains the flagella of the treated cell. The obvious conclusion is that
flagella have their origin in some cell structure deeper than the cell
wall.
4.2.8.4 Number and Arrangement of FlageUa
The number and arrangement of flagella vary with different
bacteria, but they are generally constant for each species. Some have
only one flagellum; others have two or more flagella.
In rod-shaped cells the flagella arise either at one or both poles,
or are distributed laterally with the poles being generally bare. In
some species flagella are located both laterally and at the poles. A
species may show considerable variation in the number and
arrangement of flagella. Single Alcaligenes cultures may contain forms
with a polar flagellum only; some with several lateral flagella; and
some with both lateral and polar flagella. Sometimes a species may
show cells which are flagellated in one environment and nonflagt'llated
in another.
Leifson, Carhart, and Fulton reported the presence (\f four definite
types of curvature in the flagella of Proteus vulg;lris. Individual
organisms may have more than one type of flagella, and individual
flagella may have one or two types of curves.
Environmental factors, particularly pH, may change the curvature
of the flagella on some strains, but not on all strains. In acid media
the curly curvature tends to predominate; in alkaline media the normal
predominates.
Organisms have been classified on the basis of the number and
arrangement of flagella as follows:
Monotrichous-a single flagellum at one end of the cell.
Lophotrichous-two or more flagella at one end or both ends
of the cell.
Amphitrichous-one flagellum at each end.
Peritrichous-flagella surrounding the cell.
4.2.8.5 Staining of Flagella
The staining of flagella is a difficult technique, especially in the
hands of the beginner. For this reason many methods have been
proposed. Regardless of the method employed, the film must first
be treated with a mordant to make the flagella take the stain heavily.
Mordants consist usually of a mixture of tannic acid and some metallic
salt. In some methods the mordant and stain are applied separately;
in others they are combined in one solution.
Boltjes came to the following conclusions on the staining of
bacterial flagella:
Four factors at least influence the results of staining-the skill of
the investigator, the organism studied, the culture medium on which
the organism was grown, and the staining method. Of these the first
is perhaps the most and the last the least important. The importance
of skill is shown by the repeated failure of students to stain flagella
although their teacher has no difficulty in demonstrating flagella at
the same time and with the same suspension. Further, one usually
has success with a new formula only after a number of trials; first
attempts to stain an unknown bacterium are often a failure. Another
point is that as a rule flagella can be clearly seen only in a rather
small part of the preparation. The different colours of the stained
bacteria show clearly that during the staining process conditions are
not everywhere alike, and since many flagella are tom off during
drying, we need not wonder that in most cases only a few bacteria
are successfully stained. Notwithstanding all this, it is certain that
when one has had some experience with the staining technique, the
results are so consistent that there will never be any confusion between
bacteria with true polar flagella, such, for example, a, Pseudomonas
fluorescens or Vibrio comma, and peritrichous bacteria like Proteus
mirabilis and Salmonella typhosa. I therefore consider flagella staining
to be a reliable procedure.
Photomicrographs of the arrangement of flagella on bacteria,
according to Leifson.
4.2.8.6 The Pijper Theory of Motility
In a series of investigations Pijper et al. questioned the belief
that flagella are responsible for motility. He added methyl cellulose
to a culture of a motile organism to increase the viscosity of the
medium. This treatment decreased the motility of the cells. Under
these conditions the cells exhibited a gyratory undulating movement
like other aquatic creatures. He concluded that flagella were not
organs of locomotion but only artifacts-useless appendages,
polysaccharide twirls-the result, not the cause, of bacterial motility.
To quote, "That motile bacteria always exhibit a gyratory undulating
movement was confirmed by making a slow motion cinemicrographic
film of fast-swimming bacteria in br9th, and also by examining the
same bacteria at lower temperatures, which reduced their speed.
This spirillar motion of bacteria is sufficient to propel them, and
there is no need to invoke special motor organs like flagella. There
is no evidence to show that the flagella-like appendages of bacteria
act as motile organs-in fact all the evidence when critically examined
points the other way.
Analysis of the structure of bacteria excludes the possibility that
tails, "flagella,» or the thin wavy threads are live organs, or that
they are in direct communication with the living parts of the cell.
There is no evidence from either electron pictures or stained
preparations that it is otherwise.
Not only does the visible gyratory undulating movement of motile
bacteria satisfy all requirements for locomotion, but it is possible
for bacteria grown under special conditions to swim in this fashion
without showing tails or other supposed motor organs.»
Notwithstanding the findings and conclusions of Pijper, evidence
at present appears to be overwhelmingly in favor of flagella as organs
of locomotion.
As has already been stated, Weibull reported the flagella of P.
vulgaris to be composed of 98 per cent protein. Thif. contradicts
Pijper's statement that flagella are formed from the carbohydrate slime
layer that is peeled off into a number of thin, wavy threads.
Several investigators, among them Labaw and Mosley, by means
of electron microscopy, demonstrated the presence on Brucella
bronchiseptica of uniform flagella having an external contour of a
counterclockwise or left-handed triple helix. The average periodicity
along the length of the flagella waG 19 mIL, with an average diameter
of 13.9 mIL.
4.2.9 Motion of Colonies
Several organisms have been described which exhibit colonial
motility when grown on a solid medium.
Shinn prepared lapse-time motion pictures of individual colonies
of Bacillus alvei grown on agar plates and measured their velocities.
The linear motions of colonies measuring 0.2 to 0.5 mm. in diameter
averaged about 14 mm. per hr. Comparing this figure with the speed
of individual cells of other species of motile bacteria gave the
following results:
Salmonella typhosa 65 mm. per hr.
Bacillus megaterium 27 mm. per hr.
B. alvei (colonies) 14 mm. per hr.
The colonies eXhibited not only linear motion but also a slow
rotary movement. The direction of rotation of 200 to 300 colonies
observed was counterclockwise, with the exception of two colonies
in which it was clockwise.
Turner and Eales reported that the rotation of an aerobe occurred
very early during growth. The cells segregated in small groups and
aligned themselves concentrically around a common center to form
disk-like plaques one or a few cells thick. The rate of rotation was
greater in smaller groups. As multiplication continued, successive
layers were gradually built up in terrace fashion and th colony grew
in height. Teh colonies then began to migrate. When a colony
migrated, it left a peculiar "track" on the surface of the agar. A
small number of cells were left behind, mostly at the edges of the
track, which formed two parallel lines separated by the width of the
moving colony.
F"JgUI"e 4.3 : Sketch of convoluted track of a wandering coony. showing two series of
clockwise spirals followed by a final counterclockwise spiral. The colony had increased
considerably in size after coming to rest and showed curved radial markings indicating
rotation. The total length of the track was about 2 cm.
Typical migrating colonies pursued curved or spiral paths which
were often very elaborate and of relatively great length, even 2 or 3
-em.. The direction of rotation was either clockwise or counter
clockwise. After wandering for a variable distance, a colony approached
the center of its spiral path with rapidly shortening radius, ceased to
migrate, began to rotate around its center, lost its elongated shape,
and increased in size to several times the width of the track at the end
of which it was formed.
Endospores. Endospores are bodies produced within the cells of
a considerable number of bacterial species. They are more resistant to
unfavorable environmental conditions, such as heat, cold, desiccation,
osmosis, and chemicals, than the vegetative cells producing them.
However, it is debatable if such extreme conditions -actually occur in
nature. For instance, the resistance of spores to high temperatures is
a laboratory phenomenon and probably never occurs in a natural
environment.
The bulk of evidence indicates the existence of a close relationship
between spore formation and the exhaustion of nutrients essential for
continued vegetative growth. Sporulation is a defense mechanism to
protect the cell when the occasion arises.
Spore formation is limited almost entirely to two genera of rod-
shaped bacteria: Bacillus (aerobic or facultatively anaerobic), and
Clostridium (anaerobic or aerotolerant). With one possible exception,
the common spherical bacteria do not sporulate. Some spore-bearing
species can be made to lose their ability to produce spores. When
the ability to produce spores is once lost, it is seldom regained.
SporMation is not a process to increase bacterial numbers because a
cell rarely produces more than one spore.
4.2.9.1 Morphology of Spores
Spores may be spherical, ellipsoidal, or cylindrical in shape. The
position of the spore in a cell may be central, subterminal, or terminal.
A fully grown spore may have a diameter greater than that of the
vegetative cell. This causes a bulging of the cell. The resulting forms
are known as clostridium if central, and plectridium if terminal. As
a rule, each species has its own characteristic size, shape, and position
of the spore, but this is subject to variation under different environm-
ental conditions.
Franklin and Bradley, by means of electron microscopy of carbon
replicas, reported that the spores of a majority of species of Bacillus
and Clostridium are readily distinguished by surface patterns. The
surfaces may be smooth or ribbed, with the ribs usually longitudinal.
Figure 4.4 : Surface structure of a spore of Bacillus polymyxa. From left to right:
side vIew; same rotated a quarter turn from right to left; same rotated a further quarter
turn; view,of a pole.
The sculpturing consists of a single endless ridge in the form of
two loops, similar to the marking on a tennis ball, together with
two other separate ridges terminating within the loops. An electron
micrograph of ultrathin sections of such spores, by van den Hooff
and Aninga. The spore coat consists of an outer and an inner layer
separated by a space. The outer layer is sometimes called the exine
and the inner layer the intine. The intine faintly follows the surface
relief. The central core is separated from the intine by a regular
nonosmophilic space. A peripheral spot may be observed in the core
which probably represents nuclear material.
4.2.9.2 J»arasporal lrodies
When sporulation of Bacillus laterosporus is complete, the spores
are cradled in canoe-shaped bodies. According to Hannay (1957):
"On sporulation the slender vegetative rods swell and fonn larger
spindle-shaped cells in which the spores are fonned. When the spores
mature they lie in a lateral position cradled in canoe-shaped parasporal
bodies which are highly basophilic and can be differentiated from
the surrounding vegetative cell cytoplasm with dilute basic dyes. On
completion of sporulation the vegetative cell protopla~m and the cell
wall lyse, leaving the spore cradled in its parasporal body. This
attachment continues indefinitely on the usual culture medium and
even persists after the spores have germinated. In thin sections of
sporing cells the bodies are differentiated from the cell protoplasm
by differences in structure. Whereas the protoplasm has a granular
appearance, in both longitudinal and cross-sections the parasporal body
comprises electrondense lamellae running parallel with the membranes
of the spore coat and less electrondense material in the interstices of
the lamellae. The inner surface of the body is contiguous with that
of the spore coat as if it were part of the spore, rather than a separate
body attached to the spore. The staining reactions of the parasporal
body are not consistent with those of any substance descred in
bal'ieria. "
4.2.9.3 Composition of Spores
Ross and Billing, by means of refractive index measurements on
spores and vegetative cells of B. cereus, B. cereus var. mycoides,
and B. megaterium, found the values to be very high and comparable
with that of dehydrated protein. This suggested that they contained
much less water than the vegetative cells.
Strange and Dark demonstrated the presence of a hexosamine
containing peptide in the spore coats of B. megaterillm and B. subtilis.
The breakdown of an insoluble peptide complex might well be one
of the first steps of the germination process. It was believed that the
release of the hexosamine-amino acid complex was the result of the
action of lysozyme present in the spores.
4.2.9.4 En~mes of Spores
The presence of enzyme systems in Bacillus spores has been
reported. Some of these are an inorganic pyrophosphatase that requires
manganese for activation, adenine ribosidase that hydrolyzes
adenosine, an enzyme that functions possibly to lyse the sporangium
and free the spore during germination, highly active alanine racemase
that catalyzes the conversion of L-alanine to n-alanine, several glucose
dehydrogenases, and an aldolase.
4.2.9.5 Sporulation Process
Conditions necessary for sporulation in one species do not
necessarily apply to another. The subject appears to be in such a
state of confusion that it is impossible to discuss sporulation in terms
of generalities.
The conditions which have been reported as favoring sporulation
include addition of salts of metals such as manganese, chromium,
nickel, etc., to the medium; shaking a culture of vegetative cells of
sporing aerobes with distilled water at 37°C.; addition of tomato juice
to a medium; incubating the cultures at an appropriate temperature;
addition of calcium carbonate to a carbohydrate medium to prevent
excessive accumulation of acid, and to maintain the pH at 5.5 or
above; the necessity c ygen; addition to the medium of certain amino
acids; etc.
4.2.9.6 Gen-..ination of Sp?res
With the exception of some constituents such as high concentrations
of calcium, dipicolinic aci1, and in Bacillus sphaericus, a, E-
diaminopimelic acid, sI- ..,re_ are similar to the vegetative cells in
composit:on.
When a spore prepares itself for germination, it loses its
refractility, \"nich coincides with an imbibition of water. This stage
is associated with a loss in heat resistance, stainability, and dry
weigl.,t. Later the spore coat breaks, followed by the emergence from
the spore case of a new germ cell which eventually matures into a
vegetative cell.
Spore germination has been defined in vaiiolJs ways. According
to Campbell, "Spore germinaticn may be regarded as the change
from a heat resistant spore to a heat labile entity which may not
necessarily be a true vegetative cell." Later development, leading
eventually to the formation of a mature vegetative cell, is called
outgrowth.
Some conditions which stimulate germination are as follows: (1)
Treatment at 90 to 100°C. for 1 to 2 min. stimulates germination.
(2) Spores which fail to genninate overcome this dormancy when
activated by heat. (3) The use of certain agents such as alanine,
glucose, and adenosine stimulates spore germination in most sporing
species. In some species other amino acids may be substituted for
the alanine. The same applies to glucose. (4) Yeast extract and
mixtures of vitamin-free amino acids have also been shown to
stimulate germination.
Spores genninate in a variety of ways. There is a considerable
degree of constancy in the method of spore gennination for each
species. Lamanna classified the modes of gennination as follows:
I. Spore gennination by shedding of spore coat. Characteristics
of this method. are
A. Spore does not expand greatly in volume previous to
the germ cell breaking through the spore coat. The limit
of volume increase of the spore may be considered to
be twice its original volume.
B. Spore coat does not lose all its refractive property
previous to germination.
C. After the second division of the germ cell, giving a chain
of three organisms, the original spore coat, remaining
attached to the cells, is visible for a long time after
gennination.
1. Equatorial germination.
2. Polar germination
3. Comma-shaped expansion.
o 0 0 --G-o%0~
123
000 q 0))
D~
1234 2 3
c=dJrfP~
4 5 6 . 5
o 4
ccx:::::)
5 4 5
Figure 4.5 : Methods of spore germination. From left to right: equatorial germination
without splitting along transverse axis; equatorial germination with splitting along
transverse axis; polar germination; spore germination by comma-shaped expansion.
II. Spore germination by absorption of the spore coat.
Characteristics of this method are
A. The spore expands greatly during germination. A tripling
or greater increase of the original volume occurs.
B. The spore loses its characteristic refractiveness during
germination, so that it is difficult to say when the sore
has disappeared and the genn cell appeared.
C. After the second division of the genn cell even if a thin
capsule originally remains, all traces of the spore coat
are gone.
o o o
2 3
c=J c=::J ~
4 5 6
~c "c
c=J ~
7 8
c=capsule
Figure 4.6 : Spore germination by absorption.
Some strains germinating by absorption regularly show a thin
capsule remaining about one end of the growing cell. This would
appear as a polar germination. In other cases, equatorial capsules
are seen. Yet, in all instances, the spore is considered to genninate
by absorption inasmuch as the three characteristics of the method
are still adhered to.
5
Microbiology of Viruses
A virus is a genetic element containing either DNA or RNA that
can alternate between two distinct states, intracellular and extracellular.
In the extracellular state, a virus is a submicroscopic particle
containing nucleic acid surrounded by protein and occasionally
containing other components. In this extracellular state, the virus
particle, also called the virion, is metabolic ally inert and does not
carry out respiratory or biosynthetic functions. The virion is the
structure by which the virus genome is carried from the cell in which
the virion has been produced to another cell where the viral nucleic
acid can be introduced and the intracellular state initiated. In the
intracellular state, virus reproduction occurs: the virus genome is
produced and the components which make up the virus coat are
synthesized. When a virus genome is introduced into a cell and
reproduces, the process is called infection. The cell that a virus can
infect and in which it can replicate is called a host. The virus redirects
preexisting host machinery and metabolic functions necessary for virus
replication.
Viruses may thus be considered in two ways: as agents of disease
and as agents of heredity. As agents of disease, viruses can enter
cells and cause harmful changes in these cells, leading to disrupted
function or death. As agents of heredity, viruses can enter cells and
initiate pennanent, genetic changes that are usually not harmful and
may even be beneficial. In many cases, whether a virus causes disease
or hereditary change depends upon the host cell and on the
enviromnental conditions.
Viruses are smaller than cells, ranging in size from 0.02 1Lm to
0.3 1Lm. A common unit of measure for viruses is the nanometer
(abbreviated nm), which is 1000 times smaller than a 1Lm and one
million times smaller than a millimeter.
106
MICROBIOLOGY OF VIRUSES 107
Viruses are classified initially on the basis of the hosts they infect.
Thus we have animal viruses, plant viruses, and bacterial viruses.
Bacterial viruses, sometimes called bacteriophages (or phage for short,
from the Greek phago meaning to eat), have been studied primarily
as convenient model systems for research on the molecular biology
and genetics of virus reproduction. Many of the basic concepts of
virus particle I
~ infection
I cell (host) I
disease I heredity
t t
cell altered
cell harmed
genetically
(disease or
(harm or
death)
benefit)
Figure 5.1 : Virus infection: the two-fold path.

virology were first worked out with bacterial viruses and subsequently
applied to viruses of higher organisms. Because of their frequent
medical importance, animal viruses have been extensively studied.
The two groups of animal viruses most studied are those infecting
insects and those infecting warm-blooded animals. Plant viruses are
often important in agriculture but have been less studied than animal
viruses. Although viruses are known which infect eucaryotic
microorganisms, they have been little studied. In the present chapter,
we discuss the structure, replication, and genetics of viruses infecting
bacteria and warm-blooded animals. In a nutshell:
1. The virus genome consists of either RNA or DNA. The
genome is surrounded by a coat of protein (and occasionally
other material). When the virus genome is inside the coat
it is called a virus particle or virion.
2. Viruses lack independent metabolism. They multiply only
inside living cells, using the host cell metabolic machinery.
Some virus particles do contain enzymes, however, that are
under the genetic control of the virus genome. Such enzymes
are only produced during the infection cycle.
smallpox, 200 nm
rabies, 100 x 200 n~
_ influenza, 100 nm
_ adenovirus, 70 nm
• polio, 28 nm
Figurf' S.2 : Relative sizes of some common viruses infecting humans.
DNA viruses are green, RNA viruses are red.
3. When a virus multiplies, the genome becomes released from
the coat. This process occurs during the infection process.
The present chapter is divided into three parts. The first
part deals with basic concepts of virus structure and function.
The second part deals with the nature and manner of
multiplication of the bacterial viruses (bacteriophages). In
this part we introduce the basic molecular biology of virus
multiplication. The third part deals with important groups
of animal viruses, with emphasis on molecular aspects of
animal virus multiplication.
S.! THE NATURE OF THE VIRUS PARTICLE
Virus particles vary widely in size and shape. As we have stated,
some viruses contain RNA, others DNA. We have discussed nucleic
acids in previous chapters and have noted that the DNA of the cell
genome is in the double-stranded form. Some viruses have double-
stranded DNA whereas others have single-stranded DNA (Figure 6.3).
We have also noted in Section 5.8 that the RNA of the cell is
generally in the single-stranded configuration. Interestingly, although
single-stranded RNA viruses are more common, viruses are known
in which the RNA is in the double-stranded form.
The structures of virions (virus particles) are quite diverse.
Viruses vary widely in size, shape, and chemical composition. The
nucleic acid of the virion is always located within the particle,
surrounded by a protein coat called the capsid. The terms coat, shell,
and capsid are often used interchangeably to refer to this outer layer.
The protein coat is always formed of a number of individual protein
molecules, called protein subunits, (sometimes called capsomeres)
which are arranged in a precise and highly repetitive pattern around
the nucleic acid. A few viruses have only a single kind of protein
subunit, but most viruses have several chemically distinct kinds of
protein subunits which are themselves associated in specific ways to
form larger assemblies called morphological units. It is the
morphological unit which is seen with the electron microscope.
Genetic economydictates that the variety of virus proteins be kept
small, since virus genomes do not have sufficient genetic information
to code for a large number of different kinds of proteins.
viruses
, other
f1
fungi.
protozoa.
etc.
1Ar l Irs l ~
ss ds
all SS ss ds retro- ss ss ds
viruses
Figure 5.3 : Diversity of viruses. ss: single stranded; ds: double stranded.
The information for proper aggregation of the protein subunits
into the morphological units is contained within the structure of the
subunits themselves, and the overall process of assembly is thus called
self-assembly. A single virion generally has a large number of
morphological units.
The complete complex of nucleic acid and protein, packaged in
the virus particle, is called the virus nucleocapsid. Although the virus
structure just described is frequently the total structure of a virus
particle, a number of animal viruses (and a few bacterial viruses)
have more complex structures. These viruses are enveloped viruses,
in which the nucleocapsid is enclosed in a membrane. Virus
membranes are generally lipid bilayer membranes, but associated with
these membranes are often virus-specific proteins. Inside the virion
are often one or more virus-specific enzymes. Such enzymes usually
play roles during the infection and replication process.
protein
subunits
virus RNA (capsomeres)
\ ~
;;
(a)
Figure 5.4 : Structure and manner of assembly of a simple virus. tobacco mosaic
virus. (a) Electron micrograph at high resolution of a portion of the virus particle. (b)
Assembly of the tobacco mosaic virion. The RNA assumes a helical configuration
surrounded by the protein capsomeres. The center of the particle is hollow.
Virus symmetry The nucleocapsids of viruses are constructed

in highly symmetrical ways. Symmetry refers to the way in which
the protein morphological units are arranged in the virus shell. When
a symmetrical structure is rotated around an axis, the same form is
seen again after a certain number of degrees of rotation. Two kinds
of symmetry are recognized in viruses which correspond to the two
primary shapes, rod and spherical. Rod-shaped viruses have helical
symmetry and spherical viruses have icosahedral symmetry.
A typical virus with helical symmetry is the tobacco mosaic
virus (TMV). This is an RNA virus in which the 2130 identical
protein subunits (each 158 amino acids in length) are arranged in a
helix. In TMV, the helix has 16 112 subunits per tum and the overall
dimensions of the virus particle are 18 x 300 nm. The lengths of
helical viruses are determined by the length of the nucleic acid, but
the width of the helical virus particle is determined by the size and
packing of the protein subunits.
envelope
8
caps~,me,e ~~::id~r~,. ~~~~~.
~
...-capsid
1 aCid
Cb
naked virus enveloped virus

Figure 5.5 : Comparison of naked and enveloped virus. two basic types of virus
particles.
An icosahedron is a symmetrical structure roughly spherical in
shape which has 20 faces. Icosahedral symmetry is the most efficient
arrangement for subunits in a closed shell because it uses the smallest
number of units to build a shell. The simplest arrangement of
morphological units is 3 per face, for a total of 60 units per virus
particle. The three un~ts at each face can be either identical or
different. Most viruses have more nucleic acid than can be packed
into a shell made of just 60 morphological units. The next possible
structure which permits close packing contains 180 units and many
viruses have shells with this configuration. Other known configurations
involve 240 units and 420 units.
When discussing symmetry, one speaks of axes of rotation. A
flat triangle shape, for instance, has one three-fold axis of symmetry,
since there are three possible rotations that will lead to the exact
configuration seen originally. Three dimensional objects such as
viruses can have more than one axis of symmetry. An icosahedron,
for instance, has three different axes of symmetry, two-fold, three-
fold, and fivefold. When a rod is placed through the two-fold axis
of symmetry (one of the edges) in the model, the model can be turned
once around this axis (1/2 way or 180) to obtain the same
configuration again. When the rod is placed through one of the three-
fold axes of symmetry (one of the faces), the model can be turned
three times, and if the rod is placed through one of the five-fold
axes of symmetry (one of the vertices) the model can be turned five
times.
In all cases, the characteristic structure of the virus is determined
by the structure of the protein subunits of which it is constructed.
Self-assembly leads to the fmal virus particle.
nucleic acid
Figure 5.6 : A simple icosahedral virus. Each face has three subunits. A single subunit
consists of one or more proteins. (a) Whole virus particle. (b) Virus particle opened
up; nucleic acid released.
Enveloped viruses Many viruses have complex membranous
structures surrounding the nucleocapsid. Enveloped viruses are
common in the animal world (for example, influenza virus), but some
enveloped bacterial viruses are also known. The virus envelope
consists of a lipid bilayer wi$ proteins, usually glycoproteins,
embedded in it. Although the glycoproteins of the virus membrane
are encoded by the virus, the lipids. are derived from the membranes
of the host cell. The symmetry of enveloped viruses is expressed
not in terms of the virion as a whole but in terms of the nucleocapsid
present inside the virus membrane.
Figure 5.7 : Demonstration of icosahedral symmetry.

What is the function of the membrane in a virus particle? We
will discuss this in detail later but note that because of its location
in the virion, the membrane is the structural component of the virus
particle that interacts first with the cell. The specificity of virus
infection, and some aspects of virus penetration, are controlled in
part by characteristics of virus membranes.
Complex viruses Some virions are even more complex, being
composed of several separate parts, with separate shapes and
symmetries. The most complicated viruses in terms of structure are
some of the bacterial viruses, which possess not only icosahedral
heads but helical tails. In some bacterial viruses, such as the T4
virus of Escherichia coli, the tail itself is a complex structure. For
instance, T4 has almost 20 separate proteins in the tail, and the T4
head has several more proteins. In such complex viruses, assembly
is also complex. For instance, in T4 the complete tail is formed as a
subassembly, and then the tail is added to the DNA-containing head.
Finally, tail fibers formed from another protein are added to make
the mature, infectious virus particle.
The virus genome We have stated that the virus genome consists
of either DNA or RNA, never both. Viruses differ in size, amount,
and character of their nucleic acid. Both single-stranded and
doublestranded nucleic acid is found in viruses, and the amount of
nucleic acid per virion may vary greatly from one virus type to
another. In general, in enveloped viruses the nucleic acid constitutes
only a small part of the mass of the virus particle (1-2 percent),
whereas in nonenveloped viruses the percent of the particle which is
nucleic acid is much larger, often 25-50 percent.
Interestingly, the nucleic acid in some viruses is not present in
a single molecule, the genome being segmented into several or many
molecules. For instance, retroviruses-causal agents of some cancers
and AIDS, among other diseases-have two identical RNA molecules,
influenza virus has 8 RNA molecules of sizes varying over about
three-fold, and some other animal viruses have even more RNA
molecules. The manner in which these various pieces of nucleic acid
are replicated in the cell and then assembled into mature virions is
of considerable interest-how do all these nucleic acid pieces end up
together in one particle?
Enzymes in viruses We have stated that virus particles do not
carry out metabolic processes. Outside of a host cell, a virus particle
is metabolically inert. However, some viruses do contain enzymes
which play roles in the infectious process. For instance, many viruses
contain their own nucleic acid polymerases which transcribe the viral
nucleic acid into messenger RNA once the infection process has
begun. The retroviruses are RNA viruses which replicate inside the
cell as DNA intermediates. These viruses possess an enzyme, an
RNA-dependent DNA popo called reverse transcriptase, which
transcribes the information in the incoming RNA into a DNA
intermediate. It should be noted that reverse transcriptase is unique
to the retroviruses and is not found in any other viruses or in cells.
A number of viruses contain enzymes which aid in release of
the virus from the host cells in the final stages of the infectious
process. One group of such enzymes, called neuraminadases, break
down glycosidic bonds of glycoproteins and glycolipids of the
connective tissue of animal cells, thus aiding in the liberation of the
virus. Virions infecting some bacteria possess an enzyme, lysozyme
, which hydrolyzes the cell wall, causing lysis of the host cell and
release of the virus particles. We will discuss some of these enzymes
in more detail later.
5.2 THE CLASSIFICATION OF VIRUSES
As we have noted, viruses can be classified into broad groups
depending on their hosts. For instance, there are plant viruses, animal
viruses, and bacterial viruses. A number of viruses infecting insects
are also known and although viruses are known for fungi, protozoa,
and algae, these viruses have been so little studied that no
classification has been developed. In the present chapter, we discuss
only the animal (primarily mammalian) and bacterial viruses, and
we discuss here briefly how these two groups of viruses are classified.
Classification of bacterial viruses In the bacterial viruses, a
formal classification scheme is rarely used. Rather, each bacterial
virus is designated in terms of its principal bacterial host, followed
by an arbitrary alphanumeric. Thus, we speak of T4 virus of
Eschericbia coli or P22 virus of Salmonella typhimurium. An overview
of some of the major types of bacterial viruses is given later. We
should note, however, that although a bacterial virus may be
designated in reference to its principal host, the actual host range of
the virus may be broader. Thus, bacteriophage Mu, generally studied
with Eschericbia coli, also infects Citrobacter and Salmonella.
Classification of animal viruses We should note first that
classification of animal viruses presents some major differences from
the classification of organisms. The conventional approach to
classification of organisms, involving hierarchical categories such as
species, genera, families, etc., has been applied only to animal
viruses. Even here, the higher levels of classification are not used.
The highest level of animal virus classification is the virus family.
Virus families are designated by terms ending in -viridae. Thus, the
group of poxviruses is called the Poxviridae and the herpesviruses
are called the Herpesviridae. Note that the major criteria used in
classifying animal viruses are the type of nucleic acid, the presence
or absence of an envelope, and, for certain families, the manner of
replication.
Virus genera are designated by terms ending in - virus. Thus,
among the Poxviridae those poxviruses which infect fowl are called
by the genus name Avipoxvirus. Note that frequently in the animal
viruses, the genus is defmed based on the host which the virus infects.
Except in a few cases, virus species have not been formally
designated, but would refer to specific virus entities that have been
recognized. At present, virus species are only designated by common
names, such as mumps virus, poliovirus 1, and smallpox virus. For
116 MICROBIOLOGY AJIlD BIOCHEMISTRY
instance, in the virus genus Onhopoxvirus two virus species currently
recognized are vaccinia and smallpox, but are not given Latin names.
At present, it does not appear useful to use Latin names for virus
species.
When contemplating the problem of virus classification, we can
be truly impressed with the enormous diversity of viruses.
Undoubtedly, many new viruses are awaiting discovery, although most
undiscovered viruses will probably be considered members of existing
virus families.
5.3 THE VIRUS HOST
Because viruses only replicate inside living cells, research on
viruses requires use of appropriate hosts. For the study of bacterial
viruses, pure cultures are used either in liquid or on semi-solid (agar)
medium. Because bacteria are so easy to culture, it is quite easy to
study bacterial viruses and this is why such detailed knowledge of
bacterial virus reproduction is available.
With animal viruses, the initial host may be a whole animal which
is susceptible to the virus, but for research purposes it is desirable
to have a more convenient host. Many animal viruses can be
cultivated in tissue or cell cultures, and the use of such cultures has
enormously facilitated research on animal viruses.
Cell cultures A cell culture is obtained by enabling growth of
cells taken from an organ of the experimental animal. Cell cultures
are generally obtained by aseptically removing pieces of the tissue
in question, dissociating the cells by treatment with an enzyme which
breaks apart the intercellular cement, and spreading the resulting
suspension out on the bottom of a flat surface, such as a bottle or
petri dish. The cells generally produce glycoprotein-like materials that
permit them to adhere to glass surfaces. The thin layer of cells
adhering to the glass or plastic dish, called a monolayer, is then
overlayed with a suitable culture medium and the culture incubated.
The culture media used for cell cultures are generally quite complex,
employing a number of amino acids and vitamins, salts, glucose,
and a bicarbonate buffer system. To obtain best growth, addition of
a small amount of blood serum is usually necessary, and several
antibiotics are generally added to prevent bacterial contamination.
Some cell cultures prepared in this way will grow indefmitely,
and can be established as permanent cell lines. Such cell cultures
are most convenient for virus research because continuously available
cell material can be available for research purposes. In other cases,
indefinite growth does not occur but the culture may remain alive
for a number of days. Such cultures, called primary cell cultures,
may still be useful for virus research, although of course new cultures
will have to be prepared from fresh sources from time to time.
Cancer Cancer is a cellular phenomenon of uncontrolled growth
that is sometimes induced by virus infection. Most cells in a mature
animal, although alive, do not divide extensively, apparently because
of the presence of growth-inhibiting factors which prevent them from
initiating cell division. Under a variety of pathological conditions,
among which is included infection by certain viruses, growth
inhibition is overcome and the cells begin to divide uncontrollably.
Under some conditions, this extensive cellular growth is so excessive
that the animal body is virtually consumed by cancer cells: the animal
dies. Cancerous growth is thus due to a derangement in the control
of cellular growth, and is of great medical as well as theoretical
interest.
The tumorigenic or cancer-causing ability of viruses can often
be detected by observing the induction in cell cultures of uncontrolled
growth. In cell cultures, the general arrangement of the cells is as a
monolayer, arising because growth generally ceases when the cells,
as a result of growth, come in contact with each other. Cancer cells
have altered growth requirements and continue to grow, pilmg up to
form a small focus of growth. By observing for the induction of such
foci of growth from virus infection, it is possible to observe the
tumorigenic properties of viruses.
In some cases, cell culture monolayers can not be obtained but
whole organs, or pieces of organs, can be cultured. Such organ
cultures may still be useful in virus research, since they permit growth
of viruses under more or less controlled laboratory conditions.
5.4 QUANTIFICATION OF VIRUSES
In order to obtain any significant understanding of the nature of
viruses and virus replication, it is necessary to be able to quantify
the number of virus particles. Virus particles are almost always too
small to be seen under the light microscope. Although virus particles
can be observed under the electron microscope, the use of this
instrument is cumbersome for routine study. In general, viruses are
quantified by measuring their effects on the host cells which they
infect. It is common to speak of a viru..~ infectious unit, which is the.
smallest unit that causes a detectable effect when placed with a
susceptible host. By determining the number of infectious units per
volume of fluid, a measure of virus quantity can be obtained. We
discuss here several approaches to assessment of the virus infectious
unit.
Plaque assay When a virus particle initiates an infection upon a
layer or lawn of host cells which is growing spread out on a flat
surface, a zone of lysis or growth inhibition may occur which results
in a clearing of the c!!ll growth. This clearing is called a plaque,
and it is assumed that each plaque has originated from one virus
particle.
Plaques are essentially "windows" in the lawn of confluent cell
growth. With bacterial viruses, plaques may be obtained when virus
particles are mixed into a thin layer of host bacteria which is spread
out as an agar overlay on the surface of an agar medium. During
incubation of the culture, the bacteria grow and form a turbid layer
which is visible
to the naked eye. However, wherever a successful virus infection
has been initiated, lysis of the cells occurs, resulting in the formation
of a clear zone, called a plaque.
The plaque procedure also permits the isolation of pure virus
strains, since if a plaque has arisen from one virus particle, all the
virus particles in this plaque are probably genetically identical. Some
of the particles from this plaque can be picked and inoculated into a
fresh bacterial culture to establish a pure virus line. The development
of the plaque assay technique was as important for the advance of
virology as was Koch's development of solid media for bacteriology.
Plaques may be obtained for animal viruses by using animal cell-
culture systems as hosts. A monolayer of cultured ani.JIals cells is
prepared on a plate or flat bottle and the virus suspension overlayed.
Plaques are revealed by zones of destruction of the animal cells.
In some cases, the virus may not actually destroy the cells, but
cause changes in morphology or growth rate which can be recognized.
For instance, tumor viruses may not destroy cells but cause the cells
to grow faster than uninfected cells, a phenomenon called
transfonnation. As we have noted, in a tissue culture monolayer,
these transformed cells gradually develop into a recognizable cluster
of cells called a focus of infection. By counting foci of infection, a
quantitative measure of virus may be obtained.
Efficiency of plating One important concept in quantitative
virology involves the idea of efficiency of plating. Counts made by
plaque assay are always lower than counts made with the electron
phage top bacterial

dilution agar cells
pour mixture
onto agar plate
nutrient agar
plate
sandwich of
top agar and
nutrient agar
incubate
_..,.....,~ phage
plaques
~~~5~~~~- lawn of host cells
Figure 5.8 : Quantification of bacterial virus by plaque assay using the agar overlay
technique.
microscope. The efficiency with which virus particles infect host cells
is almost never 100 percent and may often be considerably less. This
does not mean that the virus particles which have not caused infection
are inactive. It may merely mean that under the conditions used,
successful infection with these particles has not occurred. Although
with bacterial viruses, efficiency of plating is often higher than 50
percent, with many animal viruses it may be very low, 0.1 or 1
percent. Why virus particles vary in infectivity is not well understood.
It is possible that the conditions used for quantification are not
optimal. Because it is technically difficult to count virus particles
with the electron microscope, it is difficult to assess the actual
efficiency of plating, but the concept is important in both research
and medical practice. Because the efficiency of plating is rarely close
to 100 percent, when the plaque method is used to quantify virus, it
is accurate to express the titer of the virus suspension not as the
number of virion units, but as the number of plaque forming units.
Animal infectivity methods Some viruses do not cause recogn-
izable effects in cell cultures but cause death in the whole animal. In
such cases, quantification can only be done by some sort of titration
in infected animals. The general procedure is to carry out a serial
dilution of the unknown sample, generally at ten-fold dilutions, and
samples of each dilution are injected into numbers of sensitive animals.
After a suitable incubation period, the fraction of dead and live animals
at each dilution is tabulated and an end point dilution is calculated.
This is the dilution at which, for example, half of the injected animals
die. Although such serial dilution methods are much more cumbersome
and much less accurate than cell culture methods, they may be essential
for the study of certain types of viruses.
5.5 GENERAL FEATURES OF
VIRUS REPRODUCTION
The basic problem of virus replication can be simply put; the
virus must somehow induce a living host cell to synthesize all of the
essential components needed to make more virus particles. These
components must then be assembled into the proper structure and
the new virus particles must escape from the cell and infect other
cells. The various phases of this replication process in a bacteriophage
can be categorized in seven steps:
1. Attachment (adsorption) of the virion to a susceptible host
cell;
2. Penetration (injection) into the cell by the virion or by its
virus particle
/
DNA
1. attachment
(adsorption)
1
\
prote~n
cell (host)
remains
coat ---a.
...
wruDNA_me
outside _-----
) 2. penetration
(injection)
1 3. early steps: virus

enzymers synthesized
(I '
-1 4. nucleic acid
replication
~ •.•• ~
5. synthesis of
protein coats
1 6. assembly
C·rJ
7. release
(lysis)
e
e· . • •
mature
virus
particles
Figure 5.9 : The replication cycle of a bacterial virus. The general stages of virus
replication are indicated.
nucleic acid;
3. Early steps in replication of the virus nucleic acid, in which
the host cell biosynthetic machinery is altered as a prelude
to virus nucleic acid synthesis. Virus-specific enzymes may
be made;
4. Replication of the virus nucleic acid;
5. Synthesis of 'protein subunits of the virus coat;
6. Assembly of nucleic acid and protein subunits (and
membrane components in enveloped viruses) into new virus
particles;
7. Release of mature virus particles from the cell (lysis).
These stages in virus replication are recognized when virus
particles .infect cells in culture and are illustrated in Figure 6.13,
which exhibits what is called a one-step growth curve. In the tirst
few minutes after infection, a period called the eclipse occurs, in
which the virus nucleic acid has become separated from its protein
coat so that the virus particle no longer exists as an infectious entity.
Although virus nucleic acid may be infectious, the infectivity of virus
nucleic acid is many times lower than that of whole virus particles
because the machinery for bringing the virus genome into the cell is
lacking. Also, outside the virion the nucleic acid is no longer protected
from deleterious activities of the environment as it was when it was
inside the protein coat.
eclipse maturation
adsorption period
early protein
relative
virus count enzymes nucleic coats
(plaque- acid
forming units)
virus
added
~ latent period
o 30 60
time
Figure 1.10 : The one-step growth curve of virus replication. This graph displays the
results of a single round of viral multiphcation in a population of cells.
The eclipse is the period during which the stages of virus
multiplication occur. This is called the latent period, because no
infectious virus particles are evident. Finally, maturation begins as
the newly synthesized nucleic acid molecules become assembled inside
protein coats. During the maturation phase, the titer of active virus
particles inside the cell rises dramatically. At the end of maturation,
release of mature virus particles occurs, either as a result of cell
lysis or because of some budding or excretion process. The number
of virus particles released, called the burst size, will vary with the
particular virus and the particular host cell, and can range from a
few to a few thousand. The timing of this overall virus replication.
cycle varies from 20-30 minutes in many bacterial viruses to 8-40
hours in most animal viruses. We now consider each of the steps of
the virus multiplication cycle in more detail.
5.6 EARLY EVENTS OF VIRUS MULTIPLICATION
In order to discuss the stages of virus multiplication, we must
return briefly to a consideration of the virus genome. As we have
noted, virus genomes consist of either DNA or RNA, and both single-
stranded and double-stranded forms of each of these nucleic acids is
known to occur in different viruses. In the case of DNA viruses,
the nucleic acid may be in either a linear or a circular form. The
nucleic acid of RNA viruses is always in a linear form. Some virus
nucleic acids also contain covalently linked polypeptides or amino
acids which play roles in replication. In addition, in some RNA
viruses the genome is not present in a single molecule, but may be
divided over two or many nucleic acid molecules. Even more
complicated, once inside the cell, the genetic information present in
the virus genome may be transfered to another nucleic acid molecule.
To avoid confusion, we restrict the term virus genome to the nucleic
acid found in the virion (virus particle).
As we have noted, the outcome of a virus infection is the
synthesis of viral nucleic acid and viral protein coats. In effect, the
virus takes over the biosynthetic machinery of the host and uses it
for its own synthesis. A few enzymes needed for virus replication
may be present in the virus particle and may be introduced into the
cell during the infection process, but the host supplies everything
else: energy-generating system, ribosomes, amino-acid activating
enzymes, transfer RNA (with a few exceptions), and all soluble
factors. The virus genome codes for all new proteins. Such proteins
would include the coat protein subunits (of which there are generally
more than one kind) plus any new virus-specific enzymes.
Attachment There is a high specificity in the interaction between
virus and host. The most common basis for host specificity involves
the attachment process. The virus particle itself has one or more
proteins on the outside which interact with specific cell surface
comp<)nents called receptors. The receptors on the cell surface are
normal surface components of the host, such as proteins,
polysaccharides, or lipoprotein-polysaccharide complexes, to which
the virus particle attaches. In the absence of the receptor site, the
virus cannot adsorb, and hence cannot infect. If the receptor site is
altered, the host may become resistant to virus infection. However,
mutants of the virus can also arise which are able to adsorb to resistant
hosts.
In general, virus receptors carry out normal functions in the cell.
For example, in bacteria some phage receptors are pili or flagella,
others are cell-envelope components, and others are transport binding
proteins. The receptor for influenza virus is a glycoprotein found on
red blood cells and on cells of the mucous membrane of susceptible
animals, whereas the receptor site of poliovirus is a lipoprotein.
However, many animal and plant viruses do not have specific
attachment sites at all and the virus enters passively as a result of
phagocytosis or some other endocytotic_ process.
Penetratio~ The means by which the virus penetrates into the
cell depends on the nature of the host cell, especially on its surface
structures. Cells with cell walls, such as bacteria, are infected in a
different manner from animal cells, which lack a cell wall. The most
complicated penetration mechanisms have been found in viruses that
infect bacteria. The bacteriophage T4, which infects E. coli, can be
used as an example.
The particle has a head, within which the viral DNA is folded,
and a long, fairly complex tail, at the end of which is a series of
tail fibers. During the attachment process, the virus particles first
attach to the cells by means of the tail fibers. These tail fibers then
contract, and the core of the tail makes contact with the cell envelope
of the bacterium. The action of a lysozyme-like enzyme results in
the formation of a small hole. The tail sheath contracts and the DNA
of the virus passes into the cell through a hole in the tip of the tail,
the majority of the coat protein remaining outside. The DNA of T4
has a total length of about 50 JLm, whereas the dimensions of the
head of the T4 particle are 0.095 Am by 0.065 JLm. This means
that the DNA must be highly folded and packed very tightly within
the head.
With animal cells, the whole virus particle penetrates the cell,
being carried inside by endocytosis (phagocytosis or pinocytosis), an
active cellular process. We describe some of these processes in detail
later in this chapter.
Virus restriction and modification by the host We have already
seen that one form of host resistance to virus arises when there is
no receptor site on the cell surface to which the virus can attach.
Another and more specific kind of host resistance involves destruction
of the viral nucleic acid after it has been injected. This destruction
is brought about by host enzymes that cleave the viral DNA at one
or several places, thus preventing its replication. This phenomenon
is called restriction, and is part of a general host mechanism to
prevent the invasion of foreign nucleic acid.
Restriction enzymes are highly specific, attacking only certain
sequences (generally four or six base pairs). The host protects' its
own DNA from the action of restriction enzymes by modifying its
Figure S.11 : Attachment of T4 bacteriophage particle to the cell wall of E. coli and
injection of DNA: (a) Unattached particle. (b) Attachment to the wall by the long tail
fibers. (c) Contact of cell wall by the tail pin. (d) Contraction of the tail sheath and
injection of the DNA.
DNA at the sites where the restriction enzymes will act. Modification
of host DNA is brought about by methylation of purine or pyrimidine
bases.
Viruses can overcome host restriction mechanisms by modifications
of their nucleic acids so that they are no longer subject to enzymatic
attack. Two kinds of chemical modifications of viral DNA have been
recognized, glucosylation and methylation. The bacteriophages T2,
T4, and T6 have their DNA glucosylated to varying degrees, and the
glucosylation prevents or greatly reduces nuclease attack. In
bacteriophage lambda the amino groups of adenine and cytosine bases
are methylated by an enzyme- that uses S-adenosylmethionine as methyl
donor. Many other viral nucleic acids have been found to be modified
by methylation but glucosylation has been found only in the T-even
bacteriophages .(bacteriophages T2, T4, and T6). It should be
emphasized that modification of viral nucleic acid occurs after
replication has occurred and the modified bases are not copied directly.
The enzymes for methylation are actually present in the host before
infection, and hence are not virus induced functions. These host
modification enzymes probably have as their main role the modification
of host DNA so that it can be transferred without inactivation into
other cells during genetic recombination.
The ability to modify nucleic acid is not found in all strains that
support the growth of a given virus. Thus, when bacteriophage
lambda is grown on E. coli strain C it is not modified (E. coli strain
C lacks both the lambda modification and restriction enzymes), and
nucleic acid of virus grown on strain C is destroyed when it enters
E. coli strain K- 12, which does have the restriction enzyme.
However, strain K12 also has the modification enzyme, and, if lambda
is grown on K- 12, its nucleic acid is modified and it will infect
both strains K-12 and C equally well. However, if lambda is grown
on a K-12 strain made methionine deficient, methylation cannot occur
and the phage particles' released are unable to replicate in K12. In
the case of the T-even phages, glucosylation requires uridine
diphosphoglucose (UDPG), and if a T-even phage is grown on a
host deficient in UDPG its nucleic acid is not glucosylated and it is
unable to replicate in susceptible cells.
A knowledge of modification and restriction systems is of
considerable practical utility in studying DNA chemistry. So far, no
evidence exists that either modification or restriction occurs in
eucaryotic organisms.
Virus messenger RNA In order for the new virus-specific
proteins to be made from the virus genome, it is necessary for new
virus-specific RNA molecules to be made. Exactly how the virus
brings about new mRNA synthesis depends upon the type of virus,
and especially upon whether its genetic material is RNA or DNA,
and whether it is single-stranded or double-stranded. Which copy is
read into mRNA depends upon the location of the appropriate
pr~moter, since the promoter points the direction that the RNA
polymerase will follow. In cells (uninfected with virus) all mRNA is
made on the DNA template, but with RNA viruses the situation is
obviously different.
A virus-specific RNA RNA polymerase is needed, since the cell
RNA polymerase will generally not copy double-stranded RNA (and
ribosomes are not able to translate double-stranded RNA either). A
wide variety of modes of viral mRNA synthesis are outlined in Figure.
By convention, the chemical sense of the mRNA is considered to be
of the plus (+) configuration. The sense of the viral genome nucleic
acid is then indicated by a plus if it is the same as the mRNA and a
minus if it is of oppposite sense. If the virus has double-stranded
DNA (ds DNA), then mRNA synthesis can proceed directly as in
uninfected cells. However, if the virus has a singlestranded DNA
(ss DNA), then it is first converted to ds DNA and the latter serves
as the template for mRNA synthesis with the cell RNA polymerase.
If the virus has double-stranded RNA (ds RNA), this RNA serves
as a template in a manner analogous to DNA. There are three classes
of viruses with ss RNA and they differ in the mechanism by which
mRNA is synthesized. In the simplest case, the incoming viral RNA
is the plus sense and hence serves directly as mRNA, and copies of
this viral RNA are also copied to make further mRNA molecules.
In another class, the viral RNA has a minus (-) sense. In such viruses
a copy is made (Plus sense) and this copy becomes the mRNA. In
the case of the retroviruses (causal agents of certain kinds of cancers
and AIDS), a new phenomenon called reverse transcription is seen,
in which virion ss RNA is copied to a double-stranded DNA (through
a ss DNA intermediate) and the ds DNA then serves as the template
for mRNA synthesis (thus: ss RNA ss DNA ds DNA). Retrovirus
replication is of unusual interest and complexity.
Viral proteins Once mRNA is made, viral proteins (for example,
enzymes, capsomers) can be synthesized. The proteins synthesized
as a result of virus infection can be grouped into two broad categories,
the enzymes synthesized soon after infection, called the early enzymes,
which are necessary for the replication of virus nucleic acid, and
the proteins synthesized later, called the late proteins, which include
the proteins of the virus coat. Generally, both the time of appearance
and the amount of these two groups of virus proteins are regulated.
The early proteins are enzymes which, because they act catalytically,
are synthesized in smaller amounts and the late proteins, often
structural, are made in much larger amounts.
Virus infection obviously upsets the regulatory mechanisms of
the host, since there is a marked overproduction of nucleic acid and
protein in tht; infected cell. In some cases, virus infection causes a
complete shutdown of host macromolecular synthesis while in other
cases host synthesis proceeds concurrently with virus synthesis. In
either case, the regulation of virus synthesis is under the control of
the virus rather than the host. There are several elements of this
control which are similar to the host regulatory mechanisms, but there
are also some uniquely viral regulatory mechanisms. We discuss
various regulatory mechanisms when we consider the individual
viruses later in this chapter.
5.7 VIRAL GENETICS
Viruses exhibit genetic phenomena similar to those of cells.
Studies of viral genetics have played a significant role in understanding
many aspects of genetics at the molecular level. In addition,
knowledge of the basic phenomena of viral genetics has increased
our understanding of processes involved in virus replication.
Understanding these processes has also led to some practical
developments, especially in the isolation of viruses which are of use
in immunization procedures. Most of the detailed work on viral
genetics has been carried out with bacteriophages, because of the
convenience of working with these viruses. We mention here briefly
some of the types of genetic phenomena of viruses.
Mutations Much of our knowledge of viral reproduction and
how it is regulated has depended on the isolation and characterization
of virus mutants. Several kinds of mutants have been studied in
viruses: host-range mutants, plaque-type mutants, temperature-sensitive
mutants, nonsense mutants, transposons, and inversions.
Host-range mutations are those that change the range of hosts
that the virus can infect. Host resistance to phage infection can be
due to an alteration in receptor sites on the surface of the host cell,
so that the virus can no longer attach, and host-range mutations of
the virus can then be recognized as virus strains able to attach to.
and infect these virus-resistant hosts. Other host-range mutants may
involve changes in the viral and host enzymes involved in replication,
or in the restriction and modification systems.
Plaque-morphology mutations are recognized as changes in the
characteristics of the plaques formed when a phage infects cells in
the conventional agarplate technique. Characteristics of the plaque,
such as whether it is clear or turbid, and its size, are under genetic
control. The underlying basis of plaque morphology lies in processes
taking place during the virus multiplication cycle, such as the rate
of replication and the rate of lysis. Under appropriate experimental
conditions plaque morphology can bea highly reproducible
characteristic of the virus. The advantage of plaque mutants for
genetic studies is that they can be easily recognized on the agar plate,
but a disadvantage is that there is no convenient way of selecting
for them among the large background of normal particles.
Temperature-sensitive mutations are those which allow a virus
to replicate at one temperature and not at another, due to a mutational
alteration in a virus protein that renders the protein unstable at
moderately high temperatures. For instance, temperature-sensitive
mutants are known in which the phage will not be replicated in the
host at 43°C but will at 25°C, although the host functions at both
temperatures. Such mutations are called conditionally lethal, since
the virus is unable to reproduce at the higher temperature, but
replicates at the lower temperature.
Nonsense mutations change normal codons into nonsense codons.
In viruses, nonsense mutations are recognized because hosts are
available that contain suppressors able to read nonsense codons. The
virus mutant will be able to grow in the host containing the
suppressor, but not in the normal host.
Transpositions several viruses are known which act essentially
as transposons and transposition events involving viruses can lead to
their genetic change.
Genetic recombination in viruses The availability of virus
mutants makes possible the investigation of genetic recombination.
If two virus particles infect the same cell, there is a possibility for
genetic exchange between the two virus genomes during the replication
process. If recombination does occur, the progeny of such a mixed
infection should include not only the parental types, but recombinant
types as well. With appropriate mutants, it is possible to recognize
both the parental types and the recombinants and to study the events
involved in the recombination process. Genetic recombination in
viruses is an extremely complex process to analyze because
recombination does not occur as a single discrete event during mixed
infection, but may occur over and over again during the replication
cycle. It has been calculated that the T-even bacteriophages undergo,
on the average, four or five rounds of recombination during a single
infection cycle. By detailed and careful analysis of a wide variety of
virus crosses, it has been possible to construct genetic maps of a
number of bacterial viruses. Such maps have provided important
information about the genetic structure of viruses. We present a few
genetic maps when discussing specific viruses later in this chapter.
Genetic recombination arises by exchange of homologous segments
of DNA between viral genomes, most often during the replication
process. The enzymes involved in recombination are DNA
polymerases, endonucleases, and ligases, which also playa role in
DNA repair and synthesis processes.
Phenotypic mixing During studies on genetic recombination
between viruses, another phenomenon was discovered which
superficially resembles recombination but has a quite different basis.
Phenotypic mixing occurs when the DNA of one virus is incorporated
inside the protein coat of a different virus. For phenotypic mixing to
occur, the two viruses must be closely related, so that the protein coat
is of proper construction of the packaging of either viral DNA. As an
example of phenotypic mixing, in phage T2 of E. coli there is a gene
called the h gene which controls host specificity through modification
of the tail fibers of the phage. If a mixed infection is set up with two
T2 phages, mutant T2h and wild-type T2h+, tail fibers of b specificity
may be incorporated onto the particles containing DNA of h+
specificity. Since it is the h function of the tail fibers that affects
attachment, these mixed particles will show b specificity during the
next round of infection, even though they contain h+ DNA, but the
particles resulting from this second round of infection will
phenotypically become b+, because the DNA has been unchanged.
5.8 GENERAL OVERVIEW OF
BACTERIAL VIRUSES
Most of the bacterial viruses which have been studied in any
detail infect bacteria of the enteric group, such as Escherichia coli
and Salmonella typhimurium. However, viruses are known that infect
a variety of procaryotes, both eubacteria and archaebacteria. A few
bacterial viruses have lipid envelope~ but most do not. However,
many bacterial viruses are structurally complex, with head and
complex tail structures. The tail is involved in the injection of the
nucleic acid into the cell.
We now discuss some of the bacterial viruses for which molecular
details of the multiplication process are known. Although these
bacterial viruses were first studied as model systems for understanding
general features of virus multiplication many of them now serve as
convenient tools for genetic engineering. Thus, the information on
bacterial viruses is not only valuable as background for the discussion
of animal viruses, but is essential for the material presented in the
next two chapters on microbial genetics and genetic engineering.
It should already be clear from what has been stated that a great
diversity of viruses exist. It should therefore not be surprising that
there is also a great diversity in the manner by which virus
multiplication occurs. Interestingly, many viruses have special features
of their nucleic acid and protein synthesis processes that are not fomid
in cells. In the present chapter, we are only able to present some of
the major types of virus replication patterns, and must skip some of
the interesting exceptional cases.
5.9 RNA BACTERIOPHAGES
A number of bacterial viruses have RNA genomes. The best-
known bacterial RNA viruses have single-stranded RNA. Interestingly,
the bacterial RNA viruses known in the enteric bacteria group infect
only bacterial cells which behave as gene donors (males) in genetic
recombination. This restriction to male bacterial cells arises because
these viruses infect bacteria by attaching to male-specific pili. Since
such pili are absent on female cells, these RNA viruses are unable
to attach to the females, and hence do not initiate infection in females.
The bacterial RNA viruses are all of quite small size, about 26
nm in size, and they are all icosahedral, with 180 copies of coat
protein per virus particle. The complete nucleotide sequence of several
RNA phages are known. In the RNA phage MS2, which infects
Eschericbia coli, the viral RNA is 3,569 nucleotides long. The virus
RNA, although single stranded, has extensive regions of secondary
and tertiary structure. The RNA strand in the virion has the plus
( +) sense, acting directly as mRNA upon entry into the cell.
The genetic map is shown and the flow of events of MS2
multiplication. The infecting RNA goes to the host ribosome, where
it is translated into four (or more) proteins. The four proteins that
have been recognized are maturation protein (A-protein; present in
RNA
ss 0 MS2, Q13 dsO <1>6
ss DNA
o <I> X 174 ~
=======~== ffdd" M
M1133 •
~
ds DNA
T3, T7
lambda, T5
Mu
T2,T4
Figure 5.12 : Schematic representations of the main types of bacterial viruses.

Those discussed in detail are fd, M 13, <l>X 174, MS2, T4, lambda,
T 7, and Mu. Sizes are to approximate scale.
Figure 5.13 : The RNA of bacteriophage MS2. The molecule is single stranded but
there are extensive regions of complementary bases, so that pairing within the strand
leads to the secondary structure shown. Note that the start sites for three coding regions
are in the same part of the folded molecule.
the mature virus particle as a single copy), coat protein, lysis protein
(involved in the lysis process which results in release of mature virus
particles), and RNA replicase, the enzyme which brings about the
replication of the viral RNA. Interestingly, the RNA replicase is a
composite protein, composed partly of a virus-encoded polypeptide
and partly of host polypeptides. The host proteins involved in the
formation of active viral replicase are ribosomal protein Sf (one of
the subunits of the 30S ribosome), and elongation factors Tu and
Ts, involved in the translation process. Thus, the virus appears to
co-opt host proteins that normally have entirely distinct functions and
make them become part of active viral replicase.
As noted, the viral RNA is of the plus (+) sense. Replicase
synthesizes RNA of minus (-) sense using the infecting RNA as
template. After minus RNA has been synthesized, plus RNA is made
from this minus RNA. The newly mad~ plus RNA strands now serve
as messengers for virus protein synthesis. The gene for the maturation
protein is at the 5' end of the RNA. Translation of the gene coding
for the maturation protein (needed in only one copy per virus
. particle), occurs only from the newly formed plus-strand RNA as
the replication process occurs. In this way, the amount of maturation
protein needed is limited. As the virus RNA· is made, it folds into a
complex form with extensive secondary and tertiary structure. Of
the four AUG start sites, the most accessible to the translation process
is that for the coat protein. As coat protein molecules increase in
number in the cell, they combine with the RNA around the AUG
start site for the replicase protein, effectively turning off synthesis
of replicase. Thus, the major virus protein synthesized is coat protein,
which is needed in 180 copies per RNA molecule.
Another interesting feature of MS2 RNA virus is that the fourth
virus protein, the lysis protein, is coded by a gene which overlaps
with both the coat protein gene and the replicase gene. The start of
this lysis gene is not directly accessible to ribosomes. As the ribosome
passes over the coat protein gene, a frame shift occasionally occurs,
resulting in reading of the lysis gene. By restricting the efficiency of
translation in this way, premature lysis of the cell is probably avoided.
Only after sufficient coat protein is available for the assembly of
mature virus particles, does lysis commence. (In another RNA phage,
QP, the maturation protein itself also functions as a lysis protein,
and a separate lysis gene as such is not present.)
Ultimately, assembly occurs and release of virions from the cell
occurs as a result of cell lysis. The features of replication of these
simple RNA viruses are themselves fairly simple. The viral RNA
itself functions as an mRNA and regulation occurs primarily by way
of controlling access of ribosomes to the appropriate start sites on
the viral RNA.
5.10 SINGLE-STRANDED ICOSAHEDRAL
DNA BACTERIOPHAGES
A number of small bacterial viruses have genomes consisting of
single-stranded DNA in circular configuration. These viruses are very
small, about 25 nm in diameter, and the principle building block of
the protein coat is a single protein present in 60 copies (the minimum
number of protein subunits possible in an icosahedral virus), to which
are attached at the vertices of the icosahedron several other proteins
which make up spike-like structures. In contrast to the RNA viruses,
much of the enzymatic machinery for the replication of DNA already
exists in the cell. These small DNA viruses possess only a limited
amount of genetic information in their genomes, and the host cell
DNA replication machinery is used in the replication of virus DNA.
The most extensively studied virus of this group is the phage
designated cjlX174, which infects Escherichia coli. cjlX174 is of special
interest because it was the first genetic element shown to have
overlapping genes. The genomes of cells are organized in linear
fashion, with the gene coding for each protein separate from that
for all other genes. In very small viruses such as cjlX 174 there is
insufficient DNA to code for all virus-specific pfjteins. cjlX174 has
solved this problem by the use of overlapping genes. Thus, parts of
certain nucleotide sequences are read twice, in different directions
and in different reading frames. It should be noted that although the
use of overlapping genes makes possible more efficient use of genetic
information, it seriously complicates the evolution process, since a
mutation in a region of gene overlap may affect two genes simultane-
ously.
As seen in the genetic map, the sequences of genes D and E
overlap each other, gene E being contained completely within gene
D. In addition, the termination codon of gene D overlaps the initiation
codon of gene J by one nucleotide. The reading frame of gene E is
therefore in a different phase (starting point) from that of gene D.
Obviously, any mutation in gene E will also lead to an alteration in
the sequence of gene D, but whether a given mutation affects one
or both proteins will depend on the exact nature of the alteration
(because the genetic code is degenerate). Other instances of gene
overlap through use of overlapping reading frames in cjlX 174 DNA
are genes AlB, K/B, KlC, KIA, AIC, and DIE. Additionally, a small
gene A protein, called A * protein, is formed by reinitiation of
translation (not transcription) within gene A mRNA, with A' protein
being read and terminated from the same mRNA reading frame as
A protein.
The DNA of OX174 consists of a circular singlestranded molecule
of 5386 nucleotide residues. The DNA of cjlX174 was the first DNA
to be completely sequenced, a remarkable achievement when it was
accomplished by Sanger and colleagues in 1977. Now, DNA
sequencing is a routine procedure. The replication process of such a
circular singlestranded DNA molecule is of considerable general
interest, since cellular DNA replicates always in the double-stranded
configuration. The DNA strand in the virion is referred to as the
plus (+) strand and the complementary strand the minus (-) strand.
Upon infection, the viral plus strand becomes separated from the
protein coat; entrance into the cell is accompanied by the conversion
of this singlestranded DNA into a double-stranded form called the
replicative form (RF) DNA. Cell-coded proteins involved in the
conversion of viral DNA into RF consist of the enzyme RNA primase
and DNA polymerase. ligase. and gyrase. No virus-coded proteins
are involved in the conversion of single-stranded DNA to RF. The
RF is a closed, double-stranded, circular DNA which has extensive
supercoiling.
DNA replication differs between the leading strand and the
lagging strand of the DNA double helix. In cells, replication Of the
lagging strand involves the formation of short RNA primers by action
of an enzyme called RNA primase (or primase for short). Such RNA
primers are made at intervals on the lagging strand and are then
removed and replaced with DNA by DNA polymerase.
In 4>X174, however, replication begins with a single stranded
closed circle, a rather atypical situation. First, primase brings about
the synthesis of a short RNA primer, beginning at one or more
specific initiation sites on the DNA.
Once priming of DNA synthesis has been carried out, the RNA
primer is replaced with DNA through action of DNA polymerase.
Continuation of DNA replication around the closed circle leads to
the formation of the complete double-stranded RF. Once the complete
second strand has been formed, its circle is closed with DNA ligase
and a DNA gyrase introduces twists that result in supercoiling. DNA
gyrase introduces supercoils by cutting one of the two strands of the
DNA double helix, holding the two ends apart without rotation,
passing a distant region of the circle through the cut, and resealing
the ends. The degree of supercoiling is determined by the number
of twists that have been introduced into the DNA. One result of
supercoiling is that it converts the DNA into a more compact form
where it takes up less room in the cell or virion.
Once the RF is formed, nucleic acid replication occurs by
conventional semiconservative replication, resulting in the formation
of new RF molecules. As in general DNA synthesis, initiation of
the formation of a new strand begins at a unique site on the DNA,
the origin of replication. In 4>X174, the origin of replication is at
residue 4395. Formation of single-stranded viral progeny begins with
a single-stranded cleavage of the viral (Plus) strand_ of the RF at the
origin of replication. Cleavage is brought about by a protein called
gene A protein; this protein also makes a covalent bond to the 5' P
of the viral strand. Asymmetric replication by the rolling circle
mechanism results in the formation of single-stranded molecules that
will become the virus progeny. When the growing viral strand reaches
unit length (5386 residues for cl>X174), gene A protein cleaves and
then ligates the two ends of the newly synthesized single strand to
give a viral single-stranded DNA. Early in infection, viral single-
stranded progeny DNA are rapidly converted into RF by the
mechanism already described above. Later in infection, when coat
protein has accumulated, the single-stranded DNA is packaged into
-virions. . RNA primer
rNA prtmer replaced with DNA .... ' "
o-o=o-o-~
virion supercoiled
replicating
DNA form (RF) RF
(a)
~-~-~- TOning Circle

replication 000
ma,:::::::. •
mature
00 virus
(b)
o particle
capSid proteins
Figure 5.14: Events in the replication of ~X174 DNA. (a) Formation of the replicating
double-stranded DNA (RF). (b) Rolling circle replication leads to the formation of
virus progeny.
Viral mRNA synthesis is directed by the RF. Synthesis of mRNA
begins at several major promoters on the RF, and terminates at a
number of sites. The polycistronic mRNA molecules are than
translated into the various phage proteins. The strengths of the several
promoters vary, so that some proteins are made in smaller amounts
than others. Each promoter activates the transcription of a functionally
related set of genes. As we have noted, protein A and protein A'
are both made from the same gene, protein A' arising as a result of
translation of a secondary initiation site internal to the A mRNA.
Further, as we have noted, several proteins are made from mRNA
transcripts formed from different reading frames from the same DNA
sequences. One can truly be impressed by the efficiency with which
such a small genome as that of cl>X174 can have multiple uses.
Ultimately, assembly of mature virus particles occurs. Release
of virions from the cell occurs as a result of cell lysis, which involves
the participation of gene E protein.
5.11 SINGLE-STRANDED FILAMENTOUS
DNA BACTERIOPHAGES
Quite distinct from q,X174 are the filamentous DNA phages,
which have helical rather than icosahedral symmetry. 'ijJ.e most studied
member of this group is phage M13, which infects Escherichia coli.
but related phages include f1 and fd. As with the small RNA viruses,
these filamentous DNA phages only infect male cells, entering after
attachment to the male-specific pilus. Interestingly, even though these
phages are linear (filamentous) they possess circular DNA. The DNA
is not self-complementary, however, so that the two adjacent halves
of the molecule which run up and down the virus particle form loops
at the ends but exhibit very little if any base pairing. Phage M13
has found extensive use as a cloning vector and DNA sequencing
vehicle in genetic engineering. The virion of M 13 is only 6 run in
diameter but is 860 run long. These phages have the additional unique
property of being released from the cell without killing the host cell.
Thus, a cell infected with phage M13 or fd can continue to grow,
. all the while releasing virus particles. Virus infection causes a slowing
of cell growth, but otherwise a cell is able to coexist with its virus.
Plaques are thus seen only as areas of thinner cell growth in the
bacterial lawn.
Many aspects of DNA replication in filamentous phages are
similar to that of q,X 174. The unique property, release without cell
killing, can be briefly discussed. The release from the cell occurs
by a budding process in which the virus particle is always released
from the cell with the end containing the A protein first. Interestingly,
the orientation of the virus particle across the cell membrane is the
same for its entry and exit from the cell. There is no accumulation
of intracellular virus particles; the assembly of mature virus particles
occurs on the inner cell membrane and virus assembly is coupled
with the budding process.
Several features of these phages make them useful as cloning and
DNA sequencing vehicles. First, they pave single-stranded DNA, which
means that sequencing can be carried out by the Sanger
dideoxynucleotide method. Second, as long as infected cells are kept
in the growing state they can be maintained indefinitely with cloned
DNA, so that a continuous source of the cloned DNA is available.
Third, there is an intergenic space which does not code for protein
and can' ~ teplaced by variable amounts of foreign DNA.
A protein B protein
~~.D protein Gene 3: A protein

Gene 8: B protein
intergenic space
Genes 7,9: C protein

Gene 6: D protein
Figure 5.15 : The filamentous single-stranded DNA bacteriophage fd. Orientation of

the proteins and genes in the virion. Note the intergenic space which contams the
origin of DNA synthesis. Gene cloning is done in this intergenic space.
cell
cell membrane
wall
coat proteins embedded

in cell membrane
phage DNA:
ss circle
cytoplasm
environment
Figure 5.16 : Illustration of the manner by which the virion of a filamentous single-
stranded phage (such as M13 or fd) leaves an infected cell without lysis. The A protein
passes first through the membrane at a site on the membrane where coat protein
molecules have first become imbedded. The intracellular circular DNA is coated with
dimers of another protein, gp5, \\'hich is displaced by coat protein as the DNA passes
through the intact membrane.
5.12 DOUBLE-STRANDED DNA BACTERIOPHAGES
Many· bacterial viruses have genomes containing double-stranded
DNA. Such viruses were the first bacterial viruses discovered, and
have been the most extensively studied. With such a range of double-
stranded DNA viruses, a wide variety of replication systems are
present. In the present section, we discuss the best studied and most
representative of the group, T4 and 17. The simpler, 17, will be
discussed first.
Bacteriophage 1'7 Bacteriophage 17 and its close relative T3
are relatively small DNA viruses that infect Escherichia coli. (Some
strains of Shigella and Pasteurella are also hosts for phage 17.) The
virus particle has an icosahedral head and a very small tail. The
virus particle is fairly complex, with 5 different proteins in the head
and 3-6 different proteins in the tail. One tail protein, the tail fiber
protein, is the means by which the virus particle attaches to the
bacterial cell surface. Only female cells of Escherichia coli can be
infected with 17; male cells can be infected but the multiplication
process is terminated during the latent period.
The nucleic acid of the 17 genome is a linear double-stranded
molecule of 39,936 base pairs. The complete genome has been
sequenced, and the sequence information has permitted discernment
of gene structure and features of gene regulation. About 92 percent
of the DNA of 17 codes for proteins. At least 25 separate genes
have been characterized, but not all genes are separately coded on
the DNA. Gene overlap occurs for several genes. through translation
in different reading frames and through internal reinitiation with one
or more genes in the same reading frame. Further genetic economy
is achieved by internal frame shifts within certain genes to yield longer
proteins.
When the phage particle attaches to the bacterial cell, the DNA
is injected in a linear fashion, with the genes at the "left end" of
the genetic map entering the cell first. Several genes at the left end
of the DNA are transcribed immediately by a cell RNA polymerase,
using three closely spaced promoters, generating a set of overlapping
polycistronic mRNA molecules. These mRNA molecules are then
cleaved by a specific RNase of the cell at 5 sites, thus generating
smaller mRNA molecules which code for one to four proteins each.
One of these proteins is an RNA polymerase that copies double-
stranded DNA. Two other early mRNA molecules code for proteins
which stop the action of host RNA polymerase, thus turning off the
transcription of the early genes as well as the transcription of host
genes. Thus, a" host RNA polymerase is used just to copy the first
few genes and to make the mRNA for the phage-specific RNA
polymerase, and this phage specific RNA polymerase is then involved
in the major RNA transcription processes of the phage. This 17 RNA
polymerase uses a new set of promoters that are distributed along
the left-center and center portions of the genome. It is thus seen that
regulation of 17 has both negative and positive control: negative, by
means of the formation of proteins that stop host RNA polymerase
and thus shut off transcription of the early T7 genes that are
recognized by this enzyme, and positive, by means of the formation
of the new
gene disignation function
left end
Early promoters - _
0.3 _ Overcomes host restriction
0.7 • Protein kinase

Early transcription
1 • RNA polymerase
1.1 UnknOwn
Promoters - - - - - --Stert of DNA replication
1.3 DNA ligase
1.7 _ Nonessential
2 _ Inactivate host RNA polymerase
3 _ Endonuclease
3.5 - Lysozyme
4 11 Hellcase. prlmase
Protein
for DNA
I replication
promoter
____
_ 8_._ 5
7
DNA polymerase
Exonuclease
Protein in phage particle

8 • Head protein
9 • Head assembly protein

Major head protein
promoter - - - - _
Tall protein
Tall protein
promoter - - - - - .
1 Protein in phage particle
14 _ Head protein
15 11 Head protein Phage
_____161
maturation
Head protein
promoter
17 Tail protein
18 • DNA maturation
19 _ DNA maturation
Right end
Figure 5.17 : Genetic map of phage T 7, showing gene numbers, approximate sizes,
and functions of the gene products.
RNA polymerase which recognizes the rest of the T7 promoters. We
also note that T7 is an example of a virus which strongly affects
host transcription and translation processes, by producing proteins
which turn off transcription of host genes. The virus also has genes
coding for enzymes which degrade host cell DNA, and nucleotides
from such degraded DNA end up in ViI'Us.'progeny. Obviously, such
a virus has profound pathological effects on its host cell.
As seen in the genetic map, the genes after gene 1.1, transcribed
by the T7 RNA polymerase, code for proteins that are involved in
17 DNA synthesis, the formation of virus coat proteins, and assembly.
Three classes of T7 proteins are formed: class I, made 4-8 minutes
after infection, which use the cell RNA polymerase; class II, made
6-15 minutes after infection, which are made from T7 RNA
polymerase and are involved in DNA metabolism; class III, made
from 6 minutes to lysis, which are transcribed by T7 RNA polymerase
and which code for phage assembly and coat protein. This sort of
sequential pattern, commonly seen in many large double-stranded
DNA phages, results in an efficient channeling of host resources,
first toward DNA metabolism and replication, then on to formation
of virus particles and release of virus by cell lysis.
DNA replication in T7 begins at an origfn of replication at which
DNA synthesis is initiated, and DNA synthesis proceeds bidirectionally
from this origin. In both directions, an RNA primer is involved, but
the enzyme involved in the synthesis of this primer is different for
primer synthesis in the leftward and rightward direction. In the
rightward direction, the RNA primer is synthesized by 17 RNA
polymerase. whereas in the leftward direction, a virus-specific enzyme,
T7 primase (gene 4 protein) is used. Both primers are then elongated
by T7 polymerase. Replicating molecules of T7 DNA can be
recognized under the electron microscope by their characteristic
structures. Because the origin .of replication is near the left end,
Y-shaped molecules are frequently seen, and earlier in replication,
bubble-shaped molecules appear. .
A structural feature of the T7 DNA which is important in DNA
replication is that there is a direct tenninal repeat of 160 base pairs
at the ends of the molecule. In order to replicate DNA near the 5'
terminus, RNA primer molecules have to be removed before
replication is complete. There is thus an unreplicated portion of the
T7 DNA at the 5' terminus of each strand. The opposite single 3'
strands on two separate DNA molecules, being complementary, can
pair with these 5' strands, forming a DNA molecule twice as long
as the original T7 DNA. The unreplicated portions of this end-to-
end bimolecular structure are then completed through the action of
DNA polymerase and DNA ligase, resulting in a linear bimolecule,
called a concatamer. Continued replication can lead to concatamers
of considerable length, but ultimately a cutting enzyme slices each
concatamer at a specific site, resulting in the formation of virus-
sized linear molecules with repetitious ends.
We thus see that 17 has a much more complex replication scheme
than that seen for the other bacterial viruses discussed earlier.
5.13 LARGE DOUBLE-STRANDED
DNA BACTERIOPHAGES
One of the most extensively -studied groups of DNA viruses is
the group sometimes called the T-even phages, which include the
phages T2, T4, and T6. These phages are among the most
complicated in terms of both structure and manner of multiplication
replication. In the present section, we will discuss primarily
bacteriophage T4, the phage of this group for which the most
information is available.
The virus particle of phage T4 is structurally complex. It consists
of an icosahedral head which is elongated by the addition of one or
two extra bands of protein hexamers, the overall dimensions of the
. N~
~\~C~~Ylation--HOH,c6o
I
H
Figure 1.18 : The unique base in the DNA of the T-even bacteriophages,
5-hydroxymethylcytosine. The site of glucosylation is shown.
head being 85 x 110 nm. To this head is attached a complex tail
consisting of a helical tube (25 x 110 nm) to which are connected a
sheath, a connecting "neck" with "collar" and "whiskers," and a
complex base plate with pins, to which are attached long jointed tail
fibers. All together, the virus particle has over 25 distinct types of
proteins.
As we noted, the DNA of T4 has a total length about 650 times
longer than the dimension of the head. This means that the DNA is
highly folded and packed very tightly within the head.
The genome structure of T4 is quite complex. The DNA is large,
with a molecular weight of about 120 x 10, and is chemically distinct
from cell DNA, having a unique base, 5-hydroxymethylcytosine instead
of cytosine. The hydroxyl groups of the 5-hydroxymethylcytosine are
modified by addition of glucosyl residues. This glucosylated DNA is
resistant to virtually all restriction endonucleases of the host. Thus,
this virus-specific DNA modification plays an important role in the
ability of the virus to attack a host cell. Over 160 separate genes
have been recognized in T4, of which the functions of 120 are known.
These genes code not only for the complex array of coat proteins,
but for a variety of enzymes and other proteins involved in the
replication process itself.
Figure 5.19 : Simplified genetic map of T4. Late genes with morphogenetic functions
(coat proteins and assembly), and genes with functions in DNA replication are identified.
Note that although the genetic map is represented as a circle, the DNA itself is actually
linear.
The genetic map of T4 is generally represented as a circle, even
though the DNA itself is linear. This "genetic circularity" arises
because the DNA of the phage exhibits a phenomenon called circular
permutation. This arises because in different T4 phage particles, the
sequence of bases at each end differs (although for a given molecule
the same base sequence occurs at both ends). This structure, a
consequence of the way the T4 DNA replicates (see later), results in
an appearance of genetic circularity even though the DNA itself is
lineat.
mRNA synthesis and regulation in bacteriophage T4 In
bacteriophage T4, the details of regulation of replication are more
complex than those of T7, but involve primarily positive control. T4
is a much larger phage .than T7 and has many more genes and phage
functions. In addition, the DNA of T4 contains the unusual base, 5-
hydroxymethylcytosine and some of the OH groups of this base are
glucosylated. Thus enzymes for the synthesis of this unusual base
and for its glucosylation must be formed after phage infection, as
well as formation of an enzyme that breaks down the normal DNA
precursor deoxycytidine triphosphate. In addition, T4 codes for a
number of enzymes that have functions similar to those host enzymes
in DNA replication, but are formed in larger amounts, thus permitting
faster synthesis of T4-specific DNA. In all, T4 codes for over 20
new proteins that are synthesized early after in fection. It also codes
for the synthesis of several new tRNAs, whose function is presumably
to read more efficiently T4 mRNA.
Overall, the T4 genes can be divided into three groups, for early,
middle, and late proteins. The early proteins are the enzymes involved
in DNA replication. The middle proteins are also involved in DNA
replication. For instance, a DNA unwinding protein (DNA gyrase)
is formed which destabilizes the DNA double helix, forming short
single-stranded regions at which DNA synthesis can be initiated. The
late proteins are the head and tail proteins and the enzymes involved
in liberating the mature phage particles from the cell. In T4, there
is no evidence for a new phage-specific RNA polymerase, as in T7.
The control of T4 mRNA synthesis involves the production of proteins
that modify the specificity of the host RNA polymerase so that ·it
recognizes different phage promoters. The early promoter, present
at the beginning of the T4 genome, is read directly by the host RNA
polymerase, and involves the function of host sigma factor. Host RNA
polymerase m~ves down the chain until it reaches a stop signal. One
of the early proteins blocks host sigma factor action. The early protein
combines with the RNA polymerase core enzyme, and when this
protein builds up, initiation of early phage genes is stopped. The
RNA polymerase cores are now available to combine with new phage-
specific activators, which control the transcription of the middle and
late genes. The middle genes are generally transcribed along the same
DNA strand as the early genes, but the late genes are transcribed
along the opposite strand.
head
host
-==
!
®
tail
endplate IiiiiJ
l I DMA packaged
into head
Iiiiil
l
IiiiiI
® ~
head precursor
!
endplate
j
~
joined to
core
t t l
-1
1- ~l·':· sheath
protein
added
~ -1 j~ ~.=~
1 head and
tail joined host
tsJrj"
\~
fr-
stabilized
~
tail
tail ::\~\~\..J
fibers
added
complete
,'. L -I-i-
infective
particle
Figure 5.20 : Steps in the assembly

of a T4 bacterial virus.
DNA replication The process of DNA replication in T4 is similar
to that in TI, but in T4, the cutting enzyme which forms virus-sized
fragments does not recognize specific locations on the long molecule,
but rather cuts off head-full packages of DNA irrespective of the
sequence. Thus each virus DNA molecule not only contains repetitious
ends, but the nucleotide sequences at the ends of different molecules
are different, although each molecule contains the complete sequence
of viral genes. As shown, the cutting process results in the formation
of DNA molecules with permuted sequences at the ends.
Assembly and lysis In the case of T4, assembly of heads and
tails occur on independent pathways. DNA is packaged into the
assembled head and the tail and tail fibers are added subsequently.
Somehow, the DNA is packed tightly and inserted into the empty
phage head.
Exit of the virus from the cell occurs as a result of cell lysis.
The phage codes for a lytic enzyme, the T4 lysozyme, which causes
an attack on the peptidoglycan of the host cell. The burst size of the
virus (the average number of phage partides per cell) depends upon
how rapidly lysis occurs. If lysis occurs early. then a smaller burst
size occurs, whereas slower lysis leads to a higher burst size. The
wild type phage exhibits the phenomenon of lysis inhibition, and
therefore has a large burst si,?:e, but rapid lysis mutants, in which
lysis occurs early, show smaller burst sizes.
5.14 TEMPERATE BACTERIAL VIRUSES: LYSOGENY
Most of the bacterial viruses described above are called virulent
viruses, since they usually kill the cells they infect. However, many
other viruses, although also often able to kill cells, frequently have
more subtle effects. Such viruses are called temperate. Their genetic
material can become integrated into the host genome and is thus
duplicated along with the host material at the time of cell division,
being passed from one generation of bacteria to the next. Under
certain conditions these bacteria can spontaneously produce virions
of the temperate virus, which can be detected by their ability to infect
a closely related strain of bacteria. Such bacteria, which appear
uninfected but have the hereditary ability to produce phage, are called
lysogenic.
With most temperate phages, if the host simply makes a copy
of the viral DNA, lysis does not occur; but if complete virion pruticles
are produced, then the host cell lyses. In a lysogenic bacterial culture
at anyone time, a small fraction of the cells, 0.1 to 0.0001 percent,
produce virus and lyse, while the majority of the cells do not produce
virus and do not lyse. Although only rarely do cells of a lysogenic
strain actually produce virus, every cell has the potentiality. Lysogeny
can thus be considered a genetic trait of a bacterial strain.
The temperate virus does not exist in its mature, infectious state
inside the cell, but rather in a latent form, called the provirus or
prophage state. In considering virulent viruses we learned tha~ the
DNA of the virulent virus contains information for the synthesis of
a number of enzymes and other proteins essential to virus
reproduction. The prophage of the temperate virus carries similar
information, but in the lysogenic cell this information remains dormant
because the expression of the virus genes is blocked through the action
of a specific repressor coded for by the virus. As a result of a genetic
switch, the repressor is inactivated, virus reproduction occurs, the
cell lyses, and virus particles are released.
A lysogenic culture can be treated so that most or all of the
cells produce virus and lyse. Such treatment, called induction, usually
involves the use of agents such as ultraviolet radiation, nitrogen
mustards, or X rays, known to damage DNA and activate the SOS
system. However, not all prophages are inducible; in some temperate
viruses, prophage expression occurs only by natural events.
Although a lysogenic bacterium may be susceptible to infection
by other viruses, it cannot be infected by virus particles of the type
for which it is lysogenic. This immunity, which is characteristic of
lysogenized cells, is conferred by the intracellular repression mechanism
under the control of virus genes.
It is sometimes possible to eliminate the lysogenic virus (to "cure"
the strain) by heavy irradiation or treatment with nitrogen mustards.
Among the few survivors may be some cells that have been cured.
Presumably the treatment causes the prophage to detach from the
host chromosome and be lost during subsequent cell growth. Such a
cured strain is no longer immune to the virus and can serve as a
suitable host for study of virus replication.
How is it possible to determine whether a strain is lysogenic? A
sensitive host is necessary-that is, a strain closely related to the
presumed lysogenic strain but not infected with the prophage. In
practice, a large number of related strains are obtained, either from
nature or from culture collections. The presumed lysogenic strain is
cultured in a suitable medium where it grows, infrequently releasing
phage. The titer of phage particles in an induced lysogenic culture
~
~altachment
\
cell (host)
Injection
lytic infection lysogenic cycle
~I!I ~. ~ #\ viral DNA

\ : ~ '" Q ~.J
coat proteIns
synthesized;
virus particles
! rephcates
assembled
lysogenIC induction normal cell growth
Figure 5.21 : Consequences of infection by a temperate bacteriophage. The alternatives

upon infection are integration of the virus DNA into the host DNA (lysogenization) or
replication and release of mature VIruS (lysis). The lysogenic cell can also be induced
to produce mature virus and lyse.
is typically lO'-lO$/ml. Irradiation can be used to attempt to induce
the prophage to replicate. After further incubation, the culture is
filtered to remove live bacteria, and the filtrate is tested against the
various test strains using the agar overlay technique described for
use in assaying virus particles. If plaques are seen, it can be assumed
that virus particles are present and that the strain is lysogenic.
Sometimes a large number of strains must be tested to fmd a sensitive
host. Most bacteria isolated from nature are lysogenic for one or
more viruses.
Consequences of temperate virus infection What happens when
a temperate virus infects a nonlysogenic organism? The virus may
inject its DNA and initiate a reproductive cycle similar to that
described for virulent viruses, with the infected cell lysing and
releasing more virus particles. Alternatively, when the virus injects
its DNA, lysogenization may occur instead: the viral DNA becomes
incorporated into the bacterial genetic material and the host bacterium
is converted into a lysogenic bacterium. In lysogenization the infected
cell thus becomes genetically changed. Sensitive cells can undergo
either lysis or lysogenization; which of these occurs is often
determined by the action of a complex repression system, as will be
described below. We thus see that the temperate virus can have a
dual existence. Under one set of conditions, it is an independent entity
able to control its own replication, but when its DNA is integrated
into the host genetic material, replication is then under the control
of the host.
\(4'\ I":
~ "': ~ .:,
~.:~;,;-?r~ ~W1~I~ ~ ~\:;:.~ :~ :N~:'?:f;~;'·" :

.•
Figure 5.22 : Electron micrograph by negative staining of a lambda bacteriophage

particle. .
Regulation of lambda reproduction One of the best studied
temperate phages is lambda, which infects E. coli, and our knowledge
of the molecular mechanisms involved in lysogenization and lytic
processes in this phage is very adQnced. Morphologically, lambda
particles iook like those of many other bacteriophages. The virus
particle has an icosahedral head 64 nm in diameter, and a tail 150
nm which has helical symmetry. Attached to the tail is a single 23
nm long fiber. In addition to the major proteins of the coat, there
are a number of minor coat proteins.
The nucleic acid of lambda consists of a linear double-stranded
molecule of 31 x 1()6 molecular weight. At the 5' terminus of each
of the single-strands is a single-stranded tail of 12 nucleotides in length
which are complementary (the ends of the DNA are'said to be
cohesive). Thus, when the two ends of the DNA are free in the host
cell, they associate and form a double-stranded circle. In the circular
form the DNA contains 48,502 base pairs, and its complete sequence
is known.
Lysis or lysogenization? If lysogeny occurs, then the phage genes
are maintained stably in the lysogenic state until a switch occurs and
they are converted with high efficiency into a second state in which
lytic growth occurs. This process is called lysogenic induction. How
does the genetic switch from lysogeny to lysis occur?
The lambda genome has two sets of genes, one controlling lytic
growth, the other lysogenic growth. Upon infection, genes promoting
both lytic growth and lysogenic integration are expressed. Which
pathway succeeds is determined by the competing action of these
early gene products and by the influence of host factors. To
understand how this works, we need to present the genetic map of
lambda. The genetic map, although actually linear, can thus be
oriented as a circle (because of the cyclization via the cohesive ends
mentioned above). The lambda map consists of several operons, each
of which controls a set of related functions. Some of the phage genes
are transcribed by RNA polymerase from one strand of the double
helix, and others are transcribed from the other strand. Upon
injection, transcription of the phage genes which code for the lambda
repressor occurs, and if repressor builds up before lytic functions
are expressed, lytic reproduction is blocked. The repressor protein
blocks the transcription of all later lambda genes, thus preventing
expression of the genes involved in the lytic cycle.
In a lysogenic cell, a single phage gene is expressed continuously,
the gene which codes for the lambda repressor protein. This repressor
protein, which is coded for by a gene called cI, binds to two operators
on the lambda DNA and thereby turns off the transcription of all
the other genes of the phage genome. This is the negative control
function of the lambda repressor. In addition, the lambda repressor
turns on its own synthesis. This is the positive control function of
the lambda repressor. Thus, by promoting its own synthesis, the
lambda repressor ensures that no other genes except the gene coding
for itself is made. In a lysogenic cell, there will usually be only one
copy of the lambda genome, but about 100 active molecules of
"
repressor protein. Therefore, there is almost always excess repressor

to bind to lambda DNA and prevent the transcription of the genes
necessary for lambda growth and reproduction.
~
L1 §
E
.5
5'G i 3'
VIrion DNA ends

Figure 5.23 : Genetic and molecular map of lambda.
Lytic growth of lambda How, then, does lambda virus
multiplication occur? In a lysogenic cell, multiplication of lambda
occurs only after the repressor is inactivated. As we have noted,
agents which induce lysogenic cells to produce phage are agents which
damage DNA, such as ultraviolet irradiation, X rays, or DNA-
damaging chemicals such as the nitrogen mustards. Upon DNA
damage, a host defense mechanism called the SOS response is brought
into play. An array of 10-20 bacterial genes is turned on, some of
which help the bacterium survive radiation. However, one result of
DNA damage is that a bacterial protein called RecA (normally
involved in genetic recombination) is turned into a special kind of
protease which participates in the destruction of the lambda repressor.
With lambda repressor destroyed, the inhibition of expression of
lambda lytic genes is abolished. We should note that the protease
activity of RecA, brought about by DNA damage, nonnally plays
an important role in the cell's response to DNA damaging agents,
by participating in the breakdown of a host protein, LexA, which
represses a set of host genes involved in DNA repair. Induction of
bacteriophage lambda is thus an indirect consequence of the SOS
response.
Once the lambda repressor has been inactivated, the positive and
negative control exerted by this repressor are abolished, and new
transcriptional events can be initiated. The most important
transcriptional event is that involved in the synthesis of another lambda
protein called Cro. coded by a gene called ero. The gene ero is
located almost adjacent to the gene cl which codes for lambda repressor.
The key to the genetic switch lies in the close proximity of the
regulator genes for repressor and cro protein. These two genes are
transcribed in opposite directions, beginning at different start points.
DNA damage
____ ~ 1I I I IiI .-~ _ host

genome
activation of SOS response
!
activation of host RecA protein;
conversion of RecA protein to a protease
protease cleavage of lambda repressor

protein (cl)
lytic response
Figure 5.24 : Activation of the host SOS response leads to lysis of a lysogenic cell.
In the region separating these two genes are two kinds of sites,
promoters and operators, to which each of the proteins of the switch
can bind. When lambda repressor is bound to its operator, it covers
leftward transcription rightward transcription
- C~RM r-,--O-R----.I PRcro~
Figure 5.25 : Two back-to-back promoters in the region of cI and cro control the
genetic switch. When cI is present, it activates its own synthesis and blocks transcription
of cro. When cI is inactivated, transcription of cro can occur, resulting in the lytic
cycle. The cI (repressor) protein combines with the operator, OR'
the ero promoter, whereas when Cro is bound, it covers one of the
cl promoters. As we have noted, the direction in which transcription
occurs on a DNA double-strand (and hence which of the two strands
is read) depends upon the promoter. A promoter essentially points
the RNA polymerase in the proper direction. In the case of lambda,
the cl promoter points RNA polymerase "leftward," whereas ero
promoter points the polymerase "rightward."
The lambda system provides one of the best studied examples
of a genetic switch, in which one or the other of two competing
genetic functions occurs. Which of the two genetic functions gets
the upper hand will depend initially on chance events, but once one
of the two functions has become established, it prevents the action
of the other function. Only under unusual circumstances, such as
when induction occurs, would the dominant genetic function be
superseded.
Integration Integration of lambda DNA into the host chromosome
occurs at a unique site on the E. coli genome. Integration occurs by
insertion of the virus DNA into the host genome (thus effectively
lengthening the host genome by the length of the virus DNA).
The cohesive ends of the linear lambda molecule find each other
and form a circle, and it is this circular DNA which becomes
integrated into the host genome. To establish lysogeny, genes cl and
int must be expressed. As we have noted, the cI gene product is a
protein which represses early transcription and thus shuts off
transcription of all later genes. The integration process requires the
product of the int gene, which is a site-specific topoisomerase
catalyzing recombination of the phage and bacterial attachment sites.
During cell growth, the lambda repression system prevents the
expression of the integrated lambda genes except for the gene c/,
which codes for the lambda repressor. During host DNA replication,
the integrated lambda DNA is replicated along with the rest of the
host genome, and transmitted to progeny cells. When release from
repression occurs, the lambda productive cycle occurs. '
att m'
m lambda DNA
l
m'
cyclizes at
cohesive ends
m.m'
l
m'
site-specific nuclease
creates staggered ends of
. / phage and host
gal phr . / bio ch/A
--- --- ---·----==~::::::JI _~====-
":=::==.=-,== ,== -_:::::::.:.-_-=-
----------
I integration of lambda
t DNA and closing of gaps
gal phr m' by DNA ligase bio ch/A
i i
m
Figure 5.26 : Integration of lambda DNA into the host. Integration always occurs at a
specific site on the host DNA, involving a specific attachment site (att) on the phage.
Some of the host genes near the attachment site are given. A sitespecific enzyme
(integrase) is involved, and specific pairing of the complementary ends results in
integration of phage DNA.
Replication Replication of lambda DNA occurs in two distinct
fashions during different parts of the phage production cycle. Initially,
liberation of lambda DNA from the host results in replication of a
circular DNA, but subsequently linear concatamers are formed, which
replicate in a different way. Replication is initiated at a site close to
gene 0 and from there proceeds in opposite directions (bidirectional
symmetrical replication), tenninating when the two replication forks
meet. In the second stage, generation of long linear concatamers
occurs, and replication occurs in an asymmetric way by rolling circle
replication. In this mechanism, replication proceeds in one direction
only, and can result in very long chains of replicated DNA. This
mechanism is efficient in permitting extensive, rapid, relatively
uncontrolled DNA replication; thus it is of value in the later stages
of the phage replication cycle when large amounts of DNA are needed
to form mature virions. The long concatamers formed are then cut
into virus-sized lengths by a DNA cutting enzyme. In the case of
lambda, the cutting enzyme makes staggered breaks at specific sites
on the two strands, twelve nucleotides apart, which provide the
cohesive ends involved in the cyclization process.
Lambda is one of the agents of choice for use as a cloning vector
for artificial construction of DNA hybrids with restriction enzymes.
It has several features that make it an excellent system for genetic
engineering. One feature of lambda that makes it of special use for
cloning is that there is a long region of DNA, between genes Jand
au, which does not seem to have any essential functions for
replication, and can be replaced with foreign DNA.
Temperate viruses as plasmids Another class of temperate
viruses have quite a different mechanism for maintenance of the
prophage state. In this group, viruses resemble plasmids. They do
not actually become integrated into the host chromosome, but instead
replicate in the cytoplasm as circular DNA molecules. Among such
viruses is bacteriophage PI of Escherichia coli. Although in broad
features, such viruses resemble the temperate viruses such as lambda
just discussed, at the level of virus replication they are, of course,
quite different. Interestingly, although the plasmid prophage is not
physically connected to the host DNA, phage DNA replication is
closely coordinated with cell division, since only one copy of the
prophage is present per host chromosome.' The phage repressor is
somehow involved in this regulation process.
5.15 A TRANSPOSABLE PHAGE:
BACTERIOPHAGE MU
One of the more interesting bacteriophages is that called Mu,
which has the unusual property of replicating as a transposable
element. This phage is called Mu because it is a mutator phage,
inducing mutations in a host into which it becomes integrated. This
mutagenic property of Mu arises because the genome of the virus
can become inserted into the middle of host genes, causing these
genes to become inactive (and hence the host which has become
infected with Mu behaves as a mutant). Mu is a useful phage because
it can be used to generate a wide variety of mutants very easily. Mu
can be used in genetic engineering.
A transposable element is a piece of DNA which has the ability
to move from one site to another as a discrete element. Transposons
are found in both procaryotes and eucaryotes, and play important
roles in genetic variation. There are three types of transposable
elements: insenion elements, transposons, and viruses like Mu. (Also
the retroviruses discussed later, are a group of animal viruses which
have features of transposable elements). An insenion element is the
simplest type of transposable element: it carries no detectable genes
and simply moves itself around. A transposon is a genetic element
with a unique piece of DNA, usually coding for one or more proteins,
to which is attached, at each end, an insertion element. The insertion
element at each end of the transposon is identical, although the DNA
sequence may be either a direct repeat or an inverted repeat. Mu is
a very large transposable element, carrying insertion elements and a
number of Mu genes involved in Mu multiplication.
Structurally, bacteriophage Mu is a large doublestranded DNA
virus, with an icosahedral head, a helical tail, and 6 tail fibers. The
DNA of Mu is arranged inside the virus head as a linear double-
stranded molecule. It has a molecular weight of 25 X 106 (3839
kilobases). The genetic map of Mu is shown in Figure 6.37a. It can
be seen that the bulk of the genetic information is involved in the
synthesis of the head and tail proteins, but that important genes at
each end are involved in replication and immunity. At the left end
of the Mu DNA are 50-150 base pairs of host DNA and at the right
end are 500-3000 base pairs of host DNA. These host DNA sequences
are not unique and represent DNA adjacent to the location where
Mu had become inserted into the host genome.
When a Mu phage particle is formed, a length of DNA containing
the Mu genome just large enough to fill the phage head is cut out of
the host, beginning at the left end. The DNA is rolled in until the
head is full but the place at the right end where the DNA is cut varies
from one phage particle to another. For that reason, as shown on the
genetic map, there is a variable sequence of host DNA at the right-
hand end of the phage (right of the attR site) which represents the
host DNA that has become packaged into the phage head. Each phage
particle arising from a single infected cell will have a different amount
of host DNA, and the host DNA base sequence of each particle from
the same cell will be different. In some cases, completely empty Mu
heads become filled with purely host DNA. Such particles can transfer
host genes from one cell to another, a process called transduction.
posrtive acbvat90r
of late mANA synthesIS
invertible G
Immunity segment
lysis
(host range)
I
Integration
replication I
head and tail genes
rL,
variable and
(host DNA)
host rJ.., .---------'-------..IrL,
DNA
, Iys
lie AB C DEHFGITJKLMVNP Rsuu's'l
I I
allL allR
transposase
(a)
l
I - - - - , I G C C G A A G C A G C G T T G~_ _ _--1
.CGGCTTCGTCGCAAC.
L---I • 1 L---I
1-----41
L -_ _ _~CGGCTTCGTC
GC CGA A G C A G C G T T GI..._ _ _-I
GCAAC
'-----I
I----,IG C C G A A G C A G A G C A G C G T T G~_ _ _--1

CGGCTTCGTC TCGTCGCAAC
'-------' '-------'
repair of DNA leads to

formation of S-base·pair
duplication
Figure 5.27 : Bacteriophage Mu. (a) Genetic map of Mu. (Confusingly, there are two
G's, the G gene and the invertible G segment. These are different O's.) (b) Integration
of Mu into the host DNA. showing the generation of a five-base-pair duplication of
host DNA.
A~ shown in the genetic map, a specific segment of the Mu
genome called G (distinct from the G gene) is invertible, being present
in either the orientation designated SU, or in the inverted orientation
U'S'. The orientation of this segment determines the kind of tail fibers
that are made for the phage. Since adsorption to the host cell is
controlled by the specificity of the tail fibers, the host range of Mu
is determined by which orientation of this invertible segment is present
in the phage. If the G segment is in the orientation designated G+·
then the phage particle will infect Escherichia coli strain K12. If the
G segment is in the G- orientation, then the phage particle will infect
Escherichia coli strain C or several other species of enteric bacteria.
The two tail fiber proteins are coded on opposite strands within this
small G segment. Left of the G segment is a promoter that directs
transcription into the G segment. In the orientation G +, the promoter
directing transcription of S and U is active, whereas in the orientation
G-, a different promoter directs transcription of genes S' and U' on
the opposite strand.
Upon infection of a host cell by Mu, the DNA is injected. In
contrast with lambda, integration into the host genome of Mu is
essential for both lytic and lysogenic growth. Integration requires the
activity of the A gene product, which is a transposase enzyme. At
the site where the Mu DNA becomes integrated, a 5 base pair
duplication of the host DNA arises at the target site. This host DNA
duplication arises because staggered cuts are made in the host DNA
at the point Mu becomes inserted, and the resulting single-stranded
segments are converted into the double-stranded form as part of the
integration process.
Lytic growth of Mu can occur either upon initial infection, if
the c gene repressor is not formed, or by induction of a lysogen. In
either case, replication of Mu DNA involves repeated transposition
of Mu to multiple sites on the host genome. Initially, transcription
of only the early genes of Mu occurs, but after gene C protein, a
positive activator of late RNA synthesis, is expressed, the synthesis
of the Mu head and tail proteins occurs. Eventually, expression of
the lytic function occurs and mature phage particles are released.
Because Mu integrates at a wide variety of host sites, it can be
used to induce mutants at many locations. Also, Mu can be used to
carry into the ceil genes that have been derived from other host cells,
a form of in vivo genetic engineering. In addition, modified Mu phage
have been made artificially in which some of the harmful functions
of Mu have been deleted. These phages, called Mini-Mu, are deleted
for significant portions of Mu but have the ends of the phage in
- nonnal orientation. Mini-Mu phages are usually defective, unable to
form plaques, and their presence must be ascertained by the presence
of other genes which they carry. One set of Mini-Mu phages
containing the [3-galactosidase gene of the host (called Mudlac, d for
defective) can be detected in 'the integrated state if the lac gene is
oriented properly in relation to a host promoter. Under these
conditions, the hOst cell will form the enzyme [3-galactosidase, which
can be detected in colonies by a special color indicator. f3-
galactosidase-positive colonies from a agalactosidase-negative host are
thus an indication that Mud-lac infection has occurred. We thus see
that phage Mu provides a useful tool for geneticists, as well as being
an interesting bacteriophage in its own right.
5.16 GENERAL OVERVIEW OF ANIMAL VIRUSES
We have discussed in a general way the nature of animal viruses
in the first part of this chapter. Now we discuss in some detail the
structure and molecular biology of a number of important animal
viruses. Viruses will be discussed which illustrate different ways of
replicating, and both RNA and DNA viruses will be covered. One
group of animal viruses, those called the retroviruses, have both an
RNA and a DNA phase of replication. Retroviruses are especially
interesting not only because of their unusual mode of replication,
but because retroviruses cause such important diseases as certain
cancers and acquired immunodeficiency syndrome (AIDS).
Before begilming our discussion of the manner of replication of
animal viruses, we should mention first the important differences
which exist between animal and bacterial cells. Since virus replication
makes use of the biosynthetic machinery of the host, these differences
i1:t cellular organization and function imply differences in the way
the viruses themselves replicate.
Bacteria, being procaryotic, do not show compartmentation of
the biosynthetic processes. The genome of a bacterium relates directly
to the cytoplasm of the cell. Transcription into mRNA can lead
directly to translation, and the processes of transcription and
translation are not carried out in separate organelles. Animal cells,
being eucaryotic, show compartmentation of the transcription and
translation processes. Transcription of the genome into mRNA occurs
in the nucleus, whereas translation occurs in the cytoplasm. The
messenger RNA in the eucaryote is usually modified by adding to it
promoters B
c
~A
t:.::::~p:.;::L_ _ _..lI_ _ _ _ ....L_ _ _ _..........1 bacterial genome
operator cistrons (genes)l
A B c
polycistronic mRNA
formation of polyribosome
and direct translation
into protein
(a) procaryote
intron
" ~;;:.~.;:",;:
L-_ _ _...J®t..::...;:"a._ _ _ _... ......_ _ _~1 eucaryotic genome
(~----+::"::'+-----+::"::'~~----J)
,
,, ~ f§SJ
, I I I I
primary RNA transcript
,,
k
, I
, I
,
,I l
"~-....1...------L..------4(
I
I I
, I
I
I
I
I
RNA processing removes

introns
5'cap
, J
l
poly AI
capping and poly-
adenylation
--------- tail
nuclear membrane
• transfer from nucleus

to cytoplasm
• monocistronic mRNA
translated
(b) eucaryote
Figure 5.28 : Comparison of protein synthesis processes in procaryote and eucaryote.

(a) Procaryote. (b) Eucaryote.
162 MICROBIOLOGY AND 6IOCHEMISTRY
a poly (A) tail of 100 to 200 adenylic residues at the 3' end and a
methylated guanosine triphosphate, called the cap .. at the 5' end.
The cap is required for binding of the mRNA to the ribosome and
the poly A tail may be involved in subsequent RNA processing and
transfer of the mature mR:NA 'from the nucleus to the cytoplasm.
All of the protein-synthesizfqg,machinery of the eucaryotic cell, the
ribosomes, tR"NA molecules, and accessory components, is in the
cytoplasm, "~nd the mature mRNA associates with the protein-
synthesizing apparatus once it leaves the nucleus.
The genes of eucaryotes are often split, with noncoding regions
called introns separating coding regions (exons). TraPscription of both
the coding and noncoding regions of a gene occurs and an RNA,
called the primary RNA transcript, is formed and is subsequently
converted into the mature mRNA by a mechanism called RNA
processing, in which the noncoding regions are excised (the cap and
tail remain after RNA processing is complete). After processing, the
mature mRNA is translated into protein. One important distinction
between eucaryotic and procaryotic mRNA is that procaryotic mRNA
is generally polycistronic, with more than one coding region present
in a single mRNA molecule, whereas eucaryotic mRNA· is
monocistronic. In procaryotes, during the translation process the
ribosomal machinery moves down the mRNA past a stop site and
initiates translation of another gene without ever leaving the mRNA.
A-lthough eucaryotic mRNA is usually monocistronic, this does not
mean that only a single type of protein molecule results from the
translation of a eucaryotic mRNA. Frequently, the eucaryotic mRNA
codes for a single, large multifunctional protein, called a poly-protein,
which may subsequently be cleaved by a specific protease into several
distinct enzymes. In other cases, the polyprotein may remain as a
single multifunctional polypeptide.
We might also note another important difference between animal
and bacterial cells. Bacterial cells have rigid cell walls containing
peptidoglycan and associated substances. Animal cells, on the other
hand, lack cell walls. This difference is important for the way by
which the v:rus genome enters and exits the cell. In bacteria, the
protein coat of the virus remains on the outside of the cell and only
the nucleic acid enters. In animal viruses, on the other hand, uptake
of the virus often occurs by endocytosis (pinocytosis or phagocytosis),
processes which are characteristic of animal cells, so that the whole
virus particle enters the cell. The separation of animal virus genomes
from their protein coats then occurs inside the cell.
Classification of animal viruses Most of the animal viruses
which have been studied in any detail have been those which have
been amenable to cultivation in cell cultures. As seen, animal viruses
are known with either single-stranded or doublesttanded DNA or
RNA. Some animal viruses are enveloped, others are naked. Size
varies greatly, from those large enough to be just visible in the light
microscope, to those so tiny that they are hard to see well even in
the electron microscope. In the following sections, we will discuss
characteristics and manner of multiplication of some of the mqst
important and best-studied animal viruses.
uptake into .
@)~
~
whole virus
particle
animal cell
by e~OSls
and loss of
f:i;\
~
viral envelope nucleocapsid
-
uncoating
of capsid
/
virus
nucleic
acid
processes of
............ virus
multiplication
(al (en\I8Ioped)
(bl
Figure 5.29 : Uptake of an enveloped virus particle by an animal cell. (a) The process
by which the viral nucleocapsid is separated from its envelope. (b) Electron micrograph
of adenovirus particles entering a cell. Each particle is about 70 om in diameter.
Consequences of virus infection in animal cells Viruses can
have varied effects on cells. Lytic infection results in the destruction
of the host cell. However, there are several other possible effects
following viral infectioA of animal cells. In the case of enveloped
viruses, release of the viral particles, which occurs by a kind of
h¢dingprocess, may be s10w and the host cell may not be lysed.
The cell may remain alive and continue (0 produce virus over a long
/period of time. Such infections are referred to as persistent infections.
Viruses may also cause latent infection of a host. In a latent infection,
there is a delay between infection by the virus and the appearance
of symptoms. Fever blisters (cold sores), caused by the herpes simplex
virus, result from a latent viral infection; the symptoms reappear
sporadically as the virus emerges from latency. The latent stage in
viral infection of an animal cell is generally not due to the integration
of the viral genome into the genome of the animal cell, as is the
case with latent infections by temperate bacteriophages.
Viruses and cancer A number of animal viruses have the
potential to change a cell from a normal cell to a cancer cell. This
process, called transformation, can be induced by infections of animal
cells with certain kinds of viruses. One of the key differences between
normal cells and cancer cells is that the latter have different
requirements for growth factors . Rapidly growing cells pile up into
accumulations that are visible in culture as foci of infection. Because
cancerous cells in the animal body have fewer growth requirements,
they grow profusely, leading to the formation of large masses of
cells, called tumors. The term neoplasm is often used in the medical
literature to describe malignant tumors.
Not all tumors are seriously harmful. The body is able to wall
off some tumors so that they do not spread; such noninvasive tumors
are said to be benign. Other tumors, called malignant, invade the
body and destroy normal body tissues and organs. In advanced stages
of cancer, malignant tumors may develop the ability to spread to
other parts of the body and initiate new tumors, a process called
metastasis.
How does a normal cell become cancerous? The process can be
broken down into several stages. In the first step, initiation, genetic
changes in the cell occur. This step may be induced by certain
chemicals, called carcinogens, or by physical stimuli, . such as
ultraviolet radiation or X rays. Certain viruses also bring about the
genetic change that results in initiation of tumor formation. Once
initiation has occurred, the potentially cancerous cell may remain
dormant, but under certain conditions, generally involving some
environmenta1 alteration, the cell may become converted into a tumor
cell, a process called promotion. Once a cell has been promoted to
the cancerous condition, continued cell division can result in the
formation of a tumor.
Although the ability of viruses to cause tumors in animals has
been proved for many years, the relationship of viruses to cancer in
nonenveoped enveloped
o ssDNA
paNOllirus
~ dsDNA
papovavirus dsDNA
poxvirus
adenovirus
dsDNA
herpesvirus
iridovirus
~
(a) DNA viruses 100nm
-- - .... ANA
<i)
--- - -~
o ssANA
®
......-... ... orthcmpD\IIrus
@ANA
-...
@dsANA
(I
.....,...,...
•
coronaYOrus
(b)
~
........
100rwn
ANA_
®.• -.-•
....-us
<0
-... fIIII8I"V'KM'US
Figure 5.30 : The shapes and relative sizes of vertebrate viruses of the major taxonomic
groups. Bar =
100 DDl.
transfonnatlon
.(; :::=ncell~
@,' . '
. .•.
' .
_.
. $
., transformatJon
. " of normal celts
."~,' 10 turnor cells
Vi~ _(!).". _®-'. _.

.
cell
. .
,nlO tumor c e U /
_r/'.'-",)
\
... ,---_/
I
Iyticinfection
adsorptiOn penetration multiplicalron death of cell ~-. _ ._

and releasa __
.' of virus
.-.:.:~, .persistent infection
---""."'"
(i)
slow releasa ',' '~'-""::':.
of virus
without cell
death
_
-
tih -~
\iJ!!!B ~
~rnection
v'rus present
but nol caUSing
harm 10 cell: later
emerges in lytic infection
Figure 5.31 : Possible effects that animal viruses may have on ceIls they infect.
humans has, in most cases, been uncertain. It is difficult to prove
the viral origin of a human cancer because of the difficulties of
carrying out the necessary experimentation. However, it is now well
established that certain specific kinds of human tumors do have a
viral origin. A summary of some of the human cancers with definite
viral origins is given in Table.
TABLE 5.1 : SOME HUMAN CANCERS WmCH MAY BE
CAUSED BY VIRUSES
Cancer Virus Family Genome
Adult T-cell
leukemia Human T-ceU Retrovirus RNA
(type 1) leukemia virus
Burkitt's
lymphoma Epstein-Barr virus Herpes DNA
Nasopharyngeal
carcinoma Epstein-Barr virus Herpes DNA
Hepatocellular
carcinoma (liver Hepatitis B virus Not yet classified DNA
cancer)
Table Contd.
Cancer Virus Family Genome

Cervical Herpes simplex Herpes DNA
cancers (?) type 2 virus (?)
Skin and
cervical cancers Papilloma virus Papova DNA
10oor-------------------------~
,/
number of
100
infectious 10
virus
nucleic acid
I"
,I
particles /
per cell ,I
I
/
,/
hou'rs
....eclipse period-+- rise period---4

..early period+--Iate period----i
........ period necessary to
encapsidate viral genome
Figure 5.32 : One-step growth curve of animal viruses.
Replication cycle of animal viruses One-step growth curves are
also obtained for animal viruses. Such curves exhibit the same overall
features that are found in the bacterial viruses, but the rate of
multiplication is much slower. A typical one-step growth curve of an
animal virus is given in Figure. This figure also presents data on the
length of the eclipse period and the total duration of the multiplication
cycle for several well-studied animal viruses. Note that the
multiplication cycles of animal viruses range from around eight hours
to as long as 48 hours.
6
General Metabolism
6.1 CHARACTERIZATION OF METABOLISM
The vital activity of any living organism is determined by the
specific organization of biological structures, metabolic processes,
energy metabolic processes, energy metabolism, genetic information
transfer, and regulatory mechanism. Damage of any of these links
develops a pathological process and a disease in the organism. An
understanding of the molecular mechanisms involved in the vital
activity or malfunction of the organism constitutes the basis for the
search and clinical applications of biological medicinal preparations.
In the overall metabolism of the living organism distinguished
are: exogenous metabolism, which comprises extracellular
transformations of materials on the way to their uptake and excretion
by the cells, and intermediary metabolism, which occurs in the cells.
Tht' intermediary metabolism is conceived as the sum total of chemical
reactions that occur in the living cell.
Functionally, metabolism encompasses the following major
processes:
(I) accumulation of energy from decomposition of compounds
or supplied by light;
(2) utilization of energy for synthesis of essential molecular
components (monomers, macromolecules) and the performance
of work (osmotic, electric, mechanical);
(3) decomposition of renewable structural components of the cell;
(4) synthesis and decomposition of specialized biological molecules
(hormones, mediators, hormonoids, cofactors, etc.).
The sequences of chemical reactions involved form metabolic
pathWays, or cycles, each of these performing a defmite function.
168
GENERAL METABOLISM 169
Conventionally, central and special metabolic pathways are distinguished.

Central pathways are common to the decomposition and synthesis of
major macromolecules. Actually, they are much alike in all
representatives of the living world. Special cycles are characteristic of
the synthesis and decomposition of individual monomers, macromolecules,
cofactors, etc. Special cycles are extremely diversified, especially in
the plant kingdom. For this reason, the plant metabolism is conventionally
classified into primary and secondary metabolisms. The primary
metabolism includes the classical processes of synthesis and deeradation
of major macromolecules (proteins, carbohydrates, lipids, nucleic acids,
etc.), while the secondary metabolism ensuing from the primary one
includes the conversions of special biomolecules (for example, alkaloids,
terpenes, etc.) that perform regulatory or other functions, or simply are
metabolic end byproducts.
In the metabolism, two opp·ositely directed processes, or phases,
are commonly distinguished: catabolism and anabolism. Catabolism is
the sum of degradative processes leading to the cleavage of large
molecules into smaller ones. Anabolism is the sum 'of metabolic
processes leading to the synthesis of complex molecules from simpler
ones. Catabolism is accompanied by a release of energy that can be
stored as energy-rich ATP. Anabolic processes proceed through
consumption of ATP and decomposition of the latter into ADP and
H,P04. Therefore, ATP may be said to be a coupling energetic link
between the two metabolic pathways. However, ATP is not the only
linking component shared by catabolism and anabolism. Other simple
metabolites are also formed by the catabolic pathway from
macromolecules and monomers to be used as starting materials for the
subsequent synthesis of monomers and macromolecules, i.e. in the
process of anabolism. This linking pathway, or cycle, unifying
degradative and synthetic routes, is called the amphibolic pathway.
This signifies that the catabolic and anabolic pathways are coupled not
only via the energetic ATP-ADP system, but also through their common
metabolites, which renders the metabolism more versatile and
economical. At need, simple intermediates can be utilized in the
biosynthesis, without the necessity of their supply from the exterior.
The amphibolic pathways are associated with the terminal, or ultimate,
system of oxidation of the materials involved, as the latter are degraded
to the end products, CO 2 and HP, with a release of a large amount
of energy. Apart from them, urea and uric acid, which are produced
by specific metabolic reactions of amino acids and nucleotides, are
also end products of metabolism.
carbohydrat••
Proteins. Lipids,
Polynucleotida
Uric acid Urea H 20
Figure 6.1 : Scheme for catabolic and anabolic pathways (shown is their interrelation
through ATP-ADP system and amphibolic metabolite cycle)
During catabolic and anabolic processes, a renovation of the
molecular cellular components takes place. It should be emphasized
that the catabolic and anabolic pathways are independent of each other.
Be these pathways coincident and differing in the cycle direction only,
the metabolism would have been side-tracked to the so-called useless,
or futile, cycles. Such cycles arise in pathology, where a useless
turnover of metabolites may occur. To avoid this undesirable
contingency, the synthetic and degradative routes in the cell are most
commonly separated in space. For example, the oxidation of fatty
acids occurs in the mitochondria, while the synthesis thereof proceeds
extrarnitochondrially, in the microsomes.
6.2 ENERGY CYCLES IN ANIMATE NATURE
Nutrients and energy are supplied to the living organisms from
various sources. As far as the nutritional sources are concerned, living
organisms are classified into two large groups, autotrophs (from the
Greek autos, self, and trophos, food) capable of assimilating CO2 as
a startiQg nutrient for the buildup of other carbon-containing materials,
and heterotrophs (heteros, other) which utilize diverse organic compounds

as synthetized by other organisms. In a sense, the autotrophs are primary
to the heterotrophs. Reduced organic compounds (for example, gIu cose)
synthetized by the autotrophs from CO2 contain a larger amount of
energy as compared with that in the consumed carbon dioxide.
In regard to the energy sources, living organisms are divided into
phototrophs, for which the sun light is. a source of energy, and
chemotrophs, which utilize the energy of reduction-oxidation (redox)
reactions. In redox reactions, the energy that has been acquired by the
cell is released on transport of the electrons from a donor to an acceptor
(or oxidant). In these processes, the donor and the acceptor act as
partners and constitute a donor-acceptor pair, or a redox pair. If the
redox pair is made up of organic compounds, the living organisms
involved are called chemoorga 'notrophs, and if it consists of inorganic
compounds, chemolithotrophs. They are further diversified by their
relationship to oxygen as an electron acceptor. The organism cells that
utilize oxygen are referred to as aerobic ones, those capable of
dispensing with oxygen are called anaerobic. Most commonly, the
cells of higher organisms and bacteria possess both types, anaerobic
and aerobic, of energetics. For this reason, such cells and organisms
are calledfacultative anaerobes, although the degree of faculty, or the
dependence on oxygen supply, in them may vary. For example, higher
organisms are incapable of subsisting without oxygen for longer periods
of time. There are obligate anaerobes, in particular, microorganisms,
which do not need oxygen altogether and even defy it as poisonous.
The green plants possess a combined type of energetics,
phototrophic and'chemoorganotrophic (respiratory and glycolytic), which
enables them to absorb the energy of sun light at different periods of
their development; in the absence of sun light, the plants make use of
chemical energy. Microorganisms are especially remarkable for the
diversity of energetics types and combinations thereof. The energetics
types occurring in a number of representatives of the animate nature
are listed in Table.
Owing to the diversity of nutrition forms and energy consumption,
the living organisms are in nature closely related. _The interrelation
between nutrition and utilization of energy sources may be conceived
of from the standpoint of specific cycles operative in the animate
nature. Major participants of a global cycle are the Sun as a source
of energy, autophototrophs capable of acquiring the solar energy and
of synthetizing carbohydrates and other org~c materials from CO2 ,
Table 6.1 Energetics Types Encountered in Living Organisms. .....
;j
Source of energy Energetics type Representatives
1. Light Phototrophic Photosynthetic organs of higher
plants, algae, bacteria
2. Redox reactions of chemi- ChemCltrophic Animal and bacterial .cells, nonpho
cal compounds (donor-ac- tosynthetic plant cells ceptc r)
Donor: organic compounds; Chemoolrganotrophic Idem
acceptor: 02 (aerobic}
Donor and acceptor: ChemooJrganotrophic Idem
organic compounds
Donor: inorganic
compounds; acceptor: 02
(anaerobic)
Chemol ithotrophic
(aerobic:)
Bacteria -
~
("')
~
~
Donor and acceptor: Chemolithotrophic Idem
inorganic compounds
Donor: organic compounds;
(anaerobic)
Chemoorga otrophic CellS of higher animals; bacteria
§
-<
acceptor: of mixed type (facultativ anaerobic)
(organic compounds and 02)
>
~
3. Combined type: light and Photochemotropl. .c Photosynthetic plant cells at differ
redox reactions
Light and redox Photochemoorganotroph- Idem
ent (light and dark) phases g=
f§
reactions of organic
compounds
Light and redox reactions
ie
Photochemolithotrophic Photosynthetic bacteria

-
~
C l)
o-l
of inorganic compounds ~
and animals which consume organic materials and oxygen generated

by phototrophs.
Energy losses associated with the vital activity of all organisms on
the Earth are being compensated for by the energy of solar radiation.
It should be noted that the cells of man and animals utilize highly
reduced, i.e. hydrogen-containing, compounds (carbohydrates, lipids,
proteins, etc.) as energetic materials. Hydrogen is an energetically
valuable material. Its energy in a transformed form is stored in the
ATP chemical bonds in the cells of heterotrophic organisms.
6.3 ENERGETICS OF BIOCHEMICAL REACTIONS
To get a deeper insight into the metabolic and energetic processes,
knowledge of general principles of chemical energetics appears to be
of help.
In the living cell, all chemical reactions contributing to the
metabolism obey the laws of energetics. The first law of energy
conservation states that the energy of a chemical reaction can be neither
annihilated nor generated from nothing, it can merely be converted
from one form into another. In terms of this law, it becomes possible
to defme the energy balance of a chemical process.
Spontaneous chemical processes can proceed only in one direction
towards a state of equilibrium; the state of equilibrium having been
reached, the process is brought to termination. The second law of
thermodynamics (energetics) enables one to predict the direction of
biochemical processes. According to this law, any spontaneous process
takes the route corresponding to a maximum of entropy under the
given conditions until an equilibrium state for the reaction is reached.
Entropy conveys a measure of the disorder in a system. An increase
in entropy in the course of a reaction prevents the return of the reaction
to the initial state, since for that to occur a diminution in entropy
would be required. A spontaneously disordered system is never capable
of turning into an ordered one. Therefore, all reactions that proceed
with a concomitant increase in entropy are irreversible. For their
reversal, an additional energy is needed to be spent to compensate for
the losses in entropy change, i.e. to bring the system from the disordered
into the ordered state.
However, from the pract~cal standpoint it appears expedient to
use the so-called free energy which, in contrast to entropy, is amenable
to measurement in the course of a reaction. The free energy defmes
a portion of the total energy of a system that can be converted to
work at pressure and temperature kept constant. The free energy is
denoted AG. The portion of total energy of a chemical process that

cannot be converted to work at pressure and temperature kept constant
is called the bound energy and is expressed as the product TAS, where
T is the absolute temperature and AS is the entropy change of a system
under the given chemical reaction conditions. The sum of the changes
in free and bound energies is called enthalpy (internal energy of a
system) or heat energy content of a system and is denoted OH.
Enthalpy can be measured experimentally and is equal to the amount
of heat released in the given process. The enthaply change is defined
by the equation
MI = /lG + T /l S
Otherwise stated,
/lG=MI - T /l S
The energetic state of any system, including that of a cell and an
organism, can be defmed in terms of this very important equation.
The free energy is expressed in kilojoules per mole of substance, kJ/
mol.
The free energy is a very convenient parameter for defining the
spontaneity or nonspontaneity of a chemical process. For spontaneous
processes, the free energy is seen to decrease, i.e. /lG has a negative
value. Such reactions are called exergonic, i.e. proceeding with a
release of energy. These reactions supply energy to the cells. The
processes for which the free energy is increased (i.e. /lG has a positive
value) cannot proceed spontaneously. They require energy supply from
the outside. Such processes are referred to as endergonic, i.e.
proceeding with an expenditure of energy. In a state of equilibrium,
/lG = O.
The free energy of chemical reactions may be estimated both
under the standard conditions and under real, or physiological,
conditions. The standard free energy, /lGo , of a biochemical reaction
is defined as a free energy change under the standard conditions, i.e.
at the concentration of reactants I mol/litre, temperature 25°C < 298
X), and pH 7.
If water is involved either as a starting compound or as a reaction
product, its concentration is taken equal to 1.0 mol/litre, although the
true concentration of water in dilute aqueous solutions is close to 55
mol/litre.
The standard free energy is found as a difference between the
sum values of free energy for the end products and the initial reactants.
The value of /l Gp for a biochemical reaction proceeding under the
physiological conditions is estimated with allowance for the actual

concentrations of components involved.
With reference to the free energy as a characteristic of metabolism
one may say that catabolic reactions proceed with a release of energy
and anabolic ones, with a consumption of energy' The anabolic
reactions can proceed only as closely coupled to the catabolic reactions.
High-energy, or macroergic,compounds act as energetic mediators
between these two types of reactions.
6.4 HIGHT-ENEGRY AND LOW-ENERGY
PHOSPHATES: GENERAL CONSIDERATIONS
Commonly, all compounds are classified into high-energy and low-
energy ones. As a conventional borderline between these two classes,
the free energy of about 20 kJ/mol for the phosphate bond hydrolysis
has been taken. This value used for the purpose of characterization
of biochemical processes should not be confused with the bond energy
which is conceived as an energy required for disruption of the bond
between two neighbouring atoms in any molecule. Cleavage of the
phosphate bond results, alongside energy release, in the formation of
inorganic phosphates.
Energetic characteristics for some of the compounds of interest
are listed in the following Table.
TABLE 8.2 Standard Free Energies, AGo, for Hydrolysis of Some
High-energy and Low-energy Compounds, and Free Energies for
Hydrolysis of Compounds Under Physiological Conditions, AGp.
Compound - AGpI
kJ/mol
High-energy compounds
Phosphoenol pyruvate 61.7 66.7
l,3-Diphosphoglycerate 49.2 41.7
Creatine phosphate 42.5
ATP 30.4 50.0
Acetyl-CoA 30.4
(Table 8.2 Contd.)
Compound _ AGO, - AGpI

kJ/mol kJ/mol
ADP 28.3 50.0
Pyrophosphate (H4P20,) 28.3 50.0
UDP-glucose 24.2
Glucose I-phosphate 20.8
Low-energy compounds
Fructose 6-phosphate 15.8
AMP 14.1
Glucose 6-phosphate 13.8 23.8
a-Glycerol phosphate 9.2
It shouij be emphasized that ATP, the key mediator in energy
metabolism, is not the most energy-rich species. ATP is found in the
middle of. energy scale.
The most frequently occurring route is cleavage of the end
phosphate from ATP:
ATP ~ ADP + Hl04 (1)
The end phosphate adds water and is transferred onto another
compound, causing thereby the phosphorylation of the latter. An
alternative route for the phosphate bond energy release is exemplified
by pyrophosphate cleavage of ATP:
ATP ~ AMP + H4 Pp? (2)
This type of reaction is less frequent in biological processes. Its
distinctive feature is the formation of pyrophosphate, which ranks
among energy-rich materials. Hydrolysis of pyrophosphate (3) releases
roughly as much energy as hydrolysis of the ATP end phosphate
bonds. In biological processes, energy-rich pyrophosphate bonds are
seldom used for synthesis of other compounds because of the heat
energy released by pyrophosphate hydrolysis.
ADP may be used as a high-energy reactant in biochemical
processes. The cleavage of the ADP end phosphate bond (4) releases
the same amount of energy as that generated by splitting the end
phosphate bond in ATP. At first glance, it may appear that ATP can
be adequately replaced by ADP in chemical reactions, in particular
in the phosphoryla9 ion of other compounds. However, as evidenced
by the available experimental data, such a possibility has never been
realized in biological processes. At least such reactions are as yet
unknown. It is known, however, that ADP can be hydrolyzed to low-
energy AMP and phosphate with heat release. For a long time, there
, have been difficulties in determining the free energy for the ATP
phosphate bond. The standard free energy for hydrolysis of the ATP
phosphate bond, AGo, is equal to about -30.4 kJ/mol. This value has
been derived under standard conditions, i.e. at concentrations I M for

initial reactants and end products, pH 7.0, temperature 37°C, and
excess of Mg2+ ions. It stands to reason that in the cell under
physiological conditions, the concentrations of initial reactants, end
products, and Mg" ions are substantially inferior to the standard valueS;
variations in pH are also possible. Therefore, the real free energy
for hydrolysis of the end phosphate bond in ATP and ADP, and of
the phosphate bonds in pyrophosphate is close to -50.0 kJ/mol. To
be noted, the values of ~Go for other compounds differ from the
standard value, but not necessarily towards higher energies.
6.5 ENERGY TRANSFER IN
BIOCHEMICAL PROCESSES
The biological activity of the cell is closely associated with the
continuous redistribution of the energy delivered by the compounds
that enter the cell. The storage of energy in the specific phosphate
bonds of ATP constitutes the basis for the energy transfer mechanism
in the living cell. The living cell is a nonequilibrated chemical system-
the circumstance that permits storing the energy, produced by catabolic
reactions of nutrients, in the ATP phosphate bonds.
The ATP energy in the cell can be converted, via tree major
routes, to energy of chemical bonds, to thermal energy, and to energy
for performing work. We now consider in general terms the transfer
of chemical bond energy. The chemical reaction is associated with
the generation of ATP energy. If the reaction is in a state of
equilibrium, the energy transfer is accomplished to a 100% efficiency.
Nonetheless, no ATP energy will accumulate, since at equilibrium,
the free energy release is zero. Therefore, in order to provide for
energy storage in the ATP phosphate bonds, the reaction must be a
nonequilibrium one. This is accomplished as a portion of the chemical
reaction energy is lost as heat. The remaining energy is transferred
onto the ATP phosphate bonds to be stored therein. It follows,
therefore, that biological generators of energy cannot attain the 100%
efficiency.
The third route of energy conversion leads to the performance of
work. The chemical energy of ATP phosphate bonds can be spent on
osmotic, electric, mechanical, and other types of work. In doing so,
not all of the ATP energy is used for performing work; a portion of
it is dissipated as heat. Especially much heat is produced by muscle
contraction, which represents a mechanochemical mechanism for heat
generation in the living organisms. Heat is of little use for performing
work in the living system. It is only in wann-blooded animals and

man that the heat is consumed for warming and maintaining a constant
temperature of the body, equal to about 37°C.
All chemical processes in the living organism can proceed only
with the involvement of enzymes. For this reason, prior to consider
the mechanism of energy extraction and various metabolic pathways,
it appears worthwhile to discuss enzymes and their functions.
7
Metalbolism of
Saccharides
Carbohydrate metabolism in the organism tissues encompasses enzymic
processes leading either to the breakdown of carbohydrates (catabolic
-pathways), or to the synthesis thereof (anabolic pathways). Carbohydrate
breakdown leads to energy release or intermediary products that are
necessary for other biochemical processes. The carbohydrate synthesis
serves for replenishment of polysaccharide reserve or for renewal of
structural carbohydrates. The effectiveness of various routes of
carbohydrate metabolism in tissues and organs is defined by the
availability of appropriate enzymes in them.
7.1 CARBOHYDRAGE CATABOLISM IN TISSUES
A number of routes for carbohydrate catabolism in tissues are
known. They include glycolysis and its variant, glycogenolysis, which
are auxiliary pathways to energy production, respectively, by breakdown
of glucose (or other monosaccharides) and glycogen to lactate (under
anaerobic conditions) or to CO2 and Hp (under aerobic conditions).
The involvement of glycolysis and glycogenolysis in the energetic
function has been discussed in detail in the foregoing section
'Bioenergetics" .
There is known one more catabolic route for carbohydrates
commonly referred to as the pentose phosphate cycle (also called
hexose mono phosphate shunt, or phosphogluconate pathway).
As a tribute to the biochemists who have played a decisive role
in its investigation, the pentose phosphate cycle is also referred to as
the Warburg-Dickens-Horecker pathway.
The pentose phosphate cycle represents a mUltienzyme system in
179
which the important intermediates are, as the name implies, pentose

phosphates. This cycle may be regarded as a branching, or shunt, at
the glucose 6-phosphate step in the overall glycolysis.
7.1.1 Pentose Phosphate Cycle
To provide for all steps of the pentose phosphate cycle, at least
three glucose 6-phosphate molecules are required. Let us consider
separate reactions of this cycle.
1. Dehydrogenation o( g1ueose 6-phosphate is the reaction that
directs- glucose 6-phosphate via the pentose phosphate pathway; it is
catalyzed by glucose-6phosphate dehydrogenase (in the schemes below,
for a fuller description of the cyclic process, three glucose 6-phosphate
molecules are used)
H
,/ OH
~-oH I GI~hate dehydrogenaa

3 HO-C-H
~-oHI
6
·7"'-
3NADP + 3NADP·H+H+
H-C
I
H,C-oPO)H,
glucose 6-phosphate
Glucose-6-phosphate dehydrogenase is a dimer with a molecular

mass of about 135 000. Up to eight electrophoretic ally separable
isoenzymes for this enzyme are known. A specific feature of the above
reaction is the formation of NADP • Hr The reaction equilibrium is
strongly shifted to the right, since the lactone formed is liable to
hydrolysis, which is spontaneous or lactonase-assisted.
2. Hydrolysis of 6-phosphoglueonate lactone to 6-phosphogl-
ueonate:
o
H-~-OH
1
3 HO-C-H
C"
I Lactonaae
0 ~ 3 HO-C-H
I
COOH
H-b-OH
H - t - OH
H-C------!
I H-~-OH
H-C-OH
1- 1
HaC-OPOaH, H.C-OPOaH.
6-phoapbocl uconate 8-pbOllphoglueonate
lactone
METABOLISM OF SACCHARIDES 181
3. Dehydrogenation of 6-phosphogluconate to ribulose 5-

phosphate. This reaction is catalyzed by 6-phosphogluconate
dehydrogenase according to the scheme:
COOH
I
"-r-O
3 "o-y-"
"-y-o".'
H-C-OH
.1
H
. /"
3NADP'
""
6-phosphoglUCOlllte dehydrogenaR
. +lC01
H2C-oP0 3H1
tJ.phosphog!uconate D-ribulose S-phosphate
The reaction equilibrium is shifted to the right. 6-Phosphogluconate
dehydrogenase is a dimer with a molecular mass of about 100 000.
Several isoenzymes are known for this dehydrogenase. A specific
feature of this reaction is that dehydrogenation leads to an unstable
intermediate which is immediately decarboxylated on the surface of
the enzyme. This is the second oxidation reaction in the pentose
phosphate cycle that leads to NADP • H2 ; therefore, the conversion
of glucose 6-phosphate to ribulose 5-phosphate is commonly referred
to as the oxidative phase of the pentose phosphate cycle. The sequence
of reactions starting from ribulose 5-phosphate to the formation of
initial glucose 6-phosphate is called the nonoxidative, or anaerobic,
phase of this cycle.
4. Interconversion or isomerization of pentose phosphates.
Ribulose 5-phosphate is capable of a reversible isomerization to other
pentose phosphates-xylulose 5-phosphate and ribose 5-phosphate. These
reactions are catalyzed by two respective enzymes, viz., pentose-
phosphate epimerase and pentose-phosphate isomerase, according to
the scheme below:
H.C-OH H.C-OH H-C=O
I I I
c=o
-
Pmt_pllOlphate C= 0 Pentooe-pllOlp/late H-C-OH
2 HO-C-H
I
I
H-C-OH
I
HsC-OPO,H.
eplmeralll
~===-~ 3 H-C-OH
H-C-OH
I
I
I
IItC-OPO.H.
- Isomerue
I
I
H-C-OH
I
H-C-OH
H.C-OPO.H.
]).:1,1111_ ])'rlllulOlO ]).r\IIoIe
~pllOl)lllate 5-p.~OI)IIIate 5-pII01)111ate
Two other pentose phosphates (ribose 5-phosphate and xylulose

5-phosphate), which are derived from ribulose 5~phosphate, are
important for the subsequent reaction of the cycle. Two molecules of
xylulose 5-phosphate and one molecule of ribose 5-phosphate are required

for this.
S. Transfer of glycolic aldehyde from xylulose S-phosphate onto
ribose S-phosphate or the first transketolase reaction. The next
reaction, which is catalyzed by transketolase, involves the pentose
phosphates produced by the foregoing reaction (the transferable moiety
is shown in the box):
HoC-OH H.C-OH H-C=O
I I I
c=o Pent_pboopbate C= 0 Pent __pboopbate H-C-OH
I eplmerue I .........- I
2 HO-C-H ~====-:: 3 H-C-OH _~===-:: H-C-OH
I
I I
H-C-OH H-C-OH H-C-OH
I I I
BsC-OPOIH. HoC-OPOIH. HaC-OPOIHa
J>syl.l_ J>rtbal_
$-pboopbate $-p:toopbate
A ribose 5-phosphate molecule and one of the two xylulose 5-

phosphate molecules are used during the first transketolase reaction.
The other xylulose 5-phosphate molecule is consumed later, in the
second transketolase reaction.
Transketolase is a dimer with a molecular mass of 140000. Its
coenzyme is thiamine bisphosphate. Mgt+ ions are required for the
reaction. Both transketolase reaction products are used as substrates
at the next step of the cycle.
6. Transfer of dihydroxyacetone moiety from sedoheptulose
7-phosphate onto glyceraldehyde 3-phosphate. This reaction is
reversible and is catalyzed by transaldolase according to the scheme:
[H2C:'o'Hi
r-----' I I I
I H,C-OH I
I I I
... -,--_...1
I C.O I
IL __c-o
1___ .-JI HO-C-H H-C-O
HO-C-H
I
Transketolase
. H-C-OH
I
I
+
I
H-C-OH
I
H-C-OH H-C-OH H 2C-OPO)H2
I I
H 2C- OPO)H2 H-C- OH
I
H 2 C- OPO)H2
D·xylulote D·ribose D·sedoheptulose D.gIycerilldehyde
&-phosphate S.phosphllte 711hotphate 3·pllosphllte
Transaldolase is a dimer with a molecular mass of about 70 000.

The fructose 6phosphate molecule produced by this reaction enters
the glycolysis, while erythrose 4-phosphate is used as a substrate at
the subsequent steps of the cycle.
7. Transfer of glycolic aldehyde from xylulose S-phosphate onto
erythrose 4-phosphate or the second transketloase reaction. This
reaction is related to the first transketolase reaction and is catalyzed
by the same enzyme. The only distinction is that erythrose 4-phosphate
acts as an acceptor for glycolic aldehyde:
fH2C:'O"H;
r-:-----'
I H,C-OH I H-C-O
I
I
I
C.O
I
I
I I I I ... -I---.J
IL 1___ -JI
__C-O H-C-OH HO-C-H H-C .. O
I Transketolase ' I II
HO-C-H
I
+ H-C-OH
I M.H
, H- c- OH
I
+ H-1- 0H
H-C-OH H-C-OH H-C-OH H~-OPOJH2
I I I
HzC- OPOJH Z H ZC-OPO JH2 H-C- OH
I
HzC- OPOJH Z
O·xylulOll O·ribose O-sedoheptulOll O-glyceraldehyde
5-phOlPhlte 5-phosphate 7 -phosphite 3·pnosphate
Fructose 6-phosphate and glyceraldehyde 3-phosphate also enter

the glycolysis.
Thus, in the course of reactions catalyzed by the intrinsic enzymes
of the pentose phosphate cycle, two fructose 6-phosphate molecules,
one glyceraldehyde 3-phosphate molecule, and three carbon dioxide
molecules are produced from three glucose 6-phosphate molecules.
In addition, six NADP -H2 molecules are formed. The overall scheme
for the pentose phosphate cycle is:
3 Glucose 6-phosphate-6NADP+ ~ 2 Fructose 6-phosphate
+ Glyceraldehyde 3-phosphate + 6NADPeH2+ 3C02
7.1.2 Interrelation of the Pentose
Phosphate Cycle and Glycolysis
These two pathways for carbohydrate conversion are closely
related. The products of the pentose phosphate route-fructose 6-
phosphate and glyceral-dehyde 3-phosphate-are likewise glycolysis
metabolites; for this reason, they are involved in glycolysis and
undergo conversion by glycolytic enzymes. Two molecules of fructose
6-phosphate can regenerate to two glucose 6-phosphate molecules
through the agency of the glycolytic enzyme glucose-phosphate
isomerase. Here, the pentose phosphate pathway functions as a cycle.
The other product, glyceraldehype 3-phosphate, enters the glycolysis
to be either converted to lactate (under anaerobic conditions) or
oxidized to CO2 and Hp (under aerobic conditions). As can easily
be estimated, the conversion of glyceraldehyde 3-phosphate to lactate
Glucose
G
I Glucose 6-pLsphate
~
L Fructose 6-phosphate
y ' F ructose 6-phosphate
e Fructose 1.6-bisphosphate
o , Synthesis of nu-
Ly r i cleotides and nu-
" Ieosides. nucleo-
S Dihyd-~Glyceraldehyde 3-phosphate L~r--"-.tide coenzymes.
I roxyace- I ~-.;....--' polynucleotid8s.
Stone - I and histidine
I phosphate +
• Pyruvate
U
Lactate
Figure 7_1 Scheme for integration of pentose phosphate shunt and glycolysis.
leads to the formation of two ATP molecules, while the combustion
to CO2 and Hp produces 20 ATP molecules. It follows therefore
that under physiological conditions, when the pentose phosphate
pathway for carbohydrate conversion is included in the glycolysis,
the overall process of glucose 6-phosphate conversion may be
expressed via the pentose phosphate cycle. Under anaerobic conditions:
3 Glucose 6-phosphate+6NADP+ + 2P l ~ 2 Glucose 6-phosphate
+ Lactate + 2ATP + 6NADP . H2 + 3C02
Under aerobic conditions:
3 Glucose 6-phosphate + 6NADP+ + 20ADP + 20Pl
~ 2 Glucose 6-phosphate + 6NADP.H2+ 6C0 2
+ 6Hp + 20ATP
At, first glance, the energetic value of this conversion of glucose
6-phosphate via the pentose phosphate cycle appears to be inferior to
that of the aerobic glycolysis pathway, the latter providing a maximum
of 38 ATP molecules. However, it should be borne in mind that a
major portion of energy is stored in NADP - H2, and 6 NADP - H2
molecules are energetically equivalent to 18 A TP molecules.
Consequently, the energetic effect remains the same.
7.1.3 The Biological Function of
the Pentose Phosphate Cycle
The biological function of the pentose phosphate cycle involves
the production of two compounds: NADP -H 2, which is a "reductive
force" in the synthesis of various materials, and the metabolite ribose
5-phosphate, which is used as a building material in the synthesis of

various specieiS. The major functions of the pentose phosph~te cycle
~e:
(1) amphibolic function: the cycle is a route to degradation of

carbohydrates and, simultaneously, to the supply of materials
used in synthetic reactions (NADP • Hz and ribose 5-
phosphate);
(2) energetic function, since the involvement of pentose phosphate
cycle products (glyceraldehyde 3-phosphate) in the glycolysis
produces energy;
(3) synthetic function, as a major function associated with the
use of NADP • Hz and ribose 5-phosphate.
NADP • ~ is used:
(1) in the detoxification of drugs and poisons in the monooxy-
genase oxidation chain of the endoplasmic reticulum of the
liver;
(2) in the synthesis of fatty acids and other structural and reserve
lipids;
(3) in the synthesis of cholesterol and its derivatives-bile acids,
steroid hormones (corticosteroids, female and male sex
hormones), and vitamins D; and
(4) in the neutralization of ammonia under reductive amination.
Ribose 5-phosphate is used in the synthesis of histidine,
nucleosides and nucleotides (nucleotide mono-, di-, and triphosphates),
nucleotide coenzymes (NAD, NADP, FAD, and CoA), and polymeric
nucleotide derivatives (DNA, RNA, and short-chain oligonucleotides).
The pentose phosphate pathway for carbohydrate conversion is
primarily operative in the organs and tissues in which an intensive
utilization of NADP • H2 is needed for reactions of reductive synthesis
and for reactions involving ribose 5phosphate in the synthesis of
nucleotides and nucleic acids. For this reason, a high activity of this
pathway is observed in fat tissue, liver, mammary gland tissue
(especially during lactation, since the milk fat synthesis is essential
in this case), adrenal glands, gonadal glands, marrow, and lymphoid
tissue. Relatively high is the activity of pentose phosphate shunt
dehydrogenases in the erythrocytes. A low activity of the pentose
phosphate pathway is observed in muscular tissue (heart and skeletal
muscle).
7.2 BIOSYNTHESIS OF
CARBOHYDRATES IN TISSUES
In the human and animal tissues and organs, synthesis of
carbohydrates occurs. Since glucose is the starting structural unit for
producing other monosaccharides and for assembling polysaccharides,
it is expedient to consider potential routes for the glucose synthesis
in tissues and organs. Formation of glucose from nonccu:bohydrate
materials is attested by the fact that, under prolonged starv'ation (in
an extreme contingency or as applied in therapy), the polysaccharide
carbohydrate reserves are rapidly consumed, while the glucose level
in the circulating blood is maintained to supply tissues, especially brain,
with energy.
7.2.1 Gluconeogenesis
The synthesis of glucose from noncarbohydrate sources is referred
to as the gluconeogenesis. It is feasible only in certain organism
tissues. The major site for gluconeogenesis is the liver. To a lesser
extent, the kidneys and intestinal mucosa are involved in this process.
Mechanism for Gluconeogenesis. Since the glycolysis involves
three energetically irreversible steps at the pyruvate kinase,
phosphofructokinase, and hexokinase levels, the production of glucose
from simple noncarbohydrate materials, for example, pyruvate or
lactate, by a reversal of glycolysis ("from bottom upwards") is
impossible. Therefore, indirect reaction routes are to be sought for.
The Jirst indirect route in glucose synthesis involves the formation
of phosphoenolpyruvate from pyruvate without the intervention of
pyruvate kinase. This route is catalyzed by two enzymes. At first,
pyruvate is converted into oxaloacetate. This reaction occurs in the
mitochondria as the pyruvate molecules enter them, and is catalyzed
IJy pyruvate carboxylase according to the scheme
Pyruvate .arOOsyl. .
CH.-C-COOH+HCO.+ATP -
II
° .... HOOC-CH.-C-COOH+ADP+H.PO.
II
o
onlo_tate
This enzyme, similar to all CO2 assimilating enzymes, contains

biotin for a cofactor. Oxaloacetate is released from the mitochondria
into the cytoplasm to enter gluconeogenesis. In the cytoplasm,
oxaloacetate converts to phosphoenolpyruvate via a reaction catalyzed
by phosphoenolpyruvate carboxylase:
..._ _ _lpfnl...te e..~ ...
HOOC-CH.-C"":COOH+GTP(ATP) -
~
oulo_late
... CHs=C-COOH+GDP(ADPHCO.
6-POaHs
pboopboonolpfnlY.te
The reaction equilibrium is shifted to the right. The major supplier

of phosphate groups is GTP, but for this purpose, ATP may also be
available.
All of the glycolysis reactions ranging from phosphoenolpyruvate
to fructose 1,6-bisphosphate are reversible, and the phosphoenolpyruvate
IOOlecules fonned are consumed for producing fructose 1,6-bisphosphate
by making use of the same glycolysis enzymes.
The second indirect route involves the formation of fructose 6-
phosphate from fructose 1,6-bisphosphate without the intervention of
phosphofructokinase reaction. This route is catalyzed by fructose
bisphosphatase:
Fructose 1,6-bisphosphate + H20 Fructosebispoosphatase)
Fructose 6-phosphatc + H2 PO4

The reaction is irreversibly shifted to the right. Fructose 6-
phosphate is isomerized to glucose 6-phosphate by glucose-phosphate
isomerase.
The third indirect route involves the formation of free glucose
from glucose 6phosphate by circumventing the hexokinase reaction.
This route is catalyzed by
Glucose 6-phosphate + H2 0 Glucose 6-poosphatase ) Glucose + H 2 PO 4
The free glucose produced by this reaction is supplied to the blood
from the tissues. As exemplified by gluconeogenesis, one may easily
envision the economical organization of these metabolic routes, since,
apart from four special gluconeogenesis enzymes-pyruvate carboxylase,
phosphopyruvate carboxylase, fructose bisphosphatase, and glucose 6-
phosphatase-individual glycolytic enzymes are also used in the
gluconeogenesis.
Noncarbohydrate Sources for Gluconeogenesis. In addition to
pyruvate and lactate, which are delivered to the liver and kidneys,
other noncarbohydrate compounds serve as substrates for glucose
synthesis. In accordance with the gluconeogenesis scheme, it may be
anticipated that all materials of noncarbohydrate nature that are
t.
Cl
r-
e r-
(") ... ru~,...... -<
(")
o
z o
m r
o
Cl
m
z
m
i
Figure 7.2 Schematic representation of gluconeogenesis.
amenable to conversion to a glucolysis metabolite (first group of

materials), to pyruvate (second group), or to oxaloacetate (third group),
can serve as potential sources for glucose synthesis. For example,
glycerol may be included in the first group of materials, since this
triol, which can convert to dihydroxyacetone phosphate, can further
take up, depending on the reaction conditions, either gluconeogenesis
route, or glycolysis route. The involvement of glycerol in
gluconeogenesis proceeds according to the scheme:
Glycerol phosphokinase
(11 Glycerol 7 '\ · Cl-Glycerol phosphate
ATP AOP
CI-GIYtero/ phosphate
dehydrogenase O·hydr
(2) Cl -Glycerol phosphate --"';"'7"'~'-';;"~~---.. I oxyecetOne
" phosphate
NAO+ NAO-H+tf
Subsequently, dihydroxyacetone phosphate is used in glucose

synthesis.
The Krebs cycle acids convertible to oxaloacetate (third group
materials) may also be assigned to gluconeogenesis substrates.
However, amino acids that can convert the major source for
gluconeogenesis to both pyruvate and oxaloacetate and, consequently,
to glucose are the major source for gluconeogenesis. Amino acids
involved in the gluconeogenesis are referred to as glycogenic amino
acids. They encompass all of the protein amino acids, barring leucine.
7.2.2 Biosynthesis of Glycogen (Glycogenogenesis)
Synthesis of glycogen is carried out in all the cells of organism
(the erythrocytes, perhaps, being the only exception), but this process
is especially active in the skeletal muscles and in the liver. The reaction
of glycogen breakdown, which is catalyzed by glycogen phosphorylase,
is nearly irreversible; for this reason, this enzyme takes no part in
the synthesis of glycogen. Two routes for glycogen synthesis are
possible. One route involves a successive addition of glucose units to
the extant glycogen moiety (glycogen primer); the other one originates
in glucose molecules. The source of glucose residues durinr; glycogen
synthesis is the active form of glucose, uridine diphosphate glucose
(UDP-glucose), which is produced from glucose I-phosphate and
uridine triphosphate (UTP) through the agency of the enzyme glucose-
I-phosphate uridyltransferase according to the scheme:
Glucose I-phosphates-UTP p UDP-glucose+H4pp7
The next step involves a transfer of the glucose residue from
UDP-glucose onto the glycogen primer through the aid of the enzyme
glycogen synthetase:
UDP-glucose + (Glucose) ~ UDP +(Glucose). + I
To be noted, the glycogen synthetase catalyzes the formation of a-
I -.4-glycoside bonds only. The "branching" enzyme, amylo-(a-I,4
-a-I,6)-transglycolysase, transf~rs short fragments (two or three
'glucose residues) from one portion of the glycogen molecule onto
another and forms a-I -.6-glycosidic bonds (branch points). The
alternating action of these two enzymes results in the lengthening of
the glycogen molecule.
If the synthesis starts from glucose molecules, then the initial step
is the transfer of glucose residues from UDP-glucose onto an
intermediary acceptor-tiolichol phosphate (membrane-bound polyprenol
phosphate). Dolichol phosphate assists in the synthesis of an
oligosaccharide which is then transferred onto the protein. The successive

addition of oligosaccharide chains to the glycogen molecule proceeds in
the same manner as in the former route. The feasibility of the latter
route is substantiated by the fact that glycogen is always bound with
some protein.
Interrelation of Glycogen Synthesis and Degradation. Glycogen
synthetase exists in two interconvertible forms. The phosphorylated,
or inactive, form is called glycogen synthetase D; the nonphosphor-
ylated, or active, form is referred to as glycogen synthetase I. The
transition from one form to the other is accomplished by two enzymes,
glycogen synthetase kinase (1) and glycogen synthetase phosphatase
(2) according to the scheme:
ATP ADP
Glycogen synthetase I -==='>~1~L==~. Glycogen synthetase 0
(nonphosphorylated .• 7" 2 (phosphorylated
enzyme) , enzyme)
")P0 4
The processes of glycogen synthesis and degradation in the cells

are controlled via phosphorylation mechanisms involving the key
enzymes of glycogen metabolismglycogen synthetase and glycogen
phosphorylase. The activation of adenylate cyclase (for example, with
adrenal in or glucagon) leads to the production of CAMP which triggers
the "cascade" mechanism of phosphorylation of glycogen synthetase
and glycogen phosphorylase, with the resultant formation of the
inactive (phosphorylated) glycogen synthetase D and active glycogen
synthetase I. This favours glycogen synthesis.
7.2.3 Biosynthesis of Other
Oligosaccharides and Polysaccharides
Homo- and heteropolysaccharides are carbohydrate components
of plasmic and structural glycoproteins in the mammals. These
carbohydrates are made up of a small set of monosaccharides: galactose,
mannose, N-acetylglycosamine, N-acetylgalactosamine, fucose, and
sialic acid. The connective tissue polysaccharides contain glucuronic
acid, iduronic acid, xylose, and sulphated derivatives of Nacetyl-
glucosamine or N-acetylgalactosamine. These homo- and heteroglycans
can be assembled by activated monosaccharide forms, such as nucleoside
phosphate derivatives of monosaccharides. UDP-derivatives of glucose,
galactose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid
and xylose; GDP-derivatives of mannose and fucose; and CMP-
derivatives of sialic acid are used in the synthesis. Most nucleoside
phosphate saccharides are produced through reaction of monosaccharides
or their derivatives with the corresponding nucleoside triphosphates.

Occasionally, certain monosaccharides convert to other monosaccharides
as constitutive components of nucleoside diphosphate saccharides. For
example, UUPglucuronic acid and UDP-xylose are formed from UDP-
glucose:
"'. 2NAD.~
\ ......~ UDP-xylose
UDPlIlucuronic acid - -....
CO 2
GDP-fucose is formed from GDP-mannose:
GDP-mannose --7"-=--,,0::::::---" GDP-fucose

NADP+ NADP'~
For example, it is to be noted that UDP-glucuronic acid which

is formed in the tissues, is used not only for polysaccharide synthesis,
but also for neutralization and removal of toxic and useless materials
or foreign compounds from the organism.
Synthesis of oligosaccharides is carried out with the participation
of specific glyc'Osyltransjerases whose diversity and functional
specialization in each cell provide for the production of various types
of homo- and heteroglycans. In the cell, glycosyltransferases are bound
with the membranes of endoplasmic reticulum or 'Golgi apparatus,
i.e. the organelles primarily responsible for assembling oligosaccharides.
It may be presumed that initially the assembly is made on the polyprenol
phosphate molecule onto which the monosaccharide residues from
nucleoside phosphate saccharides are successively transferred by
glycosyltransferases. Subsequently, the synthetized oligosaccharide is
transferred onto proteins to form glycoproteins. Simultaneous transfer
of glycosyl residues and assembly of proteinbased oligo- and
polysaccharides are also possible.
7.3 CARBOHYDRATE METABOLISM
CONTROL IN THE ORGANISM
The carbohydrate metabolic routes in various tissues of the organism
discussed above differ in intensity, which is defined by metabolic
features specific of each tissue and organ. However, from the standpoint
of activity of the whole organism, certain specializations of the
carbohydrate metabolic routes in individual tissues are profitably
complementary. For example, strenuous muscular exertion requires
energy which is initially supplied by the breakdown ·of glycogen to
lactic acid. The latter compound is excreted into the blood to be supplied
to the hepatic tissue, where it is used for the synthesis of glucose

during gluconeogenesis. From the liver, glucose is delivered in the
blood to the skeletal muscles to be consumed for energy generation
and to be deposited as glycogen. This intertissue (or interorgan) cycle
in the carbohydrate metabolism is referred to as the Cori cycle (called
also glucoselactate cycle):
Liver Blood Muscle
Glucose _Glucose _ Glucose ...............
t ~ Glycogen
Lactate 4 - - Lactate - - Lactate""""""'-
The maintenance of a constant glucose level in the blood is of
primary importance for the organism, since glucose is the major energy
substrate for the nervous tissue. The normal glucose content in the
blood is 3.3 to 4.0 mmoIllitre. An increased concentration of glucose
in blood is known as hyperglycemia. If hyperglycemia reaches as high
as 9 to 10 mmoIllitre, the glucose excess is released into the urine,
i.e. glucosuria sets in. On the contrary, a decreased glucose percentage
in the blood is known as hypoglycemia. Hypoglycemia as low as about
1.5 mmoIllitre leads to the syncopal state, while a still lower glucose
concentration results in high excitability of the nervous system and
ultimately leads to convulsions and coma.
To gain a better understanding of the mechanism that controls
the glucose level in the blood, it is important to examine processes
that contribute to an increased or lowered glucose concentration.
Processes leading to hyperglycemia:
(1) absorption of glucose from ·the intestine (alimentary
hyperglycemia);
(2) breakdown of glycogen to glucose (commonly, in liver);
(3) gluconeogenesis (in liver and kidney).
Processes leading to hypoglycemia:
(1) transport of glucose from the blood to tissues followed by
glucose oxidation to end products;
(2) synthesis of glycogen from glucose in liver and skeletal
muscles;
(3) production of triacylglycerol from glucose in fat tissue.
The dietary intake of carbohydrates leads to a short-term (within
1 or 2 hours) hyperglycemia and, occasionally, glucosuria.
Starvation stimulates the consumption of glycogen reserves in liver
and skeletal muscles, which prevents the development of hypoglycemia,

but within the space of a few hours only. Then, under lasting starvation,
the glucose level is maintained solely owing to the gluconeogenesis,
primarily at the expense of proteinic amino acids which suffer
degradation in the tissues. In point of fact, the potential ability to
sustain starvation is determined by the protein reserves available for
glucose production.
Blood
Glycogen (liver,
Glycogen
breakdown skeletal muscle)
Absorption
from intestive ---tl-i C10+ H1 0
(numerous tissues)
Gluconeogenesis Triacylglycerides
(adipose tissue)
The glucose level in the blood is monitored by neurohormonal

mechanisms. Excitation of the sympathetic portion of the autonomic
nervous system increases the glucose level in the blood, while excitation
of the parasympathetic portion produces a reverse effect. The only
hormone capable of reducing the glucose content is insulin. It stimulates
all of the three processes of glucose assimilation (intracellular transport
and degradation of glucose, synthesis of glycogen, and synthesis of
triglyceride from glucose in fat tissue). All other hormones make the
glucose level increase; for this reason, they are occasionally referred
to as contrainsular hormones. These include adrenalin, glucagon,
thyroxin and triidothyronin, somatotropin (which stimulate glycogen
degradation), and glucocorticoids (which stimulate gluconeogenesis).
8
Metabolism of Fats
and Glycerides
Lipids are continually renewed in the organism tissues. The major
part of lipids in the human body is represented by triacylglycerides
which occur as in-clusions in most tissues; the fat tissue, which consists
nearly totally of triacyl-glycerides, is especially lipid-rich. Since
triacylglycerides play an important role in the encrgetics of the
organism, the processes of their renewal (the conversion half-time for
triacylglycerides in different organs varies from 2 to 18 days) involve
both mobilization and deposition during energy production.
Compound lipids (phospholipids, sphingolipids, glycolipids, and
cholesterol and its esters) that make part of the biomembrane are
subject to a less active renew-al as compared with triacylglycerides.
Their renewal is associated either with the restoration of an impaired
portion of the membrane, or with the replacement of a "defective"
molecule by a new one.
The renewal of tissue lipids involves their preliminary intracellular
hydrolysis by enzymes.
8.1 DEGRADATION OF LIPIDS IN TISSUES
8.1.1 Intracellular Hydrolysis of Lipids
Hydrolysis of triacylglycerides in tissues is effected by a tissue
enzyme, tri-acylglyceride lipase. which hydrolyzes triacylglycerides to
glycerol and free fatty acids. There are a variety of tissue lipases that
differ primarily in their optimum pH and their location in the cell. The
acidic lipase is contained in lysosomes; the basic lipase, in microsomes;
and the neutral lipase, in cytoplasm. A specific feature of the tissue
lipase is its sensitivity to hormones which, by activating adenylate
cyclase, elicit the transition of the inactive tissue lipase to its active
194
METABOLISM OF FATS AND GLYCERIDES 195
fonn via phosphorylation with protein kinase. This mechanism bears

resemblance to the activation of phosphorylase B. Lipases mobilize
triacylglycerides. This process is also known as the tissue lipolysis.
The cell membrane phosphoglycerides are hydrolyzed with phosp-
holipases AI' A2 , C, and D, which are located chiefly in lysosomes.
However, certain phospholi-pases also occur in other cell organelles.
Hydrolysis of phosphoglycerides yields glycerol, fatty acids, nitrogenous
alcohols, and inorganic phosphate. There are also known specific enzymes
for hydrolysis of sphingolipids and glycolipids; the enzymes are involved
in the renewal of these lipids.
As is known, hydrolysis of intracellular lipids does not lead to a
storage of glycerol and fatty acids. This indicates that the hydrolysis
rate for the lipids is balanced against the rate of their intracellular
oxidation. In the adipose tissue, glycerol and fatty acids as produced
by triacylglyceride hydrolysis are not subject to oxidation and are
released into the blood to be supplied to other organs.
8.1.2 Oxidation of Glycerol
The glycerol metabolism is closely related to the glycolysis involving
glycerol metabolites according to the following scheme:
At first, glycerol is converted to a-glycerol phosphate through the

agency of glycerol phosphokinase. a-Glycerol phosphate, by the action
of NAD-dependent a-glycerol-phosphate dehydrogenase, is converted to
dihydroxyacetone phosphate, which, as a common glycolysis metabolite,
enters glycolysis to be reduced by enzymes to lactate under anaerobic
conditions, or to CO2 and Hp under aerobic conditions. Conversion of
one glycerol molecule yields one ATP molecule under anaerobic, and
19 ATP molecules, under aerobic conditions. Glycerol is a profitable
energy sub-strate and is used as an energy source practically by all
organs and tissues.
8.1.3 Oxidation of Fatty Acids
Oxidation of higher fatty acids was first studied in 1904 by Knoop
who fed animals with phenyl-substituted fatty acids and analyzed the
products in the urine. He showed that the fatty acid oxidation results
in the successive cleavage of two carbon moieties from the carboxyl
end. Knoop coined the fatty acid oxidation mechanism as n-oxidation.
As has been established by Kennedy and Lehninger in 1948-1949, oxidation
of fatty acids o~curs in the mitochondria only. Lynen and coworkers
have outlined major enzymic processes in fatty acid oxida-tion. At the
present time, the p-oxidation of fatty acids is referred to as the
Knoop-Lynen cycle.
The fatty acids, as produced by intracellular hydrolysis of
triacylglycerides or supplied to the cell from the blood, must be brought
into a state of activation. Their activation is effected in the cytoplasm
with the participation of acyl-CoA synthetase according to the scheme:
fIIIt+
CH.-(CH.),,-CH.-CH.-COOH+ATP+CoASH acyl-COA IJIltbet":
o
-+ CH,-(CH.J,,-CH.-CH.-~ - SCoA+AMP+H.P.O,
Since the activation process is effected extramitochondrially,
transport of acyls across the membrane into the mitochondria is
necessary.
The transport is accomplished with the participation of camitine,
which takes up the acyl from acyl-CoA on the outer membrane side.
Acylcarnitine assisted by carnitine translocase diffuses to the inner
side of the membrane to give its acyl to the CoA located in the matrix.
The process of reversible acyl transfer between CoA and carnitine on
the outer and inner sides.of the membrane is effected by the. enzyme
acyl-CoA -camitine transferase.
Cytoplasm Membrane Mitochondrion Matrix
o
?
R-C-SC
I
R-C-SCoA
CoASH
Figure 8.1 Transport of fatty acids across the mitochondrial membrane.

The oxidation of fatty acids within the Knoop-Lynen cycle occurs
in the matrix. The Knoop-Lynen cycle includes four enzymes that act
successively on acetyl-CoA. These are: acyl-CoA dehydrogenase (FAD-
dependent enzyme), enoyl-CoA hydratase, 3-hydroxyacyl-CoA
dehydrogenase (NAD-dependent enzyme), and acetyl-CoA acyltranslerase.
Each turn, or revolution, of the "fatty acid spiral" produces
an acetic acid residue split from the fatty acid as acetyl-CoA to yield
one FAD • H2 molecule and one NAD • H2 molecule. The cycle
turns are then repeated until the fatty acid chain becomes shortened
to a four-carbon fra~ent, i.e. butyryl-CoA. In the last turn, butyryl-
CoA splits apart, and two, rather than one, acetyl-CoA molecules
are formed.
Figure 8.2 Scheme for oxida-tion of fatty acids in the Knoop-Ly-nen cycle.
The oxidation products of an even-numbered fatty acid are acetyl-
CoA, FAD • H2 and NAD • H2 Subsequently, acetyl-CoA enters
o
the Krebs cycle, and FAD • H2 and NAD • H2 are directly supplied
to the respiratory chain.
The specific behaviour of odd-numbered fatty acids under
oxidation is that one propionyl-CoA molecule (qH3-C~-CO-SCoA)

per oxidized fatty acid mole-cule, alongside the products acetyl-CoA,
FAD • H2 , and NAD • H2 (common to even-numbered fatty acids),
is formed. Propionyl-CoA converts to succinyl-CoA:
COz fOOH
CHJ-CHr-CO-SCOA , ) " . CHJ-CH-CO-SCoA - HOOC-CHrCHz-CO-SCoA
All' ADP+' SUCCinyf..CoA
melhylmllonyl<OA
Carboxylation of propionyl-CoA is accomplished by propionyl-
CoA carboxylase (biotin, which is the carboxyl group carrier, serves
as a coenzyme for this enzyme); the presence of ATP is also required.
The methylmalonyl-CoA formed is converted by methylmalonyl-CoA
mutase (whose coenzyme, deoxyadenosylcobalamin, is a derivative of
vitamin B12) to succinyl-CoA; the latter enters the Krebs cycle.
The specific behaviour of unsaturated fatty acids under oxidation
is determined by the position and the number of double bonds in the
fatty acid molecule. The stepwise oxidation of an unsaturated acid to
the position of a double bond in it proceeds in a manner similar to
that of saturated acid oxidation. If the double bond retains the same
configuration (trans-configuration) and position (~2, 3) as those of the
enoyl-CoA, which is produced during the oxidation of saturated fatty
acids, the subsequent oxidation proceeds via conventional route.
Otherwise, the oxidation reaction proceeds with the involvement of
an accessory enzyme, ~3, 4-CiS-~2, 3Jrans-enoyl-CoA isomerase; this
facilitates the translocation of the double bond to an appropriate
position and alters the double-bond configuration from cis to trans.
The unsaturated fatty acid oxidation proceeds at a rate higher
over that for saturated acids. For example, if the oxidation rate for
saturated stearic acid is taken as a reference value, the oxidation rate
for oleic acid is 11 times, linolic acid, 114 times, linolenic acid, 170
times, and arachidonic acid, nearly 200 times as high as that for stearic
acid.
In addition to p-oxidation, two other oxidation routes are known
for fatty acids, referred to as a- and ro-oxidation. However, they
exhibit a lower activity and initially involve the formation of a- and
o-hydroxy acids, with subsequent con-versions thereof. These oxidation
routes are of inferior energetic value as compared with p-oxidation;
presumably, they are implicated in special functions of the cell.
Energy Balance of Fatty Acid Oxidation. The energetic value
of an even-numbered fatty acid is estimated in the following manner.
Complete oxidation of a fatty acid composed of 2n carbon atoms yields
METABOLISM OF FATS A)'J1) GLYCERIDES 199
n acetyl-eoA molecules (each acetyl con-taining two carbon atoms)

and n-I FAD -Hz and NAP -~ molecules (since the last tura of the
"fatty acid spiral"-yields two acetyl-eoA molecules, one FAD -H2
molecule and one NAD -H2 molecule). Oxidation of FAD -H 2 gives
two ATP mole-cules, and oxidation of NAD -H1i three ATP
molecules, i.e. a total of 5 ATP mole-cules, or, in the general case,
5 (n-I) ATP molecules. As has been noted above, the complete
oxidation of one acetyl-eoA molecule results in the formation of 12
ATP molecules, while n acetyl-eoA molecules provide 12n ATP
molecules. One ATP molecule being used for the fatty acid activation,
12n-1 ATP molecules remain. Now, the ATP balance for the complete
oxidation of an even-numbered fatty acid may be expressed by the
formula
5 (n - I) + 12n - I = (17n - 6) ATP molecules
where n is equal to half the number of carbon atoms in a given fatty
acid. For example, the palmitic acid molecule, which contains 16
carbon atoms, produces 130 ATP molecules.
The energetic value of fatty acids is superior, for example, to
that of glucose. For example, the complete oxidation of capronic acid
(whose molecule contains the same number of carbon atoms as
glucose) yields 45 ATP molecules as compared with 38 molecules
which can ,be derived from gluco~e. However, the acetyl-eoA
molecules as produced by P-oxidation require a sufficient amount of
oxaloacetate to be degraded by the Krebs cycle. In this resp(!ct,
carbohydrates have an advantage over fatty acids, since the breakdown
of the former species leads to pyruvate serving as a source for both
acetyl-eoA and oxaloacetate (pyruvate-carboxylase reaction), i.e. the
acetyl-eoA conversion within the Krebs cycle is thus facilitated. It is
not without reason that in the older biochemical literature the notion
that "fats burn down in the carbohydrate flame" was popular, since
the ATP from glycol-ysis can be used for the cytoplasmic activation
of fatty acids, while the pyruvate-derived oxaloacetate facilitates the
insertion of fatty acid acetyl residues into the Krebs cycle.
Importance of Fatty Acids as Energy Substrates for Various
Organs and Tissues. The organism tissues differ in the extent of
utilization of fatty acids and their intermediary oxidation products,
the so-called ketone bodies, as energy substrates. Fatty acids are
actively consumed in the heart as well as in the kidneys and skeletal
muscles (under prolonged physical exertion). In these organs, the
ketone bodies undergo oxidation to yield additional energy. In the
nervous tissue, the con-sumed amount of fatty acids and ketone bodies
as sources of energy is insignifi-cant.
8.2 BIOSYNTHESIS OF LIPIDS IN TISSUES
8.2.1 Biosynthesis of Fatty Acids
In the organism tissues, fatty acids are continually renewed in
order to provide not only for the energy requirements, but also for
the synthesis of multicomponent lipids (triacylglycerides, phospholipids,
etc.). In the organism cells, fatty acids are resynthetized from simpler
compounds through the aid of a supramolecular multienzyme complex
referred to as fatty acid synthetase. At the Lynen laboratory, this
synthetase was first isolated from yeast and then from the liver of
birds and mammals. Since in mammals palmitic acid in this process
is a major product, this multienzyme complex is also called palmitate
synthetase.
Biosynthesis of fatty acids exhibits a number of ~ific features:
(1) fatty acid biosynthesis, as distinct from oxidation, is localized
in the endo-plasmic reticulum;
(2) the !;ource for the synthesis is malonyl-CoA, which is
produced from acetyl-CoA;
(3) acetyl-CoA is involved in the synthetic reactions as a primer
only;
(4) NADP .H 2 is used to reduce fatty acid biosynthesis
intermediates;
(5) all the steps of malonyl-CoA fatty acid biosynthesis are cyclic
processes that occur on the surface of palmitate synthetase.
Production of Malonyl-CoA for the Fatty Acid Biosynthesis.
Acetyl-CoA serves as a substrate in the production of malonyl-CoA.
There are several routes by which acetyl-CoA is supplied to the
cytoplasm. One route is the transfer of acetyl residues from the
mitochondrial matrix across the mitochondrial membrane into the
cyto-plasm. This process resembles a fatty acid transport and is
likewise effected with the participation of carnitine and the enzyme
acetyl-CoA-carnitine transferase. Another route is the production of
acetyl-CoA from citrate. Citrate is delivered from the mitochondria
and udergoes cleavage in the cytoplasm by the action of the enzyme
ATP-citrate lyase:
Citrate + ATP + CoA ~ Acetyl-CoA + OXaloacetate + ADP + PI
The reaction is practically irreversible, and is shifted to the right.
The acetyl-CoA supplied to the cytoplasm via the above routes is

used for the synthesis of malonyl-CoA:
CH, COOH
1- ,,1+ 1
~ = 0+ HCO.+ATP -(B--bl-ot-In"~ CHi + ATP+H.PO.,
I
SCoA C=O
acetyloCoA I
SCoA
maionyloCoA
The reaction is catalyzed by the biotin,enzyme acetyl-CoA

carboxylase (E-biotin) assisted by Mg2+ ions. This enzyme is a
tetramer with a molecular mass of 400 000-500000.
Steps of Fatty Acid Biosynthesis Assisted by Palmitate
Synthetase. Palmitate synthetase is composed of seven enzymes; of
these, each is assigned a defmite func-tion. The acyl carrier protein
(ACP) is located at the centre of the multienzyme complex; the other
six enzymes occupy peripheral positions. ACP acts both as an acceptor
and a distributor of acyl groups. ACP contains a covalently bound 4-
phos-phopantethein bearing a free SH group for accepting an acyl
moiety. In addition to this central SH group, palmitate synthetase has
a peripheral SH group. Both SH groups participate as acyl acceptors
in the synthesis of fatty acids on the sur-face of the multienzyme
complex.
The cyclic process of fatty acid synthesis may be represented by
a series of consecutive reactions (hereafter palmitate synthetase is
designated by the symbol (

SH) •
SH
1. Transfer of acetyl moiety from acetyl-CoA onto the synthetase:
o
II
SH CHI S - C-CHa
FI + ~=o- { +CoASIl
" SH ~SCoA SH"
This reaction is carried out by the first enzyme of palmitate
synthetase-acetyl-transacylase, which possesses an SH group. At this
stage of the synthesis, the acetyl acts as a primer.
2. Transfer of malonyl moiety from malonyl-CoA onto the
synthetase:
o COOH o
II
S-C-CH. ~H. II
S-C-CH.
g' + ~=O , 0
+ CoASH
~
" SH SCoA " S - C-CH.-COOH
II
The reaction is effected by the second synthetase ellzyme-
malonyltransacylase.
3. Acetyl-malonyl condensation and decarboxylation of the product
fonned:
o
II
S-C-CH. SH
/
E 0 -+ '0 0 +00.
"°11 II
" S - C-CH.-COOH
\I S - C-CH.-C-CH.
The reaction is catalyzed by the third synthetase enzyme-3-ketoacyl
synthetase. An acetoacetyl, which is bound to synthetase, is fonned
at this stage .
.4. The first reduction of the intennediate with the involvement
of NADP -H 2 :
SH 0 /SH
I _~,....-~_. \ 0
E's _ ~-CH-CH-CHl ~H+ k+ 5 - !-CH1-CH1-CH l
The reaction is catalyzed by the fourth synthetase enzyme- P-

ketoacyl reductase, to yield intennediary hydroxybutyryl.
5. Dehydration of the intermediate:
8H 8H
/ /
E 0 OH -+ E 0 + H,O
..... II I " 1\
8,.., C-CH,-CH-CH. 8,.., C-CH=CH-CH.
The reaction is catalyzed by the fifth synthetase enzyme-
hydroxyacyl hydratase, to produce crotonyl.
6. The second reduction of the intennediary product with the
involvement of
The reaction is catalyzed by the sixth synthetase enzyme-
enoylreductase, to form an enzyme-bound butyryl. The butyryl thus
synthetized is transferred, through the mediacy of the first synthetase
enzyme, acetyltransacylase, onto the SH group (the upper one in the
Scheme) initially bound to the acetyl primer. The SH group (the lower
one in the Scheme), thus freed, accepts a new malonyl residue:
o
II
SII S --- C-CH,-CH,-CH.
./
,
0
II
- I'-.
S --- C-CH,-CH,-CH. SH
The synthetic cycle is thus repeated.
Seven cycles are implicated in palmitic acid biosynthesis and,
accordingly, seven malonyl residues and one acetyl are required.
Acetyl is the end moiety in fatty acid biosynthesis. The palmitic acid
thus synthetized is either transferred onto the outer CoA to produce
acyl-CoA, or, more commonly, is hydrolyzed by the specific palmitate
deacylase to yield a free fatty acid. .
Fatty Acid Chain Elongation., The mitochondria and endoplasmic
reticulum provide the conditions for an eventual chain elongation of
the cell-synthetized or dietary fatty acids. This process is different
from the fatty acid biosynthesis in the proper sense of the term. In the
mitochondria, the chain elongation is achieved through the aid of an
enzyme complex by adding acetyl residues from acetyl-CoA. In the
endoplasmic reticulum, the chain elongation is accomplished by an
enzyme complex through making use of malonyl-CoA.
Biosynthesis of Unsaturated Fatty Acids. In the mammalian
tissues, the forma-tion of monoene fatty acids is only possible. Oleic
acid is derived from stearic acid, and palmitooleic acid, from palmitic
acid. This synthesis is carried out in the endoplasmic reticulum of
the liver cells via the monooxigenase oxidation chain. Any other
unsaturated fatty acids are not produced in the human organism and
must be supplied in vegetable food (plants are capable of generating
polyene fatty acids). Polyene fatty acids are essential food factors for
mammals.
8.2.2 Biosynthesis of Triglycerides
Triglyceride biosynthesis proceeds with the involvement of the
lipids deposited in fat tissue or· in other tissues of the organism. This
process is localized in the hyaloplasm of cells.
204 MICROBIOLOGY AND BIOCHEMISTRY·
a-Glycerol phosphate and acyl-CoA, rather than corresponding

free glycerol and free fatty acid, are utilized in the direct synthesis of
triglycerides. a-Glycerol phosphate is produced either by phosphor-
ylating the glycerol supplied to the tissue, or by reducing dibydroxy-
acetone phosphate as an intermediary product of glycolysis.
The first step of triglyceride biosynthesis is the formation of
phosphatidic acid with the involvement of glycerophosphate acyltrans-
ferase:
i
H2C-O-C-R
Hi~-OH
H-l:-OH
:;:::>'"
R-CO-SCoA
R!...CO-SCoA
cc:::::::
2 CoA SH I i
H-y-o-C-R'
HJ-o P0 3H 2 H 1C-OPO l H 2
a - glycerol phosphite
. 'Pholphltid~ ICid
Further, the phosphatidic acid is subject to an attack by phospha-

tidate phosphatase to yield diglyceride:
o
11
J=:=i_:.
H.bPQ,A
pbolpllaUtUe actd dlllJC8l'lde .
The third acyl residue is transferred onto diglyceride by means

of diglyceride acyltransferase
HIC,-O-CR
W
~ "" • H2C- O- -R
~
9 R"-CO-SCoA CoA SH 0
HC-o-~-R' H -o-!R'
HJ-oH
diaCY'g'yceride
H f-o-~R"
t~lCYIglyceride
, The triacylglyceride thus synthetized is stored as fat inclusions
in the cell cyto-plasm.
8.2.3 Phospholipid Biosynthesis
Biosynthesis of phospholipids is associated with the renewal of
.~ of p/IoIpIIagIycer
c2nd pethwrf)
.'oflIIY"theIis
tri.cylgl~
.'aeynthells
of phOlPhoglycericllll
C11t~yl---
PhoopFhat:;osito,
I.-itol
. j
CH,O-C_
LoLR'
I
CH,o--CDP
CDP-diecylgtycerol
~,of
O-CHr::HCNH')cOOH
eMP
-",. .
H R
II HQ-C-R'
j
CH,o-r-OCH'fHCOOH
OH NH,
PhoophatidylMrl...
co'1
Phoophatidylethano...... i ...
~ S-adenosylmethionine
~ 5-lIdenosylhomocystel...
Phoophatldylchollne
Figure 8.3 Two pathways for the synthesis of certain phospholipids.
membranes, This process is accomplished in the, tissue hyaloplasm.

The fIrst steps of phospholipid and trig~yceride biosyntheses coincide;
subsequently, these routes diverge at the level of phosphatidic acid
and diglyceride.
Two routes to phospholipid biosynthesis are known; in either,
the participation of CTP is necessary. The first route involves
phosphatidic acid in phosphoglyceride biosynthesis. Phosphatidic acid
reacts with CTP to yield CDP-diglyceride which, as a coenzyme,
can participate in the transfer of diglyceride onto serine (or inositol)
to produce phosphatidylserine (or phosphatidylinositol). Serine
phosphatides are liable to decarboxylation (pyridoxal phosphate acting
as a coenzyme) to yield ethanolamine phosphatides. The latter species

are subject to methylation by S-adenosylmethionine (which donates
three methyl groups), tetrahydrofolic acid and methylcobalamin acting
as methyl group carriers.
The second synthetic route involves activation of an alcohol (for
example, choline) to produce CDP-choline. The latter participates in
the transfer of choline onto diglyceride to form phosphatidylcholine.
The phospholipids thus obtained are transported by lipid-carrier
cytoplasmic proteins to the membranes (cellular or intracellular) to
replace the used or impaired phospholipid molecules.
Because of the competition between the phospholipid and
triglyceride synthetic routes for common substrates, all substances that
favour the former route impede the tissue deposition of triglycerides.
Such substances are referred to as lipotropic factors. They include:
choline, inositol, and serine, as structural components of phospholipids;
pyridoxal phosphate, as an agent facilitating the decarboxylation of
serine phosphatides; methionine, as a donor of methyl groups; and
folic acid and cyanocobalamin, involved in the formation of methyl
group transfer coenzymes (tetrahydrofolic acid and methylcobalamin).
They may be used as drugs preven-tins excessive deposition of
triglycerides in tissues (the so-called fatty infiltra-tion).
8.2.4 Biosynthesis of Ketone Bodies
Three compounds: acetoacetate, P-hydroxybutyrate, and acetone,
are known as ketone bodies. They are suboxidized metabolic
intermediates, chiefly those of fatty acids and of the carbon skeletons
of the so-called ketogenic amino acids (leucine, isoleucine, lysine,
phenylalanine, tyrosine, and tryptophan). The ketone body production,
or ketogenesis, is effected in the hepatic mitochondria (in other tissues,
ketogenesis is inoperative). Two pathways are possible for ketogenesis.
The more active of the two is the hydroxymethyl glUJarate cycle which
is named after the key intermediate involved in this cycle. The other
one is the deacylase cycle. In activity, this cycle is inferior to the
former one. Acetyl-CoA is the starting compound for the biosynthesis
of ketone bodies.
Hydroxymethyl Glutarate Cycle. At the first step of this cycle,
condensation of two acetyl-CoA molecules takes place, with the
participation of acetyl CoA acetyltransferase:
CH.-C..., SCoA+CH.-C..., SCoA .... CH.-C-CHa-c"" SCoA+CoASH
~ ~ ~ ~
acetyl-CoA
Further, acetoacetyl-CoA becomes coupled once more to an acetyl-

CoA molecule through the assistance of hydroxymethylglutaryl-CoA
synthase:
CH.
II
CH.-C-CH.-C - SCoA+CH.-C - SCoA _ HOOC-CH.-C-CH.-C _ SCoA+CoASH
II II II I
o 0 0 OH
~-Hydroxy-~-methylglutaryl-CoA is split by hydroxymethylglutaryl-

CoA lyase into acetyl-CoA and acetoacetate:
CH.
I
HOOC-CH.-C-CH.-C - SCoA ..... CH.-C- SCoA+HOOC-CH.-C-CH.
I II II II
OH 0 0 0
Acetyl-CoA is again used at the fIrst step and closes thereby the
whole process into a cycle. Acetoacetate, as a representative of the
ketone body family, is the end product of the hydroxymethyl- glutarate
cycle.
The other ketone bodies are derived from acetoacetate: P-
hydroxybutyrate, by reduction with the involvement of NAD-dependent
hydroxyburyrate dehydrogenase, and acetone, by decarboxylation of
acetoacetate with the participation of aceto-acetate decarboxylase:
i OH
HOOC-C H Z-C-CH3 7":C::::;.. H'OOC-CH -bH-CH
z 3
IIIAD'H+H+ NAD+ 11- hydroxybutyrlte
~coz
H 3C-C-CH 3
~
acetone
The Deacylase Pathway for Ketogenesis is feasible after the

formation of acetoace-tyl-CoA which is subject to hydrolysis to
acetoacetate in the liver with the involvement of acetoacetyl-CoA
hydrolase, or deacylase.
In the liver, the ketone bodies suffer no transformation, and are
excreted into the blood. The normal contents of ketone bodies (as
acetoacetate or ~-hydroxy-butyrate) amount to mere 0.1-0.6 mmol/
litre). Other tissues and organs (heart, lung, kidney, muscle, and
nervous tissue), as distinct from the liver, utilize the ketone bodies
as energy substrates. In the cells of these tissues, acetoacetate and 1-
hydroxybutyrate enter ultimately the Krebs cycle and "burn down"
to CO 2 and H,O to release energy.
8.2.5 Biosynthesis of Cholesterol

In the experiments with acetic acid labelled radioisotopically and
fed to ani-mals, it has been established that the cholesterol carbon
framework is made up entirely of the acetic acid carbon.
Biosynthesis of cholesterol from acetyl-CoA proceeds, assisted
by the enzymes of endoplasmic reticulum and hyaloplasm, in many
tissues and organs. This pro-cess is especially active in the liver of
adult humans.
Cholesterol biosynthesis is a multistage process; in general, it
may be divided into three steps:
(1) production of mevalonic acid from acetyl-CoA;
(2) synthesis of an "active isoprene" from mevalonic acid
followed by the con-densation of the former to squalene;
(3) conversion of squalene to cholesterol.
The initial reactions in the first step, prior to the formation of
P-hydroxy-p-methylglutaryl-CoA from acetyl-CoA, resemble those
involved in ketogenesis with the only distinction that ketogenesis occurs
in the mitochondria, while cho-lesterol biosynthesis is carried out
extrarnitochondrially:
2 Acetyl-CoA ~ Acetoacetyl-CoA + Acetyl-CoA ~
I3-Hydroxy-l3-methylglutaryl-CoA
Further, l3-hydroxy-l3-methylglutaryl-CoA is converted with
hydroxymethylgluta-ryl-CoA reductase to mevalonic acid:
fH l CH l
I
HOOC-CHI-C-CHI-C-SCoA 7' • HOOC-CHIC-CHl-CHIOH + CoASH
bH A 2NADP·H+H+ ~p. I
OH
This reaction is irreversible and is a rate-limiting stage of the
overall cholesterol biosynthesis.
An alternative route to mevalonic acid is also possible, which
differs from the former one in that the formation of l3-hydroxy-l3-
methylglutaryl residue occurs on the surface of an acyl carrier protein
(like in fatty acid biosynthesis). The intermediary product in this route,
P-hydroxy-p-methylglutaryl-S-ACP, is re-duced by another enzyme to
mevalonic acid.
During the second step, mevalonic acid is implicated in a number
of enzymic reactions involving ATP, and is converted to isopentyl
pyrophosphate and to its isomer 3,3-dimethylaUyl pyrophosphate.
Actually, the two compounds constitute the "active isoprene", which
is consumed in the production of squalene. During the third step,
cholesterol is generated from squalene:
Squalene ~ Lanosterol ~ Cholesterol
The steroid ring hydroxylation proceeds with the involvement of
the monooxygen-ase chain of endoplasmic reticulum membranes.
Cholesterol esters are produced by transferring an acyl moiety
from acyl-CoA or from phosphatidylcholine onto the cholesterol
hydroxyl group. The latter process is catalyzed by phosphatidylcholine
cholesterol acyltransferase:
Phosphatidylcholine + Cholesterol ~
Lysophosphatidylcholine + Cholesterol ester
Cholesterol esters are produced especially actively in the intestinal
mucosa and in the liver.
Thus, the tissue cholesterol can be synthetized from any materials
whose break-down leads to acetyl-CoA. These include carbohydrates,
amino acids, fatty acids, and glycerol.
The liver plays a decisive role in the cholesterol metabolism. The
liver accounts for 90% of the overall endogenic cholesterol and its
esters; the liver is also impli-cated in the biliary secretion of cholesterol
and in the distribution of cholesterol among other organs, since the
liver is responsible for the synthesis of apoproteins for pre-~
lipoproteins, a-lipoproteins, and l3-lipoproteins which transport the
secreted cholesterol in the blood. In part, cholesterol is decomposed
by intestinal micro-flora; however, its major part is reduced to
coprostanol and cholestanol which, together with a small amount of
nonconverted cholesterol, are excreted in the feces.
Cholesterol, mostly esterified, is utiliZed in the buildup of cell
biomembranes. Besides, cholesterol is a precursor to biologically
important steroid compounds: bile acids (in liver), steroid hormones
(in adrenal cortex, male and female sexual glands, and placenta), and
vitamin D3 , or cholecalciferol (in skin).
8.3 REGULATION OF LIPID
METABOLISM IN THE ORGANISM
The rate of lipid metabolism in the organism tissues is dependent
on the dietary supply of lipids and on the neurohormonal regulation.
An excessive intake of high-calory food (carbohydrates and triglycerides)
impedes the consumption of endogenic triglyceride reserves stored in
the fat tissues. Moreover, carbohydrates provide a very favourable
basis for the neogenesis of various lipids; for this reason, a large
dietary intake of carbohydrate-rich food exerts a significant influence on

the production of triglycerides and cholesterol in the organism.
Synthesis of endogenic cholesterol is also controlled by exogenous
cholesterol supplied in food: the more dietary cholesterol is digested,
the less endogenic cho-Iesterol is produced in the liver. Exogenous
cholesterol inhibits the activity of hydroxymethylglutaryl-CoA reductase
and the cyclization of squalene to lanosterol.
The dietary ratio of vru:ious lipids plays an important role in the
lipid metab-olism in the organism. The available amounts of polyene
fatty acids and phospho-lipids acting as solvents for fat-soluble vitamins
affect not only the absorption of the latter species, but also the solubility
and stability of cholesterol in the organism fluids (blood plasma and
lymph) and biliary ducts. Vegetable oils with a high percentage of
phospholipids and polyene fatty acids impede an excessive accumulation
of cholesterol and its deposition in blood vessels and other tissues, and
facilitate the removal of cholesterol excess from the organism. These
processes are most markedly affected by corn oil, safflower oil,
cottonseed oil, and sunflower oil. The consumption of unsaturated
fatty acids contained in vegetable oils pro-duces a favourable effect on
the synthesis of endogenic phospholipids (for which these acids are
substrates); polyene fatty acids are also needed in the production of
other materials, for example, prostaglandins. Unsaturated fatty acids
act as uncouplers for the oxidative phosphorylation and thus accelerate
oxidation processes in the mitochondria and control thereby an excessive
triglyceride deposition in the tissues.
TIle lipotropic factors exercise a marked effect on the biosynthesis
of phospho-lipids and triglycerides. As has been mentioned above,
they facilitate the phospho-lipid synthesis. The dietary deficiency of
lipotropic factors favours the triglyceride production in the organism.
Starvation elicits mobilization of triglycerides from the adipose
tissue and inhibits the endogenic cholesterol synthesis owing to the
low activity of hydroxy-methylglutaryl-CoA reductase. The latter
process provides the possibility for the active production of ketone
bodies in the liver.
The neurohormonal control of lipid metabolism chiefly affects the
mobilization and synthesis of triglycerides in the fat tissue. The
lipolysis in tissues is dependent upon the activity of triglyceride lipase.
All the regulators that favour the conversion of the inactive
(nonphosphorylated) lipase to the active (phosphorylated) one, stimulate
the lipolysis and the release of fatty acids into the blood. Adrenalin
METABOUSM OF FATS AND GLYCERIDES 211
and noradrenalin (secreted in the sympathetic nerve endings), honnones

(glucagon, adrenalin, tbyroxin, triiodothyronine, somatotropin, 3-
lipotropin, corticotropin, etc.), tissue hormones, including biogenic
amines (histamine, serptonin, etc.) act as stimulators for this process.
Jnsulin, on the contrary, inhibits the adenyl ate cyclase activity,
preventing thereby the formation of active lipase in the fat tissue,
i.e. retards the lipolysis. In addition, insulin favours the neogenesis
of triacyl-glycerides from carbohydrates, which, on the whole, provides
for lipid deposition in the fat tissues as well as for the cholesterol
production in other tissues. The thyroid hormones thyroxin and
triiodothyronine assist in the oxidation of the cholesterol side chain
and in the biliary excretion of cholesterol in the intestine.
8.4 PATHOLOGY OF LIPID METABOLISM
Most commonly, the lipid metabolism pathology is manifest as
hyperlipenaia (elevated concentration of lipids in blood) and tissue
lipidoses (excessive lipid de-position in tissues). Normally, the lipid
contents in the blood plasma are: total lipids, 4-8 g/litre; triglycerides,
0.5-2.1 mmolflitre; total phospholipids, 2.0-3.5 mmolflitre; total
cholesterol, 4.0-8.0 mmol/litre (esterified cholesterol accounts for 2/3
of total cholesterol).
Hyperlipemia may manifest itself by an increased concentration
of lipids, or certain groups thereof. For example, hypercholesterolemia
and hypertriglyceri-demia may be mentioned in this connectiori. Since
practically all the blood plasma lipids make part of lipoproteins,
hyperlipemias may be reduced to one of the hyper-lipoproteinemia
forms which differ in the varied ratios of plasma lipoproteins of
different groups.
Distinguished are exogenous, or alimentary, hyperlipemias which
are actually associated with a normally increased blood lipid
concentration after the intake of a food high in fat, and endogenic
hyperlipemias caused by impaired lipid metab-olism. The endogenic
hyperlipemia may be due to a primary hereditary defect in apoproteins
or in a lipid metabolism enzyme. However, of more frequent
occur-rence are hyperlipemias attributable to secondary causes, for
example, to regulatory disturbances of the lipid metabolism or to unfav-
ourable environmental factors. Five types of primary hyperlipopro-
teinemias are distinguished.
Hyperlipoproteinemia, Type I, is characterized by the enhanced
content of chylo-microns in the blood plasma; simultaneously, the
percentage of u- and f3-lipopro-teins may be lowered. The triglyceride
content is 8-10 times above the norm, while cholesterol does not
exceed the normal level. Presumably, Type I is associated with a
defective lipoprotein lipase that destroys chylomicrons.
Hyperlipoproteinemia, Type II. is characterized by an increased
I-lipoprotein content in the blood plasma and, respectively, by a 1.5-
2-fold higher, against the norm, cholesterol concentration. The familial
form of hyperlipoproteinemia, Type Ila, is also known, which
manifests itself in the occurrence of a defective apoprotein for P-
lipoproteins, and in a slower breakdown of these materials in the
tissues.
Hyperlipoproteinemia, Type III, is a rare hereditary disease (also
called familial dysbetalipoproteinemia) manifested by the occurrence
of an uncommon P-lipo-protein form. Cholesterol and triglyceride
contents in the patients may occasion-ally be 2-5 times superior to
the norm.
Hyperlipoproteinemia, Type IV, is characterized by increased
contents of pre-p-lipoproteins and triglycerides (2-5 fold) in the blood
plasma. Its incidence rate is higher in aged patients. Hereditary forms
of this disease (called also familial hyperprebetalipoproteinemia) have
been described.
Hyperlipoproteinemia, Type V. This pathology is manifested by
increased con-tents of chylomicrons, pre-p-lipoproteins, triglycerides,
and cholesterol in the patients' blood plasma.
Secondary hyperlipoproteinemias, which arise from a disordered
lipid tissue metabolism or its impaired control, are observed in diabetes
mellitus, thyroid gland hypofunction, alcoholism, etc.
Tissue Lipidoses. Hyperlipoproteinemias may lead to tissue
lipidoses. Lipidoses can also arise from hereditary defects of the
enzymes involved in the synthesis and breakdown of lipids in the
tissues. We now discuss certain instances of tissue lipidoses.
Atherosclerosis is a wide-spread pathology, manifested chiefly by
the deposition of cholesterol in arterial walls, which results in the
formation of lipid plaques (atheromas). Lipid plaques are specific
foreign bodies around which the connective tissue develops abnormally
(this process is called sclerosis). This leads to the cal-cification of
the impaired site of a blood vessel. The blood vessels become inelastic
and compact, the blood supply through the vessels is impeded, and
the plaques may develop into thrombi.
Atherosclerosis results from hyperlipoproteinemia. All of the
lipoproteins, ex-cepting chylomicrons, are capable of penetrating the
vessel wall. However, a-lipo-proteins, which are rich in proteins and
phospholipids, are liable to an easy break-down within the vessel Wall,
or are apt to leave it because of their small size. ~-Lipoproteins and,
partly, pre-~-lipoproteins containing much cholesterol exhibit
atherogenic properties. Elevated concentrations of lipids of these groups
and an increased vessel wall permeability are conducive to deposition
of atherogenic lipoproteins within the walls, with the subsequent
development of atherosclerosis.
Fatty infiltration of the liver. In this pathology, the triglyceride
concentration in the liver is lO-fold superior to the norm.' The
accumulation of fat in the cyto-plasm of hepatic cells leads to an
impaired liver function. The causes of this pathol-ogy are numerous;
one of these may be a deficiency in lipotropic factors and the
associated therewith synthesis of excess triglycerides.
Ketosis is a pathologic state produced by an excess, of ketone
bodies in the organism. However, ketosis may be regarded as a lipid
metabolism pathology with a certain reserve, since excessive
biosynthesis of ketone bodies in the liver is sequent upon an intensive
hepatic oxidation not only of fatty acids, but also of keto-genic amino
acids. The breakdown of the carbon frameworks of these amino acids
leads to the formation of acetyl-CoA and acetoacetyl-CoA, which are
used in
ketogenesis. The ketosis is accompanied by ketonemia and
ketonuria. which is manifested by the increased concentration of ketone
bodies in blood and their ex-cretion in the urine. In an aggravated
form of ketosis, the ketone body concentra-tion in blood may be as
high as 10-20 mmolllitre. The ketone bodies are normally present in
the daily urine in trace amounts, while in pathology, 1 to 10 g (or
even more) of ketone bodies per day is excreted in the urine. Most
commonly, ketonemia and ketonuria are observed in diabetes mellitus
(the manifest ketosis symptoms are dependent on the extent of diabetes
mellitus), as well as in prolonged starvation or in "steroid" diabetes.
8.5 APPLICA TIONS OF LIPIDS AND THEIR
COMPONENTS IN PHARMACOTHERAPY
Fat-emulgated preparations for parenteral administration have been
elaborated for clinical applications. Since these are administered to
the patients intravenously, the size of fat emulsion particles should
not exceed the size of the largest naturally occurring lipoproteins-
chylomicrons, i.e. about I JLm. Fat emulsions on the basis of corn
oil (preparation lipomaize), cottonseed oil (lipofundin, lipomo!),
soybean oil (intralipid) have been proposed. These preparations are

composed of lipids (10 to 20%), emulsifying agents (phosphatides and
other materials) and, occasion-ally, glycerol. They are prescribed to
asthenic patients for increasing energy re-sources of the organism. In
addition, lipotropic preparations (methionine, choline, and inositol),
which make part of natural phospholipids, are used in the prophy-Iaxis
of fatty infiltration of the liver.
9
Metabolism of
Nucleic Acid
The discovery of the base-paired, double-helical structure of
deoxyribonucleic acid (DNA) provides the theoretic framework for
determining how the information coded into DNA sequences is
replicated and how these sequences direct the synthesis of ribonucleic
acid (RNA) and proteins. Already clinical medicine has taken advantage
of many of these discoveries, and the future promises much more. For
example, the biochemistry of the nucleic acids is central to an
understanding of virus-induced diseases, the immune re-sponse, the
mechanism of action of drugs and antibiotics, and the spectrum of
inherited diseases.
In approaching the study of the molecular mechanisms of heredity,
this chapter first discusses the structural and functional roles of the
genetic material, DNA. This includes an analysis of its replication
and susceptibility to mutation. The health-related aspects of the use of
recombinant DNA techniques are considered, and examples of their
use in the analysis of several human genetic diseases are used to
illustrate the biochemical side of genetics.
9.1 FUNCTIONAL ROLES OF DNA
9.1.1 DNA as the Genetic Material
The nucleic acids were recognized as chemical substances more
than 70 years before DNA was found to be responsible for the
transmission of inherited characteristics. Later it was suspected that
DNA might be the genetic material because of its high concentration
in chromosomes and in some viruses. The premise was complicated,
however, because the concentration of protein in these structures was
215
also high. Furthermore, RNA but not DNA was found in some viruses.
Indirect evidence pointed to a role for nucleic acids as the transmitters
of biologic information; the wavelengths of light in the ultraviolet
region that are the 'most mutagenic are the same wavelengths at which
nucleic acids absorb the most light energy.
9.1.1.1 Constancy of DNA concentration
One property expected of the genetic material is a constancy of
amount in every cell of the body under every environmental situation.
DNA, not RNA or protein, fulfills this expectation. Its content per
nucleus is the same in every cell except the germ cells, which have
exactly half that found in the somatic cells. Again, this is expected if
progeny obtain half their characteristics from each parent. This
constancy is so dependable that the measurement of the DNA
concentration in a tissue can be used to calculate the number of nuclei
and thus the number of cells. This works well for diploid cells such
as those of the kidney" but corrections must be made for polyploid
mammalian liver or cancer cells.
9.1.1.2 Transformation of ceUs with DNA
The best evidence that exogenous DNA can produce permanent
changes in cells came from the experiments of Avery et al. DNA
from one strain of bacterial cells was used to transform a different
strain of cells so that they came to resemble the strain from which
the DNA was derived. In the original experiment, DNA was isolated
from' cells of a strain of Diplococcus pneumoniae that contained a
characteristic complex polysaccharide on their surfaces. This
polysaccharide made the cells pathogenic for mice and gave a glistening,
smooth appearance to colonies formed by these cells on nutrient agar.
When the polysaccharide was missing, as it was in some other strains
of the microorganism, the colonies were rough in appearance and the
cells were harmless when injected into mice. When DNA from the
smooth cells was added to rough cells, the DNA entered some of the
cells and became a permanent part~,f their genetic apparatus; subsequent
generations were permanently changed to pathogenic cells that formed
smooth colonies. This process is called bacterial transformation.
Subsequently, similar experiments were done with viral nucleic
acids. The pure viral nucleic acid, when added to cells, led to the
synthesis of complete virus particles; the protein coat was not required.
This process is called transfection. More recently, DNA has been
used in cell-free extracts to program the synthesis of RNA that functions
as the template for the synthesis of proteins characteristic of the DNA
METABOLISM OF NUCLEIC ACID 217
template. Considering all this evidence, DNA undoubtedly is a carrier

of genetic infonnation.
9.1.2 Cellular Location of DNA
Most of the DNA of animal cells is found in the nucleus, where
DNA is the major constituent of the chromosomes. On the other hand,
most of the RNA is located in the cytoplasm. Nuclear DNA exists as
a thin, double helix only 2 nm wide. The double helix is folded and
complexed with protein to form chromosomal strands approxim-ately
100 to 200 nm in diameter. Each chromosome contains a single DNA
duplex. The human chromosomes vary in size; the smallest contains
approximately 4.6 x 10' base pairs of DNA, and the largest 2.4 x
10' base pairs. In contrast, the Escherichia coli chromosome has 4.5
x H)6 base pairs. The DNA of the chromosomes is tightly packed and
associated with both histone and nonhistone proteins.
The amount of genomic DNA in a particular organism is roughly
proportional to the c.,omplexity of the organism. Table shows the content
of DNA in the genomes of several widely different organisms. The
data are normalized to a haploid set of chro-mosomes, since some
cells listed are haploid and others are diploid. The DNA content
TABLE 9.1 : DNA CONTENT OF SOME CELLS AND VIRUSES.
Haploid size of ge-
Source of DNA nome, base pairs
Viruses
SV40 5 x 1()3
Papilloma (wart) 8 x 1()3
Adenoviruses 2.1 x 100
Herpesviruses 1.56 x l()5
Poxviruses 2.4 x l()5
Cells
Escherichia coli 4.5 x 1()6
Yeast 1.3 x 10'
Drosophila 1.6 x loa
Human 3.2 X 109
Animal mitochondria 1.5 x lOA
of a few viruses is given for comparison. The size of DNA is often
measured in base pairs, since cellular DNA is all double stranded and
base paired. Thus the number of base pairs in a DNA molecule is a
precise measure of the number of mononucleotides that comprise the
polynucleotide chain. On the average, one base pair represents about

600 daltons; thus, to estimate the molecular weight of a DNA, the
number of base pairs is multiplied by 600.
9.1.2.1 Histones and chromosome strocture
The chromosome structure is visible only during the mitotic portion
of the cell cycle. The constituent parts of the chromosomes· are
nucleoprotein fibers called chromatin. When condensed, chromatin fonns
a microscop-ically visible chromosome-like structure. The chromosomes
are composed of DNA, RNA, and proteins. The relative amounts of
the three vary, but chromatin is primarily protein and DNA.
9.1.2.1.1 IVucleosomes
The his tones are the major proteins associated with the
chromosomes. These small, basic proteins can be separated into five
groups by polyacrylamide gel electrophoresis. All five histone groups
are found in every eukaryotic cell. These groups are called HI, H2A,
H2B, H3, and H4. Each histone is present in equimolar amounts except
for HI, which is present in approximately half the concentration of
the others. Furthermore, the HI electrophoretic band is composed of
many similar but slightly different proteins. In this respect the HI
group differs from the other histone groups, which are each single
proteins. The histone groups differ in their relative content of lysine
and arginine residues. Table lists some of the properties of these
chromosomal proteins. The sequences of histones H2A, H2B, H3, and
H4 are greatly conserved between species, even though an organism
might have several genes for the same histone. This virtual sequence
identity testifies to a very similar and essential function for the four
histones in all eukaryotic species. A clue to this function is the ability
of these histones to associate at high ionic strength to form an octomer
containing two copies of each of the four histone groups.
9.1.2.1.2 Histones of the nucleosome core
The four histone groups that are composed of ho-mogeneous proteins,
H2A, H2B, H3, and H4, make up the nucleosome core. Each core
consists of two copies of the four histones. The double-stranded DNA
is wrapped twice around each core in a left-handed superhelix. A
superhelix is the name given to the additional helix made by the double-
stranded, helical DNA as it is wrapped around the nucleosome core.
A familiar superhelix in everyday life is a twisted spiral telephone
cord. The nucleosome core of histones do not recognize specific DNA
structures; rather, they can bind to any stretch of DNA as long as it
is not too close to a neighboring nucleosome. The order of contact of
histones to the DNA is as follows:
TABLE 9.2 : PROPERTIES OF ANIMAL mSTONES.

Electrophoretic Mass (kilo-
group daltons) Lysine (%) Arginine (%)
HI 21 Z7 2
H2A 14.5 11 9
H2B 13.8 16 6
H3 15.2 10 15
H4 11.3 10 14
Protein-protein interactions between the histone subunits are
undoubtedly important in promoting formation of a nucleosome in which
146 base pairs of DNA are coiled around the outside of the histone
core. One molecule of histone HI binds to an exterior region of each
nucleosome, but histone Hi is not needed to determine nucleo-some
structure. The distance between nucleosomes is approximately 200 base
pairs; consequently, in electron micrographs, nucleosomes resemble
evenly spaced beads on a string of DNA. Neutron and x-ray diffraction
data are also consistent with this structure.
The histone core protects the DNA bound to the nucleosome from
digestion by pan-creatic deoxyribonuclease (DNase) I or micrococcal
nuclease. Nucleases, however, will cleave the linker DNA that connects
the nucleosome subunits to one another.
Nucleosornes can be reconstructed in the laboratory from DNA and
pure histones. Histone HI is not necessary for the reconstruction, which
further shows that HI is an accessory protein and not a major structural
part of the nucleosome subunit.
The primary function of the nucleosomes is to condense DNA.
Further condensation of nucleosome DNA requires nonhistone nuclear
proteins. These proteins make up a scaffoldlike structure around an
additional helix consisting of coiled nucleosomes. This produces a
structure that resembles a solenoid, with six nucleosome subunits per
turn. The solenoid structure can form large loops that give additional
structure to the incipient chromosome.
~
9.1.2.1.3 1kuuied chromosomes
Although it is not known how the characteristic banded structure of
a chromosome is related to its function, the DNA of a single chromosome

probably consists of a single DNA duplex running from one end of the
chromosome to the other. These bands, which can be seen microscopically
after staining with fluorescent dyes such as Giemsa or quinacrine, are
believed to represent regions of heterochromatin complexed with histones
and nonhistone proteins.
9.1.2.1.4 Nonhistone proteins
The nucleus contains a large number of proteins other than histones.
These so-called nonhistone proteins mayor may not be tightly associated
with the chromosomes. For example, the nucleus contains enzymes
associated with the synthesis of RNA and DNA; these are nonhistone
proteins, but they are not part of the structure of chromosomes. One
group of nonhistone proteins are the high mobility group (HMG) proteins,
named for their rapid movement on polyacryl-amide gel electrophoresis.
The HMG proteins, but not histone HI, are ~sociated with the chromatin
that is most active in RNA synthesis.
9.1.2.1.5 Mitochondrial nucleic acid
Not all the cellular DNA is in the nucleus; some is found in the
mitochondria. In addition, mitochondria contain RNA as well as several
enzymes used for protein synthesis. Interestingly, mitochond-rial RNA
and DNA bear a closer resemblance to the nucleic acid of bacterial
cells than they do to animal cells. For example, the rather small
DNA molecule of the mitochondrion is circular and does not form
nucleosomes. Its information is contained in approximately 16,500
nucleotides that func-tion in the synthesis of two ribosomal and 22
transfer RNAs (tRNAs). In addition, mitochondrial DNA codes for
the synthesis of 13 proteins, all components of the respiratory chain
and the oxidative phosphorylation system. Still, mitochondrial DNA
does not contain sufficient information for the synthesis of all
mitochondrial proteins; most are coded by nuclear genes. Most
mitochondrial proteins are synthesized in the cytosol from nuclear-
derived messenger RNAs (mRNAs) and then transported into the
mito-chondria, where they contribute to both the structural and the
functional elements of this organelle. Because mitochondria are inherited
cytoplasmically, an individual does not necessarily receive mitochondrial
nucleic acid equally from each parent. In fact, mito-chondria are
inherited maternally.
9.1.3 Clinical Comment
9.1.3.1 Leber's Hereditary Optic Myopathy
This disease is one of several myopathies caused by defects in the
mitochondrial genome; thus they are called mitochondrial myopathies.

Leber's hereditary optic neuropathy causes blindness in young males
more often than females, even though the disease is transmitted
maternally. The myopathy is caused by a single base mutation at
position 11,778 of the mitochondrial DNA that changes an arginine
codon to a histidine codon in subunit four of NADH-coenzyme Q
oxidoreductase. This enzyme is part of complex I of the respiratory
chain.
9.1.4 Other Conformations of DNA
A -DNA The Watson-Crick model of DNA is based on the x-ray
diffraction patterns of B-DNA. Most DNA is B-DNA; however, DNA
may take on two other conformations, A-DNA and Z-DNA. These
conformations are greatly favored by the base sequence or by bound
proteins. When B-DNA is slightly dehydrated in the laboratory, it takes
on the A conformation. A-DNA is very similar to B-DNA except that
the base pairs are not stacked perpendicular to the helix axis; rather,
they are tilted because the deoxyribose moiety "puckers" differently.
An A-DNA helix is wider and shorter than the B-DNA helix.
o o
11
-O-P-O-CH
n
-O-P-O-CH
I •0 I ·0
0- 0-
0- 0-
o-P~O- o-P~O-
U n
o o
Syn
Anti
Z-DNA This conformation differs more radically. It is a left-handed
helix instead of the right-handed conformation of A-DNA and B-DNA.
The Z-DNA conformation exists only along a string of alternating
purines and pyrimidines, especially several guanine-cytosine residues
in a row. An alternating dinucleotide sequence results where the
external phosphate groups zigzag, thus Z-DNA. This structure results
from alternating anti and syn conformations of the glycosidic bonds. In
A- and B-DNA the conformations of the glycosidic bonds are all anti.
In Z-DNA, guanine residues are syn, whereas cytosine and thymine

residues are anti. Eukaryotic DNA contains several alternating purine-
pyrimidine sequences consistent with the Z-DNA confonnation; however,
the biologic significance of Z-DNA is still unclear.
9.1.5 The "Central Dogma"
DNA has two broad functions: replication and expression. First,
DNA must be able to replicate itself so that the information coded
into its primary structure is transmitted faithfully to progeny cells.
Second, this information must be expressed in some useful way. The
method for this expression is through RNA intermediaries, which in
tum act as templates for the synthesis of every protein in the body.
The relationships of DNA to RNA and to protein are often expressed
in a graphic syllogism called the "central dogma.» The concept was
proposed by Crick in 1958 and was revised in 1970 to accommodate
the discovery of the RNA-dependent DNA polymerase. Crick's original
theory suggested that the flow of information was always from RNA
to protein and could not be reversed, yet it allowed for the possibility
of DNA synthesis from RNA.
,,
,
\
~
RNA - - - - - - - Protein
Figure 9.1 : The "central dogma."
Genetic expression involves the transfer of information by the
processes of transcription and translation. Transcription is the process
that transfers information using the same four-letter language of the
nucleic acids; that is, one strand of DNA serves as a template for the
synthesis of an RNA strand, the sequence of which is analogous to one
DNA strand and complementary to the other. Transcription is
"reversible" in a few cases. The dashed line in Figure represents the
synthesis of DNA from information contained in the RNA of certain
tumor viruses. Information flow in the direction of RNA to protein is
termed translation, since the four-letter language of the nucleic acids
must be converted to the different 20-letter language of the amino acids
that make up proteins. The process of translation is always unidirectional.
Single-stranded DNA templates can be translated in the laboratory, but
no evidence exists for such a function in vivo; consequently the line

between DNA and protein in Figure is dashed.
9.1.5.1 DNA synthesis
Knowing that DNA was the hereditary material gave no clues as
to how the molecule might reproduce itself until Watson and Crick
proposed their model for the structure of DNA. In this model the DNA
strands are arranged in an antiparallel fashion and are base paired along
their entire length in the form of a double helix. The molar concentration
of adenine equals that of thymine, and the cytosine concen-tration is the
same as that of guanine. Base pairing of adenine with thymine and of
cytosine with guanine yields a structure in which the sequence of one
strand can be automatically determined if the sequence of the other
strand is known. The importance of this concept in the replication of
DNA and in the synthesis of RNA strands of com-plementary sequences
was recognized immediately, but several years were required for the
enzymatic studies that gave unequivocal proof.
:" ~ . S~~) S~Conservativa~ S ':-:'.

,'~-::: ...
S
+ 4 Semiconservative +
...... . . .
,..' "" replication replicatiOn
. . "
Double-strandad
Maternal strands DNA
Daughter strands
9.1.6 Strand Separation

I1nplicit in the functioning of the Watson-Crick DNA model is the
idea that the strands of a DNA molecule must separate and new
daughter strands must be synthesized in response to the sequence of
bases in the mother strand. This is called semiconservative replication.
Still, conservative replication, in which both strands of a daughter
molecule are newly synthesized, could not be ruled out by consideration
of the structure of DNA alone.
The experiments of Meselson and Stabl proved replication to be
semiconservative. These consisted of growing E. coli cells in a medium
containing 'sNH 4Cl, so that the nitrogen atoms of the purine and
pyrimidine bases of the DNA were heavily labeled. Cells were then
transferred to a medium containing the usualligbt 14NH4Cl and grown
for one or more generations. The DNA was then prepared and separated
by density-gradient equilibrium centrifugation in a solution of cesium
chloride. After one generation the progeny DNA separated in such a

way that all of it appeared at a position midway between the very
heavy parental DNA and the light DNA of a control culture. Because
all the DNA existed as a hybrid that contained one heavy strand and
one light strand, DNA clearly was replicated by a semiconservative
mechanism.
This presented a more difficult problem: How do the double-helical
strands separate during DNA synthesis? In a rapidly growing cell such
as E. coli it has been calculated that if the strands separate by
untwisting, the molecule would have to rotate at 10,000 rpm, a rate
that is highly improbable. The answer to this problem lies in an
understanding of the mechanism of DNA replication at the enzyme
level. We will return to this subject after first considering the enzymes
involved in DNA synthesis.
9.1.7 DNA Polymerases
DNA synthesis is more complex than originally thought. One reason
is that DNA rep-lication requires many different enzymes, not just
DNA polymerase. For example, rep-lication requires enzymes that
coordinate the growth of cell membranes with DNA syn-thesis. Other
enzymes and protems initiate the synthesis of small RNA primers that
bind to single-stranded DNA. Additional enzymes are needed to remove
the RNA primers from the growing deoxyribonucleotide chain, fill in
the small regions vacated by the RNA primers, seal the strands together,
and aid in the untwisting of the DNA helix. More than one enzyme may
be required for each of these functions, and this list of functions is not
meant to be complete.
9.1.8 Bacterial DNA Polymerases
The mechanisms involved in DNA synthesis are most easily
understood by considering the DNA polymerases. The most extensively
studied are the three DNA polymerases, I, II, and III, from E. coli.
Some ambiguity still exists about the essentiality of the specific roles
played by each of the polymerases. One complicating feature is that it
_ is difficult to distinguish the polymerases from enzymes that function
exclusively to repair damaged DNA, primarily because some of the
processes that occur during DNA replication are identical to events
necessary for DNA repair. However, all the DNA polymerasees require
a DNA template and all four of the de-oxyribonucleoside triphosphates.
Synthesis proceeds from the 5' to the 3' end of the growing polynucleotide,
arid inorganic 'pyrophosphate (PP) is a product of the reaction.
Polynucleotides formed using radioactive deoxyribonucleoside
I
METABOLISM OF. NUCLEIC ACID 225
triphosphates have sequences identical to those of one strand of the
DNA template and complementary to sequences of the other strand.
Both strands are labeled in vitro.
DNA polymerase I is a nonessential enzyme, since viable E. coli
mutants lack it (pol A). This conclusion is complicated, however,
since the enzyme catalyzes three separate chemical reactions. It
polymerizes deoxyribonucleoside triphosphates, and it has two
exonucleolytic activities, a 3' to 5' activity and a 5' to 3' activity.
The pol A - mutants lack only the polymerization activity. Other mutants
lacking both the polymerase and the 5' to 3' exonuclease activity are
lethal. Thlis the exonuclease function is the more important one. This
fits with the role of this enzyme in removing damaged DNA segments
(DNA repair) and in removing covalently attached RNA from DNA
chains. We will later see that small RNAs serve as primers of DNA
synthesis.
dATP
dCTP DNA polymerase I

---------~.. DNAPoIymer + PPj
dGTP DNA template
Mg++
dTTP I
Figure 9.2 : Reaction catalyzed by DNA polymerase I. dATP

DNA polymerase I has been purified to homogeneity. When the
pure enzyme is treated with subtilisin, a proteolytic enzyme from
Bacillus subtilis. the polymerase is cleaved into two pieces. The small
fragment retains the 5' to 3' nuclease activity, whereas the larger
piece, called ,a Klenow fragment, has both polymerase activity and the
3' to 5' exonuclease activity. The Klenow fragment is sold commercially
for use in labeling DNA for use in detecting recombinant DNA.
DNA polymerase II is more likely needed for the repair synthesis
of DNA. Repair synthesis requires excision of the damaged DNA, the
synthesis of a fresh replacement segment complementary to the
remaining single strand, and the sealing of the replacement segment
to the larger polynucleotide chain. DNA polymerase II does not have
5' to 3' exonuclease activity. Mutants deficient in DNA polymerase II
activity, as determined by in vitro assay, grow well; therefore the
enzyme does not seem to have an indispensable function in the cell.
DNA polymerase III has all the enzymatic activities of DNA
polymerase I. A subunit of the enzyme is the product of the dna E gene.
Temperature-sensitive mutations of this gene testify to the importance
226 MICROBIOLOGY AND BIOCHEMISTRY -
of DNA polymerase III. A temperature-sensitive mutant is one that

grows at 30· C but fails to grow at 42° C, a temperature not lethal for
wild-type E. coli. The failure to grow at the higher temperature is
caused by a mutation in the gene for DNA polymerase III so' that a very
heat-labile enzyme is produced. Since this appears to be the only
mutation in this strain, DNA polymerase III is the only enzyme inactivated
at 42° C; thus the enzyme is essential to the organism.
9.1.8.1 Template and primer
At this point it is necessary to make a distinction between the
meanings of template and primer. The word template refers to the
structural sequence of the polymerized monomeric units of a
macromolecule that provides the pattern for the synthesis of another
macromolecule with a complementary or characteristic sequence. The
word primer, on the other hand, refers to a polymeric molecule that
contains the growing point for the further addition of monomeric units.
Glycogen is an example of a primer to which glucose units are added;
however, glycogen has no template activity.
Under certain circumstances DNA has both primer and template
activities. For example, the addition of mononucleotides is to the 3'
end of the growing DNA primer. This presents a problem with regard
to how the other strand is synthesized. Biochemists have looked hard
but unsuccessfully for an enzyme that can add deoxyribonucleotides
onto the 5' end of DNA primers. Such a primer should contain a
triphosphate on the hydroxyl group of the 5' end. Although a very
active 5'-exonuclease, actually part of DNA polymerase I, has made
the search for such an activated 5' end extremely difficult, investig-
ators conclude that a polymerase able to use such a primer probably
does not exist. On the contrary, good evidence suggests that the
synthesis of both strands is by the known DNA poly-merases.
9.1.9 Stages of DNA Synthesis
9.1.9.1 Origin of Replication
In E. coli cells, DNA replication starts at a specific site called
oriC. The oriC locus contains only 245 base pairs. Similar sequences
are responsible for initiating the synthesis of plasmid and bacteriophage
DNA. The oriC nucleotide sequence binds several units of the
tetrameric form of the dnaA protein. This protein is named for the
gene that encodes it. The dnaB and dnaC proteins then bind to the
complex. As a result of binding these proteins, a portion of the helical
DNA is unwound. This forces the rest of the DNA into a left-handed
double helix that wraps around the proteins to give a structure
resembling the histone-containing nucleosome of eukaryotic cells. The
exposed single-stranded DNA is stabilized by the binding of a 74 kDa,
single-stranded DNA-~inding protein called SSB.
9.1.9.2 RNA primers
All DNA polymerases add mononucleotides to the 3'end of an
existing primer. Consequently a special primer is needed for DNA to
replicate in its entirety. RNA polymerases can initiate polymer
synthesis without a primer; thus short RNA primers are used to initiate
DNA synthesis.
The RNA oligonucleotides are complementary to a sequence on
one of the strands of the DNA template and base pair with a portion
of the DNA molecule. Subsequently, deoxyribonuc1eotides are covalently
attached to the RNA primer. The synthesis of the primer itself is
catalyzed by a special RNA polymerase called primase. Similar RNA
polymerase-like enzymes are used to prime the synthesis of certain
viral DNAs and eUkaryotic DNA.
The dnaB and dnaC proteins, as well as at least a few other
accessory proteins, are required for the primase to initiate RNA synthesis
from the DNA template. This enzyme complex, called a primasome. is
very large, almost as large as the DNA polymerase III holoenzyme (800
kDa) , which joins the primasome and catalyzes the addition of
mono-deoxyribonuc1eotides to both growing strands. No problem exists
in visualizing the ad-dition of oligonucleotide monomers to the RNA
primer at the 5' end of the continuous strand, since this DNA polymerase
is a highly accurate, processive enzyme. Processivity means that the
polymerase can rapidly add many mononucleotides to the primer, more
than 1000 per second, before dissociating from it. On the other strand,
DNA synthesis must proceed away from the replicative fork; however,
if the template strand is looped back toward the replicative fork, subunits
of the DNA polymerase could add nucleotides to both growing strands.
Addition to an RNA primer would continue until synthesis was blocked
by the previously made primer and its attached oligodeoxyribonuc1eotide.
This stalling might trigger the synthesis of a new RNA primer and the
addition of deoxynucleotides to it.
The mechanism of DNA synthesis is known in considerable detail
so the steps illustrated in Figure are very much simplified.
9.1.10 Bidirectional Synthesis
DNA synthesis occurs in both directions at each of the rep-licating
forks. Once a DNA strand has been primed, synthesis toward the
replicating fork can be visualized as continuous. Growth of the opposite,
New DNA, continuous strand

Figure 9.3 : A single unit of DNA polymerase m complex synthesizes both new strands
of DNA. one continuously and the other in short pieces. Deoxynucleotide additon to the
daughter strands is indicated by vertical lines across the strands.
lagging strand occurs in dis-continuous bursts, each burst primed by a
short RNA segment. DNA synthesis visualized by electron microscopy
gives the appearance of an "eye" or several eyes along a DNA template
and resembles this: Eyes are thought to be regions of DNA where
recent synthesis has been initiated. Synthesis from an eye is bidirectional.
Thus as the eye enlarges, DNA synthesis along either new strand may
be considered continuous where the DNA polymerase is close to and
moving toward the replicating fork, and it may be considered discontinuous
where the DNA polymerase is close to a replicating fork but moving
away from the fork. Consequently, as an eye enlarges, DNA synthesis
is more or less continuous at one end of a growing strand but
discontinuous at the other end of the same strand.
9.1.10.1 Removal of the RNA primer and
ligation of the DNA fragments
The end result is newly synthesized DNA that is interspersed with
segments of RNA and that is discon-tinuous but base paired with an
intact parental strand. Subsequently, the 5' exonucleolytic activity of
DNA polymerase I removes the RNA segment, and either DNA
polymerase I or II fills the gap vacated by the RNA. DNA ligase
(sometimes called polynucleotide ligase) is required to join these short
pieces into phosphodiester linkage. The ligation reaction shown in the
following diagram requires that energy be supplied from ATP. This
enzyme also occurs in animal cells.
ATP + Ugase ~ Ligase - AMP + PP!
H PO:
LigaIIe - AMP + :',-'-""T'~' 0 O"",,-,,---r-,-r,:: __ I Ugase+
O·
O-P-O
I 11 I
AMP + ~~I.....
1 0 "'r---I-r--T""I-'-1
DNA ligase is not only important in DNA replication; it is also
used to seal deoxyri-bonucleotide segments in the crossover events
during gene recombination. The enzyme also functions to close breaks
in segments of DNA undergoing repair and is required to join theends
of mitochondrial DNA to form their characteristic circular structure.
9.1.10.2 Topoisomerases
Armed with this information, the unwinding problem menfioned
ear-lier can be reconsidered. By the alternating action of endonucleolytic
and ligase activities, the unwinding of DNA could be reduced to an
untwisting of only a small part of the double helix at any given time.
Both activities are part of the enzyme called topo-isomerase 1.
9.i.1O.2.1 Topoisomerase 1
This enzyme releases the torque developed during the unwinding
required for replication. This torque introduces superhelices into DNA.
A superhelix can be visualized as a helix on top of the basic DNA
helix. The enzyme first introduces a single-strand break in the superhelix.
This is not a hydrolytic cleavage but rather a transesterifica-tion of the
5' phosphoryl at one end of the broken strand to a tyrosine hydroxyl
group, thus conserving the energy of the phosphodiester bond. The
single-strand break relieves the torque as the broken strand with the
enzyme still attached rotates about the unbroken strand. When the strain
on the double helix is relaxed, the enzyme transfers the 5' phosphorylated
end back to the polynucleotide chain and dissociates from the DNA
duplex. Actually the broken strand need only pass through the neighboring
intact strand and reseal to remove one superhelical turn. To relieve the
tension of several superhelical turns, as occurs during DNA replication,
the topoisomerase catalyzes several "nicking-closing" reactions.
Topoisomerase I recognizes either positively or negatively supercolIed
DNA. Topoisomerase I activity has also been found in the nuclei of
animal cells.
9.1.10.2.2 Topoisomerase II
Another enzyme, called topoisomerase II, or DNA gyrase, also
plays a role in the unwinding of replicating DNA. Although topoisom-
erase I can relieve the positive superhelical torsion introduced into
DNA as a result of unwinding, topoisomerase II can introduce negative
superhelices ahead of the replicating fork. This relieves the twisting
pressure of DNA replication before it can develop. The enzymes also
differ in that topoisomerase I does not require high energy in the form
of adenosine S'-triphosphate (ATP), whereas topoisomerase II does,
since energy is required to make negatively supercoiled DNA. This
torsional energy is conserved in the negative superhelices found in most
naturally occurring DNA, such as the DNA of nucleosomes. The
topoisomerases also differ in that enzyme I cleaves only one DNA
strand, whereas enzyme II cleaves both. Approximately 200 base pairs
of DNA coil about topoisomerase II, much as occurs with the DNA in
a nucIeosome. Both strands are opened, and the 5' phosphoryl groups are
linked to tyrosine hydroxyl groups on the enzyme. A DNA segment is
passed through both the anchored but cleaved ends. This passage is
always in the same direction, so that only a negative superhelix forms
when the strands are resealed. A DNA gyrase-like activity has been
isolated from animal cells.
9.2 DNA SYNTHESIS IN ANIMAL CELLS
The replication process in animal cells is necessarily more complex
than in bacteria because several chromosomes must be replicated. DNA
syniliesis in animal cells also differs in that several origins of replication
occur within a single chromosome rather than the single site in E. coli.
This speeds up the duplication of the animal genome, which is
approximately 1000 times larger than that of bacteria. The eukaryotic
origins of replication have a high affinity for the nuclear matrix, the
nucleoprotein material that remains after nuclei have been washed with
a high concentration of salt.
DNA polymerases from several different animal cells have been
isolated and studied. The three DNA polymerases of animal cells,
called a, /3, and y, can be distinguished by their molecular weights,
template specificity, and sensitivity to sulfhydryl reagents. Table 14.3
compares the three in regard to these differences. DNA polymerase a
is probably the most important for DNA replication. This enzyme
shares many functional properties with DNA polymerase III of E. coli:
9.2.1 DNA Polymerase a.
Even though DNA polymerase a and its associated subunits have
not been purified to homogeneity, much is known about the function of

this enzyme complex. The concentration of DNA polymerase a is
higher than that of the other two polymerases. One of the associated
subunits has primase activity capable of making short RNA primers.
At frrst this enzyme was thought to be present in the cytoplasm of
cells, but with ~pecial precautions it can be isolated from the nuclei.
Unlike E. coli DNA polymerase I, polymerase a has no associated
nuclease acitivity. It is membrane-bound: however, and fractionates
with ribonucleotide reductase, dTMP synthase, and thymidylate kinase,
enzymes important in the synthesis of DNA precursors. The synthesis
of poly-merase a increases greatly in regenerating liver and in other
rapidly dividing cells.
9.2.2 DNA Polymerase J3
This is a smaller, stable enzyme that has been highly purified. It
is immunologically distinct from the other polymerases, indicating that
it is not merely a subunit of the larger polymerases. Polymerase 13 is
undoubtedly a repair enzyme.
9~ DNA Polymerase y
This is the enzyme responsible for the synthesis of mitochondrial
DNA and the DNA of some viruses, such as adenoviruses. Polymerase
-y is very large and consists of a tetramer of identical oligomers, each
having a molecular weight of 47,000. Synthetic ribonucleotides are very
effective· templates in the laboratory, but this mitochondrial enzyme
differs from reverse transcriptase in that natural RNAs are poor
templates.
9.2.4 Reverse Transcriptase
This enzyme is associ.ated with the virions of RNA tumor viruse&
such as the ROllS sarcoma virus (RSV). The enzyme has remarkable
enzymatic activity in that it can catalyze several seemingly diverse
steps in the synthesis of double-stranded DNA from the single-stranded
RNA viral genome. The enzyme uses a tRNA for tryp-tophan as a
, primer to make a copy of DNA that is complementary to the viral
RNA. The resulting RNA-DNA hybrid is converted to a double-stranded
DNA molecule by ribon-uclease (RNase)H and DNA-dependent DNA
polymerase activities that are intrinsic to reverse transcriptase.
9.2.4.1 Replication of linear eukaryotic chromosome
Eukaryotic chromosomes, unlike their bacterial counterparts, are
linear rather than· circular. Since RNA oligonucleotides prime both
prokaryotic and eukaryotic DNA synthesis, the 5' termini of the daughter
TABLE 9.3: COMPARISON OF PROPERTIES OF DNA POLYMERASES FROM ANIMAL TISSUES.
Property DNA polymerase a DNA polymerase ~ DNA polymerase 'Y

Molecular weight 155,000, plus three 43,000 193,000 (four oligo-
other subunits mers)
Template specificity Nicked DNA template, Nicked DNA template, Ribonucleotide template
RNA primer DNA primer and DNA primer
Deoxyribonuc1eoside triphos- All four required All four work, but single All four
phate dependence nucleotide will incorporate
Inhibition by sulfhydryl reagents Sensitive Less sensitive Sensitive
strands are incomplete in that they lack the DNA sequences that
correspond to the RNA primers.
S'
---rRNA\----
--- --
3'~
In protozoa this problem is solved by the addition of preexisting

oligodeoxynucleotide blocks to the 3' ends of DNA. These blocks are
composed of tanderoly repeated units of (T2G4)n or (TP4)n' where n is
approximately 50. The enzyme that adds these polymers requires a
primer but not a template. These oligonucleotide block polymers are
called telomers. Another DNA polymerase,
(G-G-G-G- T- T - T - 1)" -3'
which is template dependent, copies the G4T4 units, synthesizing a

complementary loop of C4A4 . This looping back allows the 5' end of
the genomic daughter strand to be finished.
DNA ligase joins both ends of the telomere to the daughter strand,
but the loop is subsequently cleaved to give flush-ended telomeres that
consist of one strand of G4T4 and another of C4A4.
9.2.5 Nucleosome Formation·

The synthesis of DNA and of histone proteins is coordinated.
Duplication of a genome requires doubling the amount of histone proteins.
During DNA synthesis the parental histones remain associated with
only the growing strand made continuously at the replicative fork. The
DNA of this new strand immediately hybridizes to one parental strand;
thus the "parental" histones tend to stay associated with the DNA
structure that remains essentially double stranded throughout the
replication· process. The other daughter strand is made in bits and
pieces, and at anyone time during replication it might contain
considerable amounts of single-stranded DNA. As the segments on the
lagging strand are fmished and ligated together, the structure now binds
newly synthesized "daughter" histones.
The association of histones to one or the other of the strands can
be distinguished using electron microscopy of material from cells grown
under conditions where protein (i.e. histone) synthesis is inhibited but
DNA synthesis is not inhibited.
9.2.5.1 Information stored in eukaryotic genes
Most of the E. coli genome codes for mRNAs that are translated
into proteins, but this is not the case for the animal genome. Since
animals are more complex, they probably require 50 to 100 times
more protein than bacteria, but their genomes are more than 500 times
larger. For example, the single chromosome of E. coli contains 4.5
million base pairs, whereas the 23 haploid human chromosomes contain
2400 million base pairs. The animal genome does have duplicate genes
for many proteins, and the genes for ribosomal RNA and for some
tRNAs are repeated many times, but the function of most animal
DNA is still unknown.
Even the genes that code for proteins are more complex in
vertebrates than in bacteria. Most, but not all, are expressed as long
RNA molecules that are reduced in size by splicing together the coding
segments. This yields a continuous template RNA that is sequentially
decoded by protein-synthesizing enzymes.
DNA that has no apparent function as a template for cellular
RNAs is sometimes called non genetic DNA. Nongenetic DNA includes
the pseudogenes. Pseudo genes are genes that cannot be expressed
because they lack sequences necessary for RNA modification or protein
synthesis initiation or because they contain protein synthesis "stop"
signals in the middle of coding sequences. Nongenetic DNA also includes
much repetitive DNA; about 30% of human DNA is repetitive. An
example is the human Alu sequence. This 300-base pair sequence is

repeated almost a million times at many places throughout the genome.
Mouse satellite DNA is composed of a repeated sequence of similar
size and number, but its sequence is tandemly repeated. The Alu sequence
is named for the restriction endonuclease that cleaves at a single site
within each repeated segment to yield many, almost identical copies of
300 base pairs each. Individual Alu sequences are homologous to one
another by about 85 %. Alu sequences are sometimes transcribed into
RNA. A small cytoplasmic RNA, called 7SL RNA, that functions as
part of a protein-secreting system is homologous with the Alu sequences
at its 3' and 5' end; consequently the Alu sequences originally may have
been derived from the 7SL RNA gene. What role, if any, they now play
is unknown.
9.2.6 Transposable Genetic Elements
Mobile genetic elements further complicate the or-ganization of
the chromosome. Mobile genetic elements are relatively small pieces
of DNA that have characteristic sequences at either end. These pieces
of DNA can move from one gene, or larger piece of DNA, to other
locations, even on a different chromosome. The short sequences that
flank the genetic elements are cleaved by an endonuclease to give
staggered ends that base pair with complementary strands, a result of
the nucleolytic cleavage of the target DNA by the same or a similar
enzyme. A recombinational event (i.e., a crossing-over reaction) serves
to transfer the small genetic element to its new location.
Many examples of mobile elements are found in bacteria, where
they are called transpo-sons. Bacterial transposons have terminal repeat
sequences that both code for the enzymes catalyzing the process of
transposition (transposases) and physically interact with these enzymes
to bring them to the DNA target site. At this site the DNA-bound
transposase presumably ca~alyzes the endonucleolytic cleavage of the
terminal repeat sequence of the trahsposon and also catalyzes a similar
sequence in the target DNA.
Perhaps the best examples of genetic transposition in animal cells
are the integration and subsequent removal of DNA programmed by
RNA retroviruses to and from any number of sites on eukaryotic
chromosomal DNA. The single-stranded RNA retrovirus uses the enzyme
reverse transcriptase to make a complementary DNA copy of itself.
The RNA present in the DNA-RNA hybrid is rapidly degraded, leaving
a single-stranded DNA, which integrates into the host genome when
copied. Integration is random in respect to the host DNA. Transcription
of the integrated viral genome produces many copies of viral RNA,

which can be packaged into virus particles; the whole process is repeated
in other cells or other organisms (horizontal transmission). Sometimes
the pro-virus is carried in germ line cells, where the sequences might
be transmitted to new generations (vertical transmission). In the case of
the retroviruses, the enzymes needed for the movement of the proviral
DNA are coded within the transposon, not in the repeated terminal
sequences, as they are in bacteria.
The animal transposons, as with other transposons, can carry along
pieces of the host DNA. For example, cellular oncogenes (abbreviated
c-onc) are sometimes carried along with the proviral DNA when it is
excised. These host sequences are maintained and carried, as RNA, in
the retrovirus, where over time and after many passages they are
extensively modified. Oncogenes isolated from retroviruses are
• abbreviated v-onc. More than 20 different v-onc genes have been found
in the retroviruses of different experimental animals. These oncogenes
code for proteins that lead to the transformation of normal cells to
cancer cells. The various oncogene proteins have diverse functions and
may be found in different parts of the cell. Several are tyrosine-specific
protein kinases or other protein kinases, some bind guanine nucleotides
and have guanosine triphosphatase (GTPase) activity, whereas others
may be derivatives of normal hormone receptors or protein growth
factors.
9.3 MOLECULAR BASIS OF MUTATION
On rare occasions a base may be changed or modified in the DNA
sequence. When protein synthesis is considered, such a change in the
structural gene for a protein could lead to the insertion of the wrong
amino acid. If changed at a crucial position, the resulting protein will
be unable to function. If the amino acid replacement occurs at a less
important position, activity may be diminished or not affected at all.
Mutations are responsible for dozens of known genetic diseases and
undoubtedly for many more yet to be discovered. Usually these changes
are subtle so they cannot be detected cytologically at the level of the
chromosome. Gross chromosomal abnormalities do occur and are very
important in the health sciences, but generally they are not inherited in
the classic mendelian way. Rather, most are caused by nondisjunction,
that is, a failure of either the egg or the sperm to receive an exact set
of haploid chromosomes or of a mitotic cell to receive an exact diploid
set early in development. Many others are caused by translocations that
are also difficult to predict.
Mutations are caused by both chemical and physical agents, although
the action of even the physical agents, (e.g., ionizing radiation) can
usually be explained by a chemical mechanism. Regardless of the agent
used to produce a mutation, none is selective in the sense that it can
specifically mutate one gene and not another. Because all genes are
composed of only four different types of purine or pyrimidine bases, an
agent that may react specifically with only one of the four could
potentially cause mutations in every gene. Mutations are essentially
random events. During our evolution the selective pressures of nature
eliminated an astronomic number of deleterious mutations. The smaller
number of beneficial mutations gave primitive life a survival advantage
over competitors and allowed for the eventual emergence of intelligent
beings. Consequently, in a highly evolved species· such as humans, most
mutations produce deleterious effects.
9.3.1 Mutagens
9.3.1.1 Purine and pyrimidine analogues
Mutations may be produced in many ways. Bases may be deleted
or new ones may be inserted; more frequently an existing base may be
chemically modified so that on replication, improper base pairing will
cause a different base to appear at the modified position. The latter type
of mutation is called a replacement. When a purine is replaced by
another purine or a pyrimidine by a different pyrimidine, the change is
called a transition. A transversion is a change from pyrimidine to purine
or purine to pyrimidine.
Many of the mutations caused by artificially produced base analogues
are transitions. Mutations are produced by base analogues in one of two
different ways. On entering the cell, a base analogue is converted to a
nucleoside triphosphate that base pairs, perhaps incorrectly, with a
DNA template and is inserted into the nucleotide chain. This is one
way in which the mutation can be produced. The other requires an
additional round of replication so that an improper base pair forms as
a result of the previously incorporated analogue. The result in both
cases is a permanently modified DNA.
As might be expected, base analogues can also inhibit DNA
synthesis and cell multiplication. It is this feature that has stimulated
organic chemists to create hundreds of different base analogues in the
hope that some may be useful for inhibiting rapidly proliferating cancer
cells. Examples of base analogues that have some usefulness in cancer
chemotherapy and that are also mutagenic are 6-mercaptopurine and
2-aminopurine.
SH
0:) N H
6-Mercaptopurine 2-Amlnopurlne
Not all analogues become active against cancer cells through
incorporation into nucleic acid. Some analogues block the synthesis of
normal purine and pyrimidine nucleotides; for example, 8-azaguanine
blocks guanosine monophosphate (GMP) synthesis and 6-mercaptopurine
inhibits adenosine monophosphate (AMP) syn-thesis.
9.3.1.2 Alkylating agents
Alkylating agents are also mutagenic substances that have been
used in cancer chemotherapy. Alkylating agents such as nitrogen or
o N .... CH,CH.CI
p .... ,
?! ~
C I ~o CH,CH,CI
NH CH, -fl-O-(CH,J. -O-S-CH,
o ~
Cyclophosphamide Busulfan
sulfur mustards chiefly cause transversions. Bifunctional compounds
such as those shown next produce cross-links between DNA strands or
between a DNA strand and any other reactive group in the vicinity.
The mechanism of action of alkylating agents is complex. Adenine
and guanine are easily alkylated. Guanine is alkylated primarly at
position 7 and adenine at position 3. The reaction produces an
exceedingly labile glycosidic bond. Splitting of this bond leads to
depurination.
o
A:J=
HN
~
N
H.N
I N>N
OCH.
DNAChain~
o
~DNAChain
In .those cases where alkylation does not lead to depurination, it

is more likely that the mutation will be of the transition type. However,
when depurination does occur, on replication the position opposite the
gap might be filled by anyone of the four bases. This accounts for the
transversions often caused by these agents.
9.3.1.3 Dyes
Acridine dyes such as the antimalarial agent quinacrine (Atabrine)
shown next are large planar aromatic compounds that intercalate or
sandwich themselves between the stacked bases of the helix.
CH.
I /CH,CH.
H, N-CH-CH,CH.N,
~
CH,CH.
-::r -::r I ""'" OCH.
a : : :,. "'N #
On replication, insenion or deletion of bases may occur. Chain

scission and chromosome breaks are also possible. Quinacrine is useful
in human cytogenetics, since it intercalates significantly into the
heterochromatin of the Y chromosome, making it fluoresce and rendering
it identifiable cytologically. Detection of the Y chromosome is important
in prenatal sex determination. Other dyes present in our environment
are potentially mutagenic. For example, some hair dyes were shown
to be mutagenic for E. coli.
9.3.2 Physical Agents
Growing tissues are most sensitive to ionizing radiation. DNA
synthesis is inhibited, yet the action of x-rays is indirect. They produce
free radicals, which in turn react with DNA and thus produce point
mutations or chromosomal breaks.
Large doses of ultraviolet light can damage DNA. In humans this
damage is confmed to the skin, since, unlike x-rays, ultraviolet light is
easily absorbed. The chemical lesion in this case is the formation of
dimers between adjacent thymine residues on the same DNA strand.
Unless corrected or removed, these dimers will stop DNA synthesis.
DNA repair Because most mutations are very damaging, even the
simplest organisms have enzyme systems that repair DNA. These DNA
repair systems are important because genetic defects in them can cause
some human diseases.
9.3.3 Excision Repair
The excision repair system consists of several enzymes, each involved
in several steps. First, the error must be recognized. For example, an
endonuclease binds regions of the DNA that contain thymine dimers and
cleaves at the 5' sides of the dimers. A DNA polymerase activity
replaces that portion of the DNA strand that had contained the thymine
dimer. An exonuclease then removes the piece of DNA containing the
dimer, and a DNA ligase rejoins the repaired and restored DNA
strand. These reactions are diagrammed in Figure 14.5. This form of
nucleotide repair also acts on other types of damaged DNA, such as
carcinogen-DNA adducts, and removes them by chain scission, patching,
and ligation.
Some damaged bases, particularly alkylated purine bases, are
removed by N-glyco-sylases. The gapped chain is cleaved by apurinic
endonucleases and the defective strand patched and ligated. Single-
strand breaks are repaired by analogous excision repair mech-anisffiS.
Mitomycin D and platinum complexes used in cancer therapy can cause
DNA-DNA cross-links between bases on opposite strands. These cross-
linked bases can be excised and repaired, first on one strand and then
on the other. The repair is error free unless the drugs have cross-linked
directly opposing bases.
9.3.4 Postreplication Repair .
Sometimes damaged DNA 'is replicated before it can be repaired.
When this happens, the replicating strand stops at the site of damage,
skips over the damaged base, and completes synthesis of the new strand.
The new daughter and old maternal strands separate, and eventually the
missing base is added, postreplicatively. The complemen-tary maternal
strand still contains the damaged DNA, so this mechanism is not,
strictly speaking, a repair mechanism, even though it allows synthesis
of normal DNA. Eventually the damaged DNA is repaired by another
mechanism.
9.3.4.1 Photoreactivation
This system acts directly 'on DNA damaged by ultraviolet light to
restore the damaged base to its original state without actually replacing
it. Because this system operates only on ultraviolet light-damaged DNA,
it plays a limited role in repairing human DNA. A light-activated
DNA photolyase catalyzes the conversion of thymine dimers to
monomers.
9.3.4.2 DNA glycosylases
The DNA bases that contain amino groups tend to de aminate
spontaneously. In particular, cytosine significantly deaminates to uracil,
but adenine and guanine can also deaminate to hypoxanthine and xanthine,
respectively. If not corrected, the new bases can cause serious mutations
---I
===~
---: )
___ I
Endonuclease
.. DNA polymerase I ..
--- ( - - - : 4 ,',
., I
---I New
DNA
---I ---I
- --I
- --', ---. ,
..
---I ---I
DNA ligase
- --I
,, ---,,
---,
- --I
,, ,
___ I
---I
Figure 9.4 : Repair of DNA inactivated by ultraviolet light. Light causes the dimerization
of adjacent thymine residues that block DNA replication. The four enzymes shown are
involved in removal and replacement of a portion of the DNA that contains the dimer.
on replication. Fortunately, highly specific enzymes recognize these
bases as being foreign to DNA, and they catalyze the hydrolysis of the
N-glycosyl bonds that connect the bases to the DNA polymer. This
produces DNA polymers with a few skipped bases. Another repair
enzyme, an endonuclease, recognizes the skipped bases and cleaves the
chain to leave a 3' hydroxyl group on the 5' adjacent nucleotide. A
DNA polymerase now fills in the missing mononucle-otide, taking its
instructions from the intact complementary strand.
Defects have been found in these mechanisms that cause various
human diseases. For example, patients with the genetic disease xeroderma
pigmentosum are especially sensitive to ultraviolet light and develop
skin cancer. Skin fibroblasts cultured from these patients have been
shown to be defective in DNA repair.
9.4 CHEMICAL CARCINOGENESIS
Most human cancer is caused by substances in the environment,
chemicals, viruses, or radiation. All these carcinogens affect DNA.

Sometimes we can relate a substance to a specific type of cancer; for
example, cigarette smoking to lung cancer. In other cases it is more
difficult to draw a cause-and-effect relationship. Nevertheless, the
evidence is overwhelming that carcinogens cause cancer by interacting
with DNA. When the DNA modified by these agents cannot be repaired,
cancer often results.
Carcinogenesis develops in three stages: initiation, promotion, and
progression. Chem-ical substances can act at the initiation stage or at
the promotion stage. Moreover, some chemicals have both initiating
and promoting activities.
9.4.1 Initiating Agents
Initiating agents alter the native molecular structure of DNA. They
- may cause an accu-mulation of somatic mutations over the lifetime of
an individual. Initiating agents may be physical, biologic, or chemical-
for example, ionizing radiation, tumor viruses, and cyclophos-phamide.
Chemical initiating agents have been extensively studied. These substances
either interact directly with the DNA or are enzymatically modified to
produce a metabolite that interacts with the DNA. The interaction is
often covalent, although noncovalent reactions are possible. The resultant
action of the initiating agent with DNA causes an irreversible change
similar to a mutation; however, an observable mutational event, that is,
a phenotype, is not an obligatory step in the initiation process.
Furthermore, initiation does not in itself cause cancer. Instead it programs
the cell so that subsequent reaction at a later time with a promoting
agent starts the formation of cancerous cells.
9.4.2 Promoting Agents
Unlike initiating agents, pr6moting agents do not interact directly
with DNA, but rather influence the expression of the genetic information
coded in DNA. Promoting agents include a variety of substances, such
as hormones, protein growth factors, drugs, and plant products. Asbestos,
cigarette smoke, alcohol, and phorbol esters are examples of promoting
agents. These substances influence genetic expression by binding to
receptors on cell surfaces or in the cytoplasm or nucleus. In contrast
to initiating agents, .the action of promoting agents is reversible. Some
promoting agents, (e.g., estrogen and prolactin) are very specific so that
they promote the formation of a Hunor only in their target tissues. Other
promoting agents do not act through a receptor mechanism, are
nonspecific, and can promote tumor formation in a variety of tissues
(e. g., iodoacetate).
Phorbol esters are promoters that interact with cellular receptors

and activate protein kinase C. Usually protein kinase C is activated
by Ca++ and diacylglycerol, both of which result from the hydrolysis
of phosphoinositides catalyzed by phospholipase C. Phospholipase C is
normally activated by several different growth factors. Thus phorbOl
esters bypass a tightly regulated step in the control of cell growth.
Since protein kinase C phosphorylates various proteins, it is not known
how this activity participates in establishing a cancerous line of cells.
Following promotion, cells go through a stage in carcinogenesis
called progression. During this stage, there is a karyotype change
from diploid to aneuploid that is associated with metastasis and
morphologic changes.
9.4.3 Oncogenes
Oncogenes are genes involved in the transformation of normal cells
to tumor cells. They do this by affecting cell growth and differentiation.
Oncogenes were discovered as part of the genomes of RNA tumor
viruses. Recall that RNA tumor viruses are propagated through DNA
intermediates that are synthesized in reactions mediated by reverse
transcriptase, a RNA-dependent DNA polymerase that is part of the
virion. Thus RNA tumor viruses are called retroviruses. Not all
retrovinses transform normal cells into tumor cells; for example, the
human immunodeficiency virus (HIV) causes acquired immuno-deficiency
syndrome (AIDS).
~~o~ ; / VI~e~ ~
Q.e / tumor' \
/ Q_~ /,""g~tu.....
~ \:J f19 ~
~~,-<eJ I
I
u~
Initiating
agent
Euploid cell population ~ Increasing aneuploidy •
Figure 9.S : Karyotypic changes during initiation. promotIon. and progression stages of
carcinogenesis.
The ROllS sarcoma virus (RSV) is a retrovirus that contains an

oncogene called v-src. Oncogenes that are part of the genomes of
retroviruses are abbreviated v-one whereas oncogenes of cellular genomes
are designated c-one. Cellular oncogenes are often called proto-oncogenes
to distinguish them from their viral counterparts and to emphasize that
the viral oncogenes originated at one time from cellular oncogenes.
Evidence for the first oncogene was found when certain mutants of
RSV failed to transform cells at high temperature but did transform
cells at low temperature. The mutant virus replicated well at both
temperatures. This means that the virus contained a tem-perature-sensitive
mutation in a gene that coded for a sarcoma-producing protein, tllat is,
for v-src.
Normal cells also were found to contain src-like genes when their
DNA was hybridized with labeled nucleic acid probes from the v-src
gene. As with other cellular genes, the c-onc genes were interrupted
with introns. v-One genes lack introns. Consequently, in the distant past
c-one genes must have been transferred to the retroviruses. If the
transfer had been from the virus to the cell, e-one genes probably would
not contain introns.
The retroviral one gene products have diverse functions, depending
on the particular oncogene. A few representative oncogenes and the
functions of their protein products. Notice that the oncogene proteins
are all involved in some aspect of cellular growth promotion. For
example, the tyrosine kinase activity of sre is a property shared by
several receptors for hormones (e. g., insulin) and for growth factors
(e.g., epidermal growth factor, or EGF). Some oncogenes code for
subunits of growth factors; an example is the v-sis protein, which is
homologous to a subunit of platelet-derived growth factor (PDGF).
The oncogene v-erbB, unlike e-erbB, codes for a shortened form
of the EGF receptor protein. Usually the binding of EGF is required
to turn on the tyrosine kinase activity of the EGF receptor protein,
the one coded by e-erbB. However, the tyrosine kinase activity of the
receptor derived from v-erbB is switched on permanently, even in the
absence of EGF. The uncontrolled grqwth Cllaracteristic of cancer cells
results.
Other oncogenes code for proteins that bind guanine nucleotides,
and others code for nuclear proteins. The guanine nucleotide-binding
proteins, the so-called G proteins, affect several key reactions. Some
G proteins are stimulatory, whereas others are inhibitory. For example,
they link hormone receptors to adenylate cyclase, they translocate
TABLE 9.4 : EXAMPLES OF PROMOTERS WITH THEIR
ASSOCIATED NEOPLASMS IN HUMANS.
Agent Neoplasm
Excessive dietary fat (calories) General increase in incidence of

cancer
Alcoholic beverages Oral, liver, and esophageal
cancer
Cigarette smoke Bronchogenic carcinoma, esoph-
ageal, and bladder cancer
Synthetic estrogens Liver adenomas
Asbestos Bronchogenic carcinoma and
mesothelioma
Not yet proved In humans
Saccharin Bladder
Tetradecanoylphorbol acetate Skin (a phorbol ester)
molecules in protein synthesis, and they regulate cell proliferation. The

func-tions of the nuclear oncogene proteins are not as well characterized,
but they also are probably involved in key reactions of gene expression
that regulate growth.
TABLE 9.5 : ONCOGENES OF RNA TUMOR VIRUSES.
Virus Oncogene Oncogene protein
product
Abelson murine leukemia virus abi Tyrosine kinase
Rous sarcoma virus src Tyrosine kinase
Avian erythroblastosis virus erbB Receptor for
growth factor;
tyrosine kinase
activity
Simian sarcoma virus sis Growth factor,
subunit of PDGF
Harvey murine sarcoma virus Ha-ras Guanine nucleotide-
binding protein
Avian myelocytomatosis virus myc Nuclear protein
A unifying theme of the role of oncogene function in cell transfo-

rmation is that the expression of the proto-oncogenes is normally tightly
regulated. Anything that disrupts the regulation of oncogene expression
could potentially cause cancer. For example, a chromosome break or
a mutation, in either somatic or germinal cells, at or near an oncogene
might lead to aberrant expression of the oncogene (see case 5). Similarly,
a tumor virus could introduce an unregulated oncogene into cells and
also cause cancer.
9.5 DNA SEQUENCE ANALYSIS
Some of the chemical reactions that cause mutations by modifying
purine and pyrimidine bases can be used in the laboratory for sequencing
DNA. One of the most commonly used methods for sequencing DNA
is the chemical technique developed by Maxam and Gilbert. Since
DNA molecules are extremely large, they are first broken into pieces
more easily sequenced. This is done with restriction endonucleases, and
the fragments are separated one from the other. A double-stranded
DNA fragment is then labeled at its 5' end using ATP (y_32P) and a
polynucleotide kinase derived from bacte-riophage T4-infected cells.
The DNA fragment is denatured to yield single-stranded pieces, and
these are separated from one another by electrophoresis on neutral gels
of polyacrylamide:
Large DNA ...=e, DoubIe-stranded (ds) DNA fragments; resolve on polyacrylamide gel
T4~
dsDNA fragment 7" _"" dsDNA _.
I \(32Pal5' ends)
ATP (y-32P) ADP
5' 3'
32p-------
dsDNA PoIy~ 11"\
(32P al5' ends) - . , p o -
+
3'
--------~5'32P
Resolved dsONA, labeled al 5' end
Each 5'-labeled single-stranded DNA is then sequenced. The method

is based on two principles:
(1) polyacrylamide gel electrophoresis in 7 mollL urea is capable
of separating oligo-deoxyribonucleotides that differ in size by just a
single base.
(2) DNA can be chemically reacted so that chain cleavages occur
at specific bases.
In a typical experiment a portion of the 5' -labeled single-stranded
DNA is reacted with dimethylsulfate under conditions where many but
not all of the purine bases react. This reagent alkylates the N7 position
of guanine and the N3 position of adenine. Depurination of methylated
guanine residues predominates when an aliquot is heated at neutral pH.
Methylated adenine bases are preferentially removed by treatment with
dilute acid. Both samples are then cleaved at the depurinated sites by
alkaline hydrolysis. One now has two samples: one that contains DNA
fragments all 3' -terminated at adenine residues and another consisting
of DNA fragments all terminated at guanine residues. In practice,
however, a few adenines are retOOved with the guanines. The deadenylation
reaction is more specific. Since the 32p label is at the 5' end of the
DNA and the depurination reactions are not allowed to go to completion,
the mixture of labeled oligonucleotides will range in size from the very
largest, cleaved at the 3' purine residue most distal to the label, to the
smallest, where the purine residue most proximal to the label has been
retOOved.
An analogous ~ries of reactions is used to produce depyrimidinated
DNA fragments. Hydrazine is used in these reactions, since both cytosine
and thymine react with hydrazine. The bases are cleaved to yield urea
and a pyrazole ring. The deoxyribose moiety is left as a hydrazone.
Piperidine, which reacts with the hydrazone, is used to cleave the
nucleotide chain. Cytosines react specifically with hydrazine in 5 mol!
L NaCI, but no specific reaction eXIsts for thymines. Consequently, one
aliquot yields labeled oligonu-cleotides 3'-terminated at cytosines,
whereas a second aliquot contains nucleotides cleaved in the absence of
NaCI at both cytosine and thymine residues.
Each of four aliquots terminated at A, G, C, and C + T are
denatured and loaded onto slabs of polyacrylamide gel. After
electrophoresis, the radioactively labeled nu-cleotides are visualized by
exposing the gel to an x-ray fIlm. The resultant autoradiogram shows
the position of each labeled oligonucleotide. The longer polynucleotides
are shown at the top of the diagram, and the shorter, faster moving
polynucleotides are at the bottom. Because the chemical reactions do
not go to completion, the autoradiogram displays a whole spectrum of
polynucleotides, some of which have been cleaved at only a single base
and others that have been cleaved at several. Each band differs from
the adjacent band by one nucleotide. Consequently, the DNA sequence,
5' to 3', can be read starting from the bottom. Figure 14.8 shows only
a portion of the gel.
Sequences of more than 350 bases can be obtained from some gels.
The sequence of the complementary DNA strand is often determined as
a check on accuracy of the first analysis. The primary structure of
thousands of deoxyribonucleotides can be deduced by the analysis of
overlapping DNA fragments generated by different restriction

endonu-cleases. This technology is useful in determining the proximity
of one gene to another and in studying mechanisms of gene expression.
9.6 RECOMBINANT DNA
TECHNOLOGY IN MEDICINE
Thirty years ago the thought of sequencing all the genes in the
-human genome seemed as impossible as a trip to the moon. Now it is
in the realm of possibility, and scientists are seriously talking about
sequencing the entire human genome, all 23 chromosomes, amounting
to 2,400 million base pairs (2.4 x 1()6 kb). Such plans are not based
on the physical chemical separation of thousands of genes from human
tissue, but rather on the isolation of genes or short pieces of DNA
from a relatively small number of cells. These pieces of DNA can be
cleanly separated from one another and then amplified to obtain
sufficient material by using the powerful biologic methods of
recombinant DNA tech-nology.
S'End 3' End
o
II 32
-o-p-o - - - - - - - - - - - - - - - - - - - - - O H
I Single-stranded polydeoxyribonucleotide
-0 IDim~thYI sulfate H,..yd_r_a_Zi_ne--,-_ _...,
rl Heat. pH 7 SM NaCI
Adenines Guanines Cytosines Cytoslnes and

modified modified modified thymines modified
Ad,,'"
1-Vo::""riO~ if G"",..
OH-
Chain cleaves Chain cleaves

Cytosine
~P"rid" --i 0'\,.,...,
~ -_.
Chain cleaves
f
Chain cleaves
thymine
at at al at
adenine gaps guanine gaps cytosine gaps cytosine and
thymidine
gaps
Figure 9.6 : Summary of reactions used to prepare oligonucleotides specifically cleaved

at a particular base.
9.6.1 Restriction Endonucleases
Restriction endonucleases are at the core of recombinar,.t DNA
technology. The specificity of some of these enzymes is described in
Chapter 13. Fortunately the _restriction endo-nucleases cleave DNA at
relatively few sites. The DNA fragments that result are usually long
enough to be useful but short enough to analyze and even sequence. The
mixture of DNA fragments following restriction nuclease digestion are
resolved by electrophoresis on gels of polyacrylamide or agarose. The
dye ethidium bromide is often used to detect the resolved fragments on
the gel. For further analysis, individual fragments can be cut out of the
gel and separated from the gel matrix and dye.
9.6.2 Restriction Maps
Now commercially available are many restriction enzymes, named
for the bacterial species from which they are isolated and possessing
a W\de range of specificity. Because the enzymes recognize such different
nucleotide sequences, they can be used individually and in combination
to develop "maps" of a particular DNA. For example, a 12 kb viral
DNA might be cut into two pieces of 2 kb and 10 kb by endonuclease
"A"; however, endonuclease "B" might cleave it into 5 kb and 7 kb
fragments. All these can be resolved from one another by gel
electrophoresis. Figure 14.9 illustrates these cleavages and shows that
there are two different arrangements for the cleavages catalyzed by
each nuclease. When endonuclease "A" and endonuclease "B" are
mixed together during the digestion and the products separated by
elec~ophoresis, the size of the fragments allows one to determine the
relative position of the cleavage sites produced by each of the restriction
enzymes. In this example the 2 kb fragment is assumed to be located
at the left end of thee original DNA molecule.
9.6.3 Cloning of Recombinant DNA
The most useful restriction enzymes cleave both strands of duplex
DNA, but the nucleotide breaks are not directly opposite one another;
the cleavages occur a few base pairs from a point of symmetry. These
staggered ends can base pair to hold the DNA together weakly, or they
can base pair with other DNA preparations that have been cut with the
same type of nuclease. In the laboratory the broken strands can be
covalently joined together by treating them with DNA ligase. In this
way foreign DNA can be linked to the DNA of plasmids or bacteriophages
(bacterial viruses). Which shows some of the genes and restriction sites
of the commonly used plasmid pBR322. ApR is a gene for ampicillin
resistance, Tc R is the gene for tetracycline resistance, and on denotes
the origin o'f replication sequences necessary for the plasmid to replicate
its DNA separately from the bacterial DNA.
The bacteriophages or plasmids that have foreign DNA built into
them in this manner are called vectors. Usually bacteriophages insert
DNA very efficiently into their host bacteria. A single bacteriophage

DNA can be replicated approximately lOO-fold or more per cell
depending on the type of phage and its host.
9.6.3.1 Plasmid vectors
Plasmids are small, circular DNAs that range in size from 2 to
approximately 100 kb. They also can be amplified to approximately 20
copies per cell. In addition, plasmids can be introduced into bacterial
cells, although less efficiently than phage vectors. Some plasmids can
be used to introduce recombinant DNA into yeast cells.
9.6.4 Clones
One usually tries to adjust experimental conditions so that only one
plasmid or phage is introduced into a single cell. When that cell
replicates on a plate of nutrient agar, it will produce millions of cells
until a colony becomes visible to the naked eye. If cells are sufficiently
diluted before they are spread onto the plate, each colony will consist
of a clone derived from a single ancestral cell. If each cell originally
contained only one copy of the phage or plasmid vector, each colony
will contain recombinant DNA that is homogeneous. That is, each
colony will contain cloned DNA.
12 kb DNA
Endonuclease 'A' Endonuclease 'B'
Ll 5kb 11 7kb R (b)
L ~l 10 kb R (aI
or
LI 7kb 11 Skb R (e)
LwC!J17 I R (a,b)
or •
Figure 9.8 : The construction of restriction maps. The relative position of cleavage
sites for two hypothetic restriction enzymes is illustrated for a 12 kb piece of DNA.
9.6.4.1 Clone selection using antibiotic resistance

Plasmids found in nature often carry genes that code for proteins
that make their hosts resistant to antibiotics. These genes are used to
good advantage to select bacterial colonies that contain recombinant
plasmids. For example, a restriction site may occur within a gene for
tetracyline resistance. A Sall site exists within the tetracycline resistance
gene of plasmid pBR322. When foreign DNA is inserted at this site,
it interrupts the gene for tetracycline resistance so that the cells are
now sensitive to tetracycline.
Tetracycline sensitivity can be detected by plating the vector-
containing cells onto agar containing ampicillin but not tetracycline. All
colonies on such plates will contain either the original plasmid or
plasmids containing recombinant DNA. Colonies are then replicate-
plated onto agar that contains tetracycline. Colonies that are sensitive
to this antibiotic can be determined from the differences in the pattern
of colonies appearing on the two plates. The antibiotic-sensitive colonies
can be picked off and used for further experiments. These colonies will
contain plasmids carrying foreign DNA .
......G-G-A-T-c-c..... .
......C-C-T-A-G-G..... .
BamH
endonuclease
......a G-A-T-c-c.... ~
......c-c-T-... -a a......
0NA1Igaae G-A-T-o-")_ .... (

G C-C-T-A-G
......O-G-A-T-C-C' ForaisJ'! DNA I

G-Q-A-T-C-C......
......C-C-T-A-G-G. I C-C-T-A-G-G._..
Figure 5.9 : The formation of recombinant DNA using the restriction endonuclease
BamHI.
9.6.4.2 Phage and cosmid vectors

Plasmids usually are able to carry much less foreign DNA than a
bacteriophage such as lambda. Thus plasmid vectors mainly are used to
obtain pieces of DNA (up to 3 kb in size) suitable for sequencing, to
subclone larger DNAs carried by phage vectors, or to clone single
expressible genes derived from complementary"DNAs (cDNAs). On the
other hand, vectors of bacteriophage lambda can accommodate up to 23
kb of foreign DNA. The amount of DNA that can be inserted depends
on the amount of nonessential lambda genes that can be replaced and
the total amount of DNA that can be packaged into a particle able to
be covered with phage coat proteins. Some eukaryotic genomic genes
have such long intervening sequences that their DNA may not fit into
one phage head. For cloning these large genes, cosmid vectors are often
used. Cosmids can accommodate very large pieces of genomic DNA.
They contain only a small part of the lambda DNA, the two cohesive
Eco RI
lSa11
DNA ligase 1 4
Sal I foreign DNA
"--.. One of many
Eco RI· different
pieces
+Original circular
pBR322 plasmid
+Circular fragments of
foreign DNA
Figure 12.10 : Some genes and restriction sites on the E. coli plasma pBR322. Eco RI,
Pst I, and Sal I are restriction endonucleases and the sites cleaved by these enzymes.
to be packaged into particles in vitro. As with bacterioph~ges, these
ends (cos gene) that allow any DNA particles are very efficient vep.icles
for inserting DNA into E. coli cells; however, unlike phages; they
cannot make infectious particles in vivo. Cosmids can carry up to 40
kb of recombinant DNA. They are engineered to cOIitain an origin of
replication, so the recombinant DNA can be amplified, and an antibiotic
resistance gene, so they can be easily selected. Once in the cell,
cosmids behave more as plasmids do.
cDNA is made in the laboratory from mRNAs using the enzyme
reverse transcriptase. Unlike eukaryotic genomic DNA, cDNA does
not contain intervening sequences. A complete cDNA may contain all
the information needed to synthesize a mRNA for a specific protein.
If the cDNA or its vector is modified so that the recombinant DNA
has all the sequences necessary for RNA and protein synthesis, one
can synthesize in bacterial cells the protein encoded in the recombinant
cDNA. Such a vector is called an expression vector. Expression vectors
can be used to obtain large quantities of a protein that might be very
difficult to isolate by conventional techniques. This is possible because
bacterial cells contain many copies of the recombinant gene and because
RNA and protein synthesis occurs very rapidly in bacteria.
9.6.5 Libraries of Genomic DNA
A recombinant gene library is made by digesting cDNA or genomic
DNA with one or more restriction enzymes, ligating the fragments to
a vector, and introducing the vector into appropriate bacterial or yeast
cells so that each cell will contain a single recombinant DNA. A
culture of millions of cells may contain fragments of all the cDNAs or
all the fragments of a genome. These libraries can be screened to
isolate a clone of interest by using a variety of techniques.
Genomic DNA is much more complex than cDNA. Since cDNAs
are synthesized in the laboratory from mRNAs using reverse transcriptase,
they are no more diverse than the number of mRNAs present at the
time of isolation. However, only about 2% of the mammalian genome
codes for the synthesis of proteins and their mRNAs. Thus genomic
DNA libraries are at least 50 times more diverse than cDNA libraries.
cDNA libraries are not easier to make, however, both because of the
inherent instability of mRNA compared to DNA and because reverse
transcriptase prematurely terminates when copying long mRNAs.
The otherwise impossible task of analyzing the 4 to 8 X 109 base
pairs of human genomic DNA can be simplified. Many of the 23 human
chromosomes can be physically separated from each other, their DNA
isolated, and genomic libraries made from them. Also, a technique
called pulsed-field electrophoresis allows the separation of DNAs as
large as 1()6 base pairs. This procedure uses short pulses of current at
alternating angles to the direction of movement of the DNA. The long
DNA molecules take longer to reorient in the agarose gel matrix than
the shorter molecules, and thus migrate more slowly.
9.6.6 Detection of Recombinant DNA
Recombinant DNA in bacterial colonies or in bacteriophage plaques
is most frequently detected using labeled hybridization probes. The
hybridization probes may be composed of RNA, DNA, or oligonucle-
otides that have been labeled. Often a DNA restriction fragment is
labeled in vitro using commercially available enzymes and radioisot-
opes. Smaller deoxyribonucleotides can be synthesized chemically using
automated machines called DNA synthesizers. Methods are available
for labeling either end of a nucleotide with 32p substrates. Heavily
labeled DNA probes can be made using a (l2P)-deoxyribo-nucleoside
triphosphates and the Klenow fragment of the E. coli DNA polymerase
I. Small, random oligonucleotides are added as primers to the denatured
DNA template so that large segments of cDNA are labeled with 32P,
not just the ends of the oligonucleotides.
A Petri dish containing bacterial colonies is blotted with nitrocellu-
lose paper. This transfers a large portion of each colony to the paper.
which is saturated with a solution that lyses (breaks open) the cells. The
DNA of the lysed colonies is denatured with alkali. The nitrocellulose
paper is neutralized, washed, and the paper either baked in an oven or
treated with ultraviolet light to immobilize the denatured DNA. The
DNA on the paper is hybridized with the labeled probe of interest, and
the excess label is washed off. The dried paper is exposed to photographic
film and the film developed. The exposed spots on the film can be
matched with the colonies on the master plate and colonies picked off
for further study.
A very similar protocol can be used to detect a particular DNA
or RNA that has been resolved by electrophoresis. E.M. Southern
developed a method for detecting individual DNAs that had been resolved
by gel electrophoresis. The gel was blotted to nitrocellulose paper and
the DNA on the paper hybridized with a labeled DNA probe. This
procedure of analyzing DNA resolved by electrophoresis is called
Southern blotting. Procedures wefe developed later for transferring by
blotting RNAs that had been resolved by electrophoresis. This procedure
was called Northern blotting. Western blotting is the analogous transfer
of electrophoretically rec;olved proteins to nitrocellu-Iose paper. In this

case the proteins are usually detected immunolo-gically.
9.6.6.1 Restriction fragment length polymorphism
The Southern blotting of genomic DNA is used to detect restriction
fragment length polymorphisms (RFLPs). Polymorphism is a genetic
term indicating variation of a gene product within a population. Such
variation may result in observable phenotypes; if serious, these are
called mutations. In humans polymorphisms usually are not associated
with an easily identifiable phenotype. Many polymorphisms occur in
clinically normal people as part of their natural diversity. It has been
estimated that a normal individual may be heterogeneous at 20% of his
or her genes. The resulting enzymes are different but normal. Some
loci, such as a-haptoglobin, are so diverse that no single allele can
serve as the "normal" standard. Usually a single gene product must be
represented in less than I % of the cases examined for it to be
considered polymorphic.
With the advent of electrophoresis, many polymorphisms were
detected by the abnormal migration of a protein or enzyme. Many
more polymorphisms now can be detected by the gel electrophoresis
of DNA restriction fragments followed by Southern blotting. These
DNA fragments show different mobilities on the gel, and thus they
differ in length. For this reason they are called length polymorphisms.
Length differences can result either from a mutation at an existing
restriction site so that it is no longer recognized by the enzyme or
from a mutation at another position to create a new restriction site.
In the former case the restriction fragment would be longer; in the
latter case it would be shorter.
RFLPs are often a reflection of individual genetic diversity and
are not related to a clinical phenotype, but occasionally they can be
diagnostic of an inherited disease. This technique is relatively new;
yet, it has been applied to the prenatal detection of sickle cell anemia,
thalassemia, phenylketonuria, a,-antitrypsin deficiency, Huntington's
chorea, Duchenne muscular dystrophy, hemophilia A and B, cystic
fibrosis, and several other. diseases.
9.6.7 Clinical Comment
9.6.7.1 Prenatal diagnosis of sickle ceU disease
Sickle cell disease is caused by a mutation that results in the
substitution of a valine residue for a glutamate residue in the sixth
position of the hemoglobin ~-chain. This results from the substitution of
a T for an A in the glutamate codon. When (1) DNA from a patient
with sickle cell disease is digested with the restriction enzyme MstII,
(2) the fragments separated by gel electrophoresis, (3) the gel blotted
under denaturing conditions to nitrocellulose paper, and (4) the resolved
DNA fragments hybridized with radioactive human globin DNA, one
finds a new 376-base pair fragment instead of the normal 175-base pair
DNA fragment. This indicates that a site usually recognized by the
enzyme MstII has been changed as a result of the mutation. MstII
requires the following sequence for its endonucleas~-;action:
.... C-C T-N-A-G-G ....
The letter N indicates that any nucleotide at that position satisfies
the specificity of MstII. The nonnaI sequence of the DNA of the 13-
chain of hemoglobin A near the site of the sickle cell mutation is:
.... C-C T-G-A-G-G ....
This sequence is cleaved by MstII; however, the mutation to sickle
cell anemia results in a change to the sequence:
.... C-C-T-G~T-G-G ....
This sequence cannot be cleaved by MstII.
Restriction analysis can be used to detect sickle cell disea!!e
prenatally, since the DNA of all cells, including amniotic cells, carries
the mutant DNA. It is much more difficult to obtain fetal blood for
the analysis of the mutant hemoglobin A l3-chain. Furthermore, fetal
blood is composed mo~tly of fetal hemoglobin, sinu-hemoglobin A is
madelaler in development.
9.6.8 Reverse Genetics
. Originally this term applied to the modification in vitro of a piece
of DNA of unknown function, with the subsequent identification of its
function by introducing it baCkmto cells. In human genetics the term
is now used to describe the determination of the cause of an inherited
disease by starting with the responsible gene and tracing it back to a
defective enzyme. In the past, human geneticists first recognized the
defective enzyme and then located the responsible gene.
Reverse genetics has been applied to diseases such as Duchenne
muscular dystrophy and cystic fibrosis, in which the responsible enzymes
are unknown and the disease. results from a significant deletion. By
combining RFLP analysis with cytogenetics, it has been possible to
increasingly narrow the location of the defective genes to small regions
on the affected chromosomes.
9.6.9 Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a technique for amplifying
small amounts of DNA when only small amounts of human tissue or

cells are available. One needs to know the sequence of the DNA in
question. Using the sequence, one chemically synthesizes two
oligodeoxyribonucleotides of approximately 20 to 25 residues that are
complementary to each of the two ends of the DNA of interest. These
are annealed (heated, then cooled slowly) to the ends and serve as
primers for the in vitro copying of each DNA strand using a heat-stable
DNA polymerase from Thermus aquaticus (Taq polymerase). The
chemically synthesized primers are in excess so that when the reaction
mixture is heated to 63 0 C, the DNA strands separate and reanneal
with more primer on cooling. This process of heating, cooling, and
synthesis can be repeated many times, and in the process the DNA
fragment of interest is greatly amplified. So much of a particular DNA
can be made that it can be detected by procedures that do not require
radioisotopes. The poly-merase chain reaction has been applied to the
detection of carriers of hemophilia A, to the prenatal diagnosis of sickle
cell disease, and to fetal sex determination, among others.
9.6.10 Cloning and Sequencing the Human Genome
Molecular biologists around the world have proposed the long-
term goal of sequencing every gene on every human chromosome.
Such a project will require international co-operation on a grand scale,
as well as the development of automated machines to do most of the
time-consuming work. At present such a project is theoretically possible,
but the necessary machines have not yet been built. The goal is a
worthy one because it will provide the framework to analyze every
human genetic disease. Literally thousands of DNA probes would be
available for screening patients, carriers, and others at potential risk,
'The sequences would also provide the information needed for genetic
therapy when such procedures become available.

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Microbiology And Biochemistry

Dr. Madan Lal Bagdi

Microbiology And Biochemistry

First Edition: 2009

All rights reserved no part ofthis work may be reproduced, stored

Published by D. P. Yadav for Manglam Publications, Delhi-110053,

The present title Microbiology and Biochemistry is an

3.5.1 Aureobasidium Spp ................................ 70

8.2 Biosynthesis of Lipids in Tissues .......................... 200

removed with a comer of the cover slip or an applicator stick. Mounts

Material from suspected

f'iiure 2.1 : Hooke's compound microscope: drawn by himself.

Figure 2.2 : Drawing of "white mould".

,. : Note, that, unlike Hooke, Leeuwenhoek made many of his

,:,'<:THE' ORIGIN OF LIFE" ", - ,",.' ( "

~2 ..4 ~ "QI~~A~ES~' OF. ;~S, ;;:jL,J.

t~·... t;'lh '_.j:; r.')j{l'.:'" ll1i

. . :JrlI innu . .~ni.~:}UIL;

Ir '}';I noLc. .1 ..-L .:d

-' ' qe~~:-v~)_a~ tl1~t. t~. ~~,;~; ffi~S1?-J3ttJ~J;r pe~~ ~'~i1i~ce.

, : IIqbe same "Y~y~, wille$ could be pn:;l!ervedbybeat~g irom

Geologic EVOLUlJONARY 'SUCCESSION Of liFE' IQRM$

(Duration of Eras "MOdem" M a n ' '

'~S-:AtI*" - ' - '500,000 -

Figure 3.1 : Alternaria alternata. Taken by phase-contrast microscopy after 2 weeks on

Figure 3.2 : Aureobasidium pullulans. Taken by phase-contrast microscopy after 2

Figure 3.3 : Cladosporium carrionii. Taken by phas~:-dlltJ:ast microscopy after 2 weeks

Figure 3.4 : Cladosporium bantianum. Taken by phase-contrast microscopy after 2

Figure 3.5 : Curvularia lunata. Taken by phase-contrast microscopy after 2 weeks on

narrow septa. cells and thickness of septa and outer

lageniform. Conidia one to several celled bearing annellations. E. jeanselmei has

with distinct scars, and sympodial in

Wangiella Conidiophores hyphalike, subhyaline to W dermatitidis is differentiated from E. 16, 19,24

Figure 3.6 : Drechslera spicifera. Taken by phase-contrast microscopy after 2 weeks

Figure 3.7 : Helm inthosporiurn solani. Taken by phase-contrast microscopy after 2

Figure 3.8 : Exophiala jeanselmei. Taken by phase-contrast microscopy after 2 weeks

Figure 3.9 : Exophiala moniliae. Taken by phase-contrast microscopy after ;2 weeks

Figure 3.10 : Exophiala spinifera. Taken by phase-contrast microscopy after 2 weeks

Figure 3.11 : Exophiala werneckli. Taken by phase-contrast microscopy after 2 weeks

Figure 3.12 : Fonsecaea IJedrosoi. Taken by phase-contrast microscopy after 2 weeks

Figure 3.13 : Fonsecaea compacta. Taken by phase-contrast microscopy after 2 weeks

Figure 3.14 : Phaeococcomyces exophialae. Taken by phasecontrast microscopy after

Figure 3.15 : Phialophora parasitIca. Taken by phase-contrast microscopy after 2 weeks

Figure 3.16 : PhIalophora repens. Taken by phase-contrast mIcroscopy after 2 weeks

Figure 3.17 : Phialophora richardsiae. Taken by phase-contrast Illicroscopy after 2

Figure 3.18 : Phialophora verrucosa. Taken by phase-contrast microscopy after 2 weeks

Figure 3.19 : Lecythophora mutabilis. Taken by phase-contrast microscopy after 2

Figure 3.20 : Lecytbophora mutabilis. Note the presence of chlamydoconidia. Taken

Figure 3.21': Lecythophora hoffmannll Taken by phase-contrast rrucroscopy after 2

Figure 3.22 ; RhinocIadiella aquaspersa. Taken by phase-contrast microscopy after 2

Figure 3.23 : Scedosporium apIOspermum. Taken by phase-contrast microscopy after

Figure 3.24 : Scytalidium Iignacola. Taken by phase-<:ontrast microscopy after 2 weeks

Figure. 3.25 : ScytahdlUm hyahnum. Taken by phase-contrast Il1Icroscopy after 2 weeks

Figure 3.26 : Sporothrix schenckii. Taken by phase-contrast microscopy after 2 weeks

Figure 3.27 : Wangiella dermatitidis. Taken by phase-contrast microscopy

Some bacteria measure as large as 80 p. in length; others as small

Figure 4.2 : Composite representation of a metachromatic granule.

Figure 5.1 : Virus infection: the two-fold path.

rabies, 100 x 200 n~

Virus symmetry The nucleocapsids of viruses are constructed

naked virus enveloped virus

Figure 5.7 : Demonstration of icosahedral symmetry.

phage top bacterial

~5~~- lawn of host cells