HW Case Study Bergeys Difftest

Your name/ section: Abigail Vogt, Ryan T/TH 12pm
1 Bacillus megaterium
2 Bacillus subtillis
3 Citrobacter freundii
4 Enterobacter aerogenes
5 Enterobacter cloacae
6 Escherichia coli
7 Serratia liquefaciens
8 Serratia marcescens
9 Staphylococcus epidermidis
10 Staphylococcus saprophyticus
UNKNOWN (Contaminate A)
UNKNOWN (Contaminate B)
6 Escherichia coli
UNKNOWN (Contaminate A)
UKNOWN (Conaminate B)
Cell morphology Cell arrangement
Gram
staining
Straight Rod
Straight Rod
Straight Rod
Straight Rod
Straight Rod
Straight Rod
Straight Rod
Straight Rod
Cocci
Cocci
Straight Rod
Straight Rod
Paired or chained with round or square ends

Single and Paired
Single and Paired
Single and Paired
Single and Paired
Single and Paired
Single and Paired
Single and Paired and irregular clusters
Single and Paired and irregular clusters
Single and Paired
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Negative
Positive
Endospore
(formation/
position)
Capsulte (production/type)
Catalase
test
Yes
Yes
Yes
No
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
No
Yes
Yes
Positive
Positive
N/A
N/A
N/A
N/A
N/A
N/A
Positive
Positive
N/A
Positive
Any other important characterisitcs 5) Burgey's Group 6)

or info.
6 Escherichia coli
18
18
5
5
5
5
5
5
17
17
7)
Cell wall structure
Requiring Requiring Colony morphology

catalase IMViC
on TSA
test?
tests?
Growth on MAC
Single Membrane
Single Membrane
Double Membrane
Double Membrane
Double Membrane
Double Membrane
Double Membrane
Double Membrane
Single Membrane
Single Membrane
Double Membrane
Single Membrane
Yes
Yes
No
No
No
No
No
No
Yes
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
0.5 - 2.5 diameter

0.5 - 2.5 diameter
1um diameter
0.3 - 1.5um diameter
1.1 - 1.5 by 2 - 6 um
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
1)
N/A
N/A
Negative
Negative
Negative
Positive
Varies
Varies
N/A
N/A
Negative
N/A
N/A
N/A
Positive
Varies
Varies
Positive
Positive
Positive
N/A
N/A
Positive
N/A
N/A
N/A
Negative
Positive
Positive
Negative
Negative
Negative
N/A
N/A
Negative
N/A
N/A
N/A
Positive
Positive
Positive
Positive
Negative
Negative
N/A
N/A
Negative
N/A
Phenylalalnine Deaminase - Negative

Phenylalalnine Deaminase - Positive
Ornithine Carboxylase Test- Negative
Lactose Fermentation - Positive
Lactose Fermentation - Positive
8)
9)
Lactose Fermentation - Negative

NO3 reduced to NO2- - Positive
NO3 reduced to NO2- - Negative
Mixed acid fermentation (VP) - negative
Mixed acid fermentation (VP) - negative
Growth on PEA
Oxygen requirment
Motility
Yes
Yes
No
No
No
No
No
No
Yes
Yes
No
Yes
Aerobic or facultative anaerobe

Aerobic or facultative anaerobe
Facultative anaerobe
Motile with peritrichous flagella

Motile with peritrichous flagella or nonmotile
Non-motile
Non-motile
Motile
2)
3)
4)
VP - Negative
VP - Positive
Lysyne Decarboxylase - Negative
Lysyne Decarboxylase - Positive
Lysyne Decarboxylase - Negative
Lysyne Decarboxylase - Positive
Arabinose Fermentation - Positive
Arabinose Fermentation - Negative
MR - Negative
MR - Negative
Arabinose Fermentation- Negative
Abigail Vogt, Ryan T TH 12:00
Purpose
Discription
Isolates and differentiate members of the a selective and differential medium containing
Enterobacteriaceae based on the ability to lactose, bile salts, neutral red, and crystal violet.
MacCon ferment lactose.
key agar
(MAC)
media
Isolates staphylococci and streptococci
Phenylet from specimens containing mixtures of
hyl bacterial flora
Alcohol
Agar
(PEA)
media
an undefined, selective medium that encourages

growth of Gram + organisms and inhibits growth of
most Gram - organisms. Digests of casein and
soybean meal provide nutrition while sodium
chloride provides a stable osmotic environment
suitable for the addition of sheep blood if desired.
Phenylethyl alcohol is the selective agent that
inhibits Gram - organisms.
SIM media is used to test for the possesion a media used for the determination of indole
of the enzyme tryptophanase in bacteria
production from tryptophan
Indol
Mixed
Acid
Ferminta
tion
Used to differentiate Enterobacteriaceae

and to distingiush them from othe gramnegative rods.
Included in base medium are peptone and the pH

indicator phenol red. Phenol red is yellow at a pH
lower than 6.8, pink to magenta above 7.4, and red
in between. During preperation the pH is adjusted
to 7.3 so it appears red and a Durham tube is added
to each tube to trap a portion of any gas produced.
Purpose
Discription
Used to distinguish between member of the Designed for organisms that are able to ferment
Enterobacteriaceae and differentiate them glucose, but quickly convert their acid priducts to
acetoin and 2,3-butanediol.
Butanedi from other Gram-negative rods
ol
Ferminta
tion
Citrate
Used to determine the ability of an

Designed to differentiate members of
organism to use citrate as its sole source of Enterobacteriaceae, all of which are faculative
carbon.
anaerobes. The Citric Acid
Used to differentiate organisms in the

family Enterobacteriaceae and to
distinguish them from other gram negative
Ornithin
rods
e
Decarbo
xylase
The media used contain peptone, glucose, the pH

indicator bromcresol purple and the conenzyme
pyridoxal phosphate, Bromcresol purple is purple at
6.8 and above, and yellow at 5.2 or below. After
inoculation fermenation is promoted by overlaying
mineral oil to seal the medium form external
oxygen.
Used to differentiate organisms in the

The media used contain peptone, glucose, the pH
family Enterobacteriaceae and to
indicator bromcresol purple and the conenzyme
distinguish
them
from
other
gram
negative
pyridoxal phosphate, Bromcresol purple is purple at
Lysine
Decarbo rods because the media use can contain any 6.8 and above, and yellow at 5.2 or below. After
inoculation fermenation is promoted by overlaying
xylase one of several amino acids.
mineral oil to seal the medium form external
oxygen.
Sucrose
Ferment
ation
Purpose
Discription
The purpose is to see if the microbe can

ferment the carbohydrate sucrose as a
carbon source.
An inoculum from a pure culture is transferred

aseptically to a sterile tube of phenol red sucrose
broth. The inoculated tube is incubated at 35-37 C
for 24 hours and the results are determined. A
positive test consists of a color change from red to
yellow, indicating a pH change to acidic.
Nutrient gelatin is a differential medium

Gelatina that tests the ability of an organism to
se
produce an exoenzyme, called gelatinase,
that hydrolyzes gelatin.
Gelatin is commonly known as a component of

gelled salads and some desserts, but it's actually a
protein derived from connective tissue. When
gelatin is at a temperature below 32C (or within a
few degrees thereof), it is a semisolid material. At
temperatures above 32C, it is a viscous liquid.

ferment the carbohydrate (sugar) arabinose
Arabinos as a carbon source.
e
Ferminta
tion
If arabinose is fermented to produce acid end

products, the pH of the medium will drop. A pH
indicator in the medium changes color to indicate
acid production. If arabinose is fermented to
produce acid end products, the pH of the medium
will drop. A pH indicator in the medium changes
color to indicate acid production.

ferment the carbohydrate xylose as a
Xylose carbon source.
Ferminta
tion
An inoculum from a pure culture is transferred

aseptically to a sterile tube of phenol red xylose
Purpose
Discription
This test determines whether the microbe An inoculum from a pure culture is transferred
reduces sulfur-containing compounds to aseptically to a sterile triple sugar iron agar (TSIA)
H2S sulfides during the process of metabolism. slant. The inoculated tube is incubated at 35-37 C
for 24 hours and the results are determined. Present
producti
in TSIA is an iron compound. The iron ions (Fe2+)
on
have a high affinity (strong attraction) for sulfide
ions. The result is that H2S combines with the iron
to make FeS, a black compound. In tubes of TSIA
containing
bacteria
hydrogen
sulfide, the
An
inoculum
from aproducing
pure culture
is transferred
agar turns black
from the
ferment the carbohydrate (sugar) mannitol aseptically
to a sterile
tubeFeS.
of phenol red mannitol
Mannitol as a carbon source.
Ferminta
tion
The purpose is to see if the microbe has
catalase, a protective enzyme capable of
destroying the dangerous chemical
Catalase hydrogen peroxide.
An inoculum from a pure culture is streaked on a

sterile plate of general purpose growth medium The
inoculated plate is incubated at 35-37 C for 24
hours. Growth from the plate is smeared on a
microscope slide and a drop of hydrogen peroxide
is placed on the smear. Copious bubbles liberated in
the hydrogen peroxide by the growth indicates
presence of catalase.
Differentiates bacteria based on their

The agar is stabbed through the oil to "plant" the
ability to oxidize or ferment certain sugars. bacteria in the butt of the tube. The inoculated tube
is incubated at 35-37 C for 24 hours. A positive
result for fermentation of glucose is indicated by the
Glucose
medium turning yellow.
Oxidatio
nFerment
ation
Purpose
Nitrate broth is used to determine the

ability of an organism to reduce nitrate
(NO3) to nitrite (NO2) using the enzyme
Nitrate nitrate reductase. It also tests the ability of
Reductio organisms to perform nitrification on
n
nitrate and nitrite to produce molecular
nitrogen.
Determine the ability of a microbe to
produce gelatinases.
Gelatin
Hydrolys
is
This test determines whether the bacterium

is either sensitive (susceptible) to
novobiocin or resistant to the drug.
Novobio Knowledge about novobiocinsusceptibility
cin is valuable in identification of Gram
Sensitivi positive cocci, some of which are
susceptible and others of which are
ty
resistant.
Purpose
Discription
If the organism has reduced nitrate to nitrite, the

nitrites in the medium will form nitrous acid. When
sulfanilic acid is added, it will react with the nitrous
acid to produce diazotized sulfanilic acid. This
reacts with the -naphthylamine to form a redcolored compound.
Gelatin is a protein derived from collagen.

Gelatinases comprise a family of extracellular
enzymes produced and secreted by some
microorganisms to hydrolyze gelatin. Therefore, the
cell can take up individual amino acids and use
them for metabolic purposes.
Novobiocin is an antibiotic interfering with the
unpackaging and repackaging of DNA during DNA
replication and the bacterial cell cycle. Different
types of bacteria have different degrees of
susceptibility to novobiocin. Susceptibility to
novobiocin is determined by placing a novobiocinimpregnated paper disk on a nutrient agar plate
seeded with the microbe under investigation. As the
microbe multiplies during incubation to produce a
lawn of confluent growth, cells are exposed to the
antibiotic diffusing into the agar from the paper
disk.
Discription
Used to distinguish between member of the Designed for organisms that are able to ferment
Enterobacteriaceae and differentiate them glucose, but quickly convert their acid priducts to
from other Gram-negative rods
acetoin and 2,3-butanediol.
Voges
Proskaue
r
Motility
SIM medium is used to observe growth

patterns from a stab line
D MORE TESTS AS NEEDED HERE
a semisolind fluid that is used to observe growth

patterns
Reading results
The positive result indicates:
Use of Media /reagent
Growth + = Gram - bacteria; colorless =

pH>6.8; red = pH<6.8; red = lactose is
fermented
Gram negative bacteria and

lactose fermentation occurs
To differentiate
Enterobacteriaceae based
on lactose fermentation
poor or no growth = probable Gram organism; good growth = probable

Staphylococcus, Streptococcus,
Enterococcus, or Lactococcus
Bacteria is Gram +
to isolate Gram +
bacteria from a mixed
culture
red in aclohol of kovacs' reagent = +;

reagent color is unchanged = -
Tryptophan is broken down into

indole and pyruvate
to determine if a bacteria
uses tryptophanase to
produce indole
The broth will change color from yellow to Fermentation has occurred as well Phenol Red Broth
magenta depending on pH. There may or as deanimation of peptome amino
may not be a bubble in the Durham tube if acids which produces ammonia.
fermentation has occurred.
Reading results
Broth may change color to red or copper

color
2, 3-butanediol fermentation has MR-VP broth, Methyl

occured. In other words, acetoin is Red Reagent, VP
oxidized to diacetyl which in turn Reagant A, VP Reagent
reacts with guanidine nuclie to
B
produce red color.
Media will change from green to blue.
Bacteria are using citrate as a

Simmons Citrate
nuntrient source using the enzyme Medium
citrate permease.
The medium will change to either yellow or Yellow indicates that fermentation Moller's Lysine
purple or they may be no change. Results has occurred however, no
Decarboxylase media.
are positive even if the medium turns
decarboxylation. A purple color
slightly purple.
means decarboxylation has
occurred and produces the
decarboxylase enzyme.
The medium will change to either yellow or Yellow indicates that fermentation Moller's Lysine
purple or they may be no change. Results has occurred however, no
Decarboxylase media.
are positive even if the medium turns
decarboxylation. A purple color
slightly purple.
means decarboxylation has
occurred and produces the
decarboxylase enzyme.
Reading results
The culture will have changed to yellow in The microbe uses sucrose as a
the presence of acids (indicating a positive carbon source.
test) or magenta or hot pink in the presence
of bases/alkali (indicating a negative test).
If an organism can break down gelatin, the The organism has the ability to
areas where the organism has grown will poroduce an exoenzyme,
remain liquid even if the gelatin is
geltinase, that hydrolyzed gelatin.
refrigerated. If gelatin solidifies it is a
negative test.
Phenol red sucrose broth.

The medium is a nutrient
broth to which 0.5-1.0%
sucrose is added. The pH
indicator phenol red is
red at neutral pH but
turns yellow at pH < 6.8.
It also changes to
magenta
or hot pink at
Nutrient gelatin.
pH >8.4.
A positive test consists of a color change The microbe can ferment the
Phenol Red Arabinose
from red to yellow, indicating a pH change carbohydrate (sugar) arabinose as Broth
to acidic.
a carbon source.
If xylose is fermented to produce acid end The microbe can ferment the
products, the pH of the medium will drop. carbohydrate xylose as a carbon
A pH indicator in the medium changes
source.
color to indicate acid production.
Phenol Red Xylose

Broth
Reading results
black in the medium = +; no black in the

medium = -
sulfur is reduced and H2S is

produced
SIM medium
If mannitol is fermented to produce acid

end products, the pH of the medium will
drop. A pH indicator in the medium
changes color to indicate acid production.
The microbe can ferment the

Phenol Red Mannitol
carbohydrate (sugar) mannitol as a Broth
carbon source.
Growth from an overnight culture of the

microbe is smeared on a microscope slide.
A drop of 3% hydrogen peroxide is added.
If copious bubbles are observed, the
microbe is positive for catalase.
The microbe has catalase, a

General purpose
protective enzyme capable of
medium, reagant:
destroying the dangerous chemical hydrogen peroxide
hydrogen peroxide.
OF glucose medium with oil overlay is

The bactium can use glucose in Oxidation- Fermentation
used. This consists of a nutrient medium to both oxidation and fermentation. glucose medium with oil
which 1% glucose has been added. Mineral
overlay is used.
oil lighter in density that the medium is
added to provide an airtight barrier. The pH
indicator is brom thymol blue, which is
green at neutral pH and turns yellow at pH
<6.0. It gets its name because it turns blue
at pH >7.6.
Reading results
If the medium turns red after the addition of The organism has the ability to
the nitrate reagents, it is considered a
reduce nitrate to nitrite using the
positive result for nitrate reduction.
enzyme nitrate reductase.
Nitrate broth, Reagants:

sulfanilic acid and naphthylamine
There will be gelatin either present or non- Gelatinase is present.

present in the medium. Gelatin production
is a postive result.
Nutrient Gelatin tubes.
If the bacteria are susceptible tonovobiocin, The microbe is suseptable to the

there will be a visible zone of inhibition
antiobiotic.
forming around the disk, representing an
area where the antibiotic concentration has
prevented bacterial growth. Should the
microbe be resistant, the lawn of cells will
form visible growth up to the margin of the
disk.
The medium used for

growing the bacterial
lawn is typically either
blood agar or a nutrient
rich, general-purpose
medium like nutrient
agar.
Reading results
Broth may change color to red or copper

color
2, 3-butanediol fermentation has MR-VP broth, VP

occured. In other words, acetoin is Reagant A, VP Reagent
oxidized to diacetyl which in turn B
reacts with guanidine nuclie to
produce red color.
growth radiating from the stab mark =

motile; no radiating growth = mon motile
motile bacteria
to determine the motility

of a bacterial sample
Biochemical pathways (How

media/reagents and bacterial
products/enzyme works?)
Bile salts and CV+ inhibit Gram +

bacteria growth while the medium
contains lactose allowing fermentation to
change the pH.
PEA inhibits Gram - organisms by

breaking down their membreane
permeability barrier, allowing influx of
substances ordinarily blocked and
leakage of large amounts of cellular
potassium. This ultimately disrupts or
halts DNA synthesis.
tryptophanase uses tryptophan from the
medium and hydrolizes it into ammonia,
pyruvate, and indole
Deanimation of peptone amino acids

produces ammonia, which raises pH and
turns the broth pink. Gas production will
occur if the broth fermentates.

VP reagants oxidizes the acetoin to

diacetyl, which in turn reacts with
guanidine nuclei from peptone to
produce a red color.
Ammonium dihydrogen phosphate

provides the sole nitrogen source in the
medium. Un the process of pulling
nitrogen from the ammonium salt, citrate
positive bacteria produce ammonia and
ammonium hydroxide, both of which
alkalinize the medium and turn it blue.
Decarboxylation of the amino acid
results in accumulation of alkaline end
products that turn the medium purple.
Glucose fermentation in the anaerobic
medium initially turns yellow due to the
accumulation of acid end products.
Decarboxylation of the amino acid

results in accumulation of alkaline end
products that turn the medium purple.
Glucose fermentation in the anaerobic
medium initially turns yellow due to the
accumulation of acid end products.
Deanimation of peptone amino acids

produces ammonia, which raises pH and
turns the broth pink. Gas production will
occur if the broth fermentates.
Gelatinase allows the organisms that

produce it to break down gelatin into
smaller polypeptides, peptides, and
amino acids that can cross the cell
membrane and be utilized by the
organism.
The medium is a nutrient broth to which

0.5-1.0% arabinose is added. The pH
indicator phenol red is red at neutral pH
but turns yellow at pH <6.8 It also
changes to magenta or hot pink at pH
>8.4.
The medium is a nutrient broth to which 0.51.0% xylose is added. The pH indicator phenol
red is red at neutral pH but turns yellow at pH
<6.8. It also changes to magenta or hot pink at
pH >8.4.

If an organism can reduce sulfur to

hydrogen sulfide, the hydrogen sulfide
will combine with the iron to form ferric
sulfide, which is a black precipitate. If
there is any blackening of the medium, it
indicates the reduction of sulfur and is a
positive result.
The medium is a nutrient broth to which
0.5-1.0% arabinose is added. The pH
indicator phenol red is red at neutral pH
but turns yellow at pH <6.8 It also
changes to magenta or hot pink at pH
>8.4.
Catalase mediates the breakdown of
hydrogen peroxide H2O2 into oxygen
and water. The function of this enzyme is
to detoxify hydrogenperoxide (H2O2),
which is formed from the superoxide
radical by superoxide dismutase.
OF medium includes a high peptone to

sugar ratio to reduce the possibilty that
alkaline products from peptone
utilization will nutralize weak acids
produced by oxidation of the
carbohydrate. In this medium, oxidative
organisms oxidize the carbohydrate to
CO2, H2O and energy. Fermentative
organisms convert the carbohydrate to
pyruvate, but because oxygen is not
available, the pyruvate and other organic
intermediates are reduced to organic
Biochemical
acids, gas, or pathways
alcohol. (How
When sulfanilic acid is added, it will

react with the nitrous acid to produce
diazotized sulfanilic acid. This reacts
with the -naphthylamine to form a redcolored compound. Therefore, if the
medium turns red after the addition of
the nitrate reagents, it is considered a
positive result for nitrate reduction.
When a tube is stabbed with gelatinase
positve organsims, secreted gelatinase
will liquify into the medium. Gelatin
hydolysis occurs by a family of enzymes.
It takes water and gelatin and turns it
into gelatinase which makes polypetides.
Then those polypetides and water are
also made in gelatinase and creates
amino
acids.
Coagulase
negative staphylococci can
either be resistant or susceptible to
novobiocin. The one most commonly
isolated novobiocin resistant coagulase
negative staphylococcus from urine is
Staphylococcus saprophyticus. So, when
coagulase negative staphylococcus is
isolated from urine of a woman of this
age novobiocin susceptibility test can be
performed for presumptive identification
of Staphylococcus saprophyticus.
VP reagants oxidizes the acetoin to

diacetyl, which in turn reacts with
guanidine nuclei from peptone to
produce a red color.
the medium is semisolid and therefore

growth can spread through the medium
if it is motile.
Names (in your group) :

Given Mixed culture number:
if
continued,
from what
number?
step Test
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Why
Prediction (if +/- result, what is the

next step?)
Media/reagent to request to TA

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Your name/ section: Abigail Vogt, Ryan T/TH 12pm

Cell morphology Cell arrangement

Paired or chained with round or square ends

Any other important characterisitcs 5) Burgey's Group 6)

Cell wall structure

Requiring Requiring Colony morphology

0.5 - 2.5 diameter

Phenylalalnine Deaminase - Negative

Lactose Fermentation - Negative

Aerobic or facultative anaerobe

Motile with peritrichous flagella

Arabinose Fermentation- Negative

Abigail Vogt, Ryan T TH 12:00

an undefined, selective medium that encourages

Used to differentiate Enterobacteriaceae

Included in base medium are peptone and the pH

Used to determine the ability of an

Used to differentiate organisms in the

The media used contain peptone, glucose, the pH

Used to differentiate organisms in the

The purpose is to see if the microbe can

An inoculum from a pure culture is transferred

Nutrient gelatin is a differential medium

Gelatin is commonly known as a component of

The purpose is to see if the microbe can

If arabinose is fermented to produce acid end

The purpose is to see if the microbe can

An inoculum from a pure culture is transferred

An inoculum from a pure culture is streaked on a

Differentiates bacteria based on their

Nitrate broth is used to determine the

This test determines whether the bacterium

If the organism has reduced nitrate to nitrite, the

Gelatin is a protein derived from collagen.

SIM medium is used to observe growth

D MORE TESTS AS NEEDED HERE

a semisolind fluid that is used to observe growth

The positive result indicates:

Use of Media /reagent

Growth + = Gram - bacteria; colorless =

Gram negative bacteria and

poor or no growth = probable Gram organism; good growth = probable

red in aclohol of kovacs' reagent = +;

Tryptophan is broken down into

The positive result indicates:

Use of Media /reagent

Broth may change color to red or copper

2, 3-butanediol fermentation has MR-VP broth, Methyl

Media will change from green to blue.

Bacteria are using citrate as a

The positive result indicates:

Use of Media /reagent

Phenol red sucrose broth.

Phenol Red Xylose

The positive result indicates:

Use of Media /reagent

black in the medium = +; no black in the

sulfur is reduced and H2S is

If mannitol is fermented to produce acid

The microbe can ferment the

Growth from an overnight culture of the

The microbe has catalase, a

OF glucose medium with oil overlay is