In the name of God
Yasuj University of Medical Sciences
Department of Microbiology
By: Dr. S. S. Khoramrooz
Department of Microbiology, Faculty of Medicine,
Yasuj University of Medical Sciences, Yasuj, Iran
Intended Use

Bile Esculin Agar is used to differentiate enterococci and the Streptococcus
bovis group from other streptococci.
Principles of the Procedure

Enterococci and certain streptococci hydrolyze the glycoside esculin to
esculetin and dextrose.

Esculetin reacts with an iron salt to form a dark brown or black
complex.

Ferric citrate is incorporated into the medium as an indicator of esculin
hydrolysis and resulting esculetin formation.

Oxgall is used to inhibit gram-positive bacteria other than enterococci.
Dr. S. S. Khoramrooz
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Expected results
 Any
blackening of the plated medium indicates a
positive result; if no blackening occurs, the test is
negative.
 For
slants, if more than half of the slant is
blackened within 24-48 hours, the test is positive;
if less than half is blackened or no blackening
occurs within 24-48 hours, the test is negative.
Dr. S. S. Khoramrooz
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 Intended
Use
 DNase Test Agar, DNase Test Agar with Methyl
Green and DNase Test Agar with Toluidine Blue
are differential media used for the detection of
deoxyribonuclease activity to aid in the
identification of bacteria isolated from clinical
specimens.
Dr. S. S. Khoramrooz
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Summary and Explanation

The DNase test is used to detect the degradation of
deoxyribonucleic acid (DNA).

The test is useful for differentiating Serratia from
Enterobacter, Staphylococcus aureus from coagulase-negative
staphylococci, and Moraxella catarrhalis from Neisseria
species.

DNase Test Agar with Toluidine Blue contains a metachromatic
dye to eliminate the necessity of reagent addition to the agar
following incubation.

Toluidine blue may be toxic to some gram-positive cocci and,
therefore, should be used primarily with Enterobacteriaceae.
Dr. S. S. Khoramrooz
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 Principles
 DNA is
of the Procedure
the substrate for DNase activity.
 DNase
is an extracellular enzyme that breaks the
DNA down into subunits composed of
nucleotides.
 The
depolymerization of the DNA may be detected
by flooding the surface of the medium with 1 N
HCl and observing for clear zones in the medium
surrounding growth.
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 In
the absence of DNase activity, the reagent reacts
with the intact nucleic acid, resulting in the
formation of a cloudy precipitate.
 The
HCl reagent is not needed to detect DNase
activity on DNase Agar with Methyl Green.
 Methyl
green forms a complex with intact
(polymerized) DNA to form the green color of the
medium.
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 DNase
activity depolymerizes the DNA, breaking
down the methyl green-DNA complex, which
results in the formation of colorless zones around
colonies of the test organism.
A
negative test is indicated by the absence of a
colorless zone around the colonies.
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 For
detection of deoxyribonuclease activity in
microorganisms including staphylococci
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 The
HCl reagent is not needed to detect DNase
activity on DNase Agar with Toluidine Blue.
 Toluidine
blue forms a complex with intact
(polymerized) DNA.
 In
the intact DNA complex, the toluidine blue has
the normal blue color.
 DNase
activity depolymerizes the DNA, breaking
down the dye-DNA complex.
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 In
the presence of nucleotides produced from the
DNase depolymerization, the dye takes on its
metachromatic color, forming pink to red zones
around bacterial growth.
 A negative
test is indicated when the medium
remains blue.
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Procedure
 Inoculate by making a single streak line using
inoculum from an agar slant or plate.
 One
plate may be inoculated with up to eight
isolates by spot inoculation (1/8 to 1/4 inch) or
streak inoculation (a single 1- to 2-inch line).
 Incubate
at 35 ± 2°C for 24-48 hours.
Dr. S. S. Khoramrooz
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 Plates
should be incubated in an inverted position.
Incubate tubes with loosened caps.
 Following
incubation, flood DNase Test Agar
plates with 1N HCl reagent and observe for
reaction.
 Reagent
addition is not required with DNase Test
Agar with Methyl Green or with DNase Test Agar
with Toluidine Blue.
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
Expected Results

A clear area surrounding growth (band/spot inocula) on DNase Test Agar
after the addition of 1N HCl indicates a positive reaction, DNase
activity.

A negative reaction is indicated by no clearing and a cloudy precipitate
around colonies and throughout medium due to precipitated salts in the
medium.

A positive reaction on DNase Test Agar with Methyl Green is a distinct
clear zone surrounding growth in an otherwise green-colored medium.

The color of the medium remains unchanged if the test is negative.

On DNase Test Agar with Toluidine Blue, DNase activity is indicated by
pink to red zones surrounding growth.

The color of the medium remains unchanged if the test is negative.
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Intended Use
 Mannitol Salt Agar is used for the selective
isolation and enumeration of staphylococci from
clinical and nonclinical materials.
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Principles of the Procedure
 The
7.5% concentration of sodium chloride results
in the partial or complete inhibition of bacterial
organisms other than staphylococci.
 Mannitol
fermentation, as indicated by a change in
the phenol red indicator, aids in the differentiation
of staphylococcal species.
 Agar
is a solidifying agent.
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Procedure
plates at 35 ― 2•
‹C in an aerobic
atmosphere for 24-48 hours, or as instructed in the
standard reference.
 Incubate
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Expected Results

Coagulase-positive staphylococci produce growth of
yellow colonies with yellow zones.

Coagulase negative staphylococci produce small red
colonies with no color change to the medium.

Micrococcus produce large, white to orange colonies, with
no color change to the medium.

Most other bacteria will be inhibited.
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 Intended
Use
 Bismuth Sulfite Agar is a highly selective medium
used for isolating Salmonella spp., particularly
Salmonella Typhi, from food and clinical
specimens.
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Principles of the Procedure

Dextrose is an energy source.

Bismuth sulfite indicator and brilliant green are
complementary in inhibiting gram-positive bacteria
and members of the coliform group, while allowing
Salmonella to grow luxuriantly.

Ferrous sulfate is included for detection of H2S
production.

When H2S is present, the iron in the formula is
precipitated, giving positive cultures the characteristic
brown to black color with metallic sheen.
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 For
isolation of Salmonella spp. from clinical
specimens, inoculate fecal specimens and rectal
swabs onto a small area of one quadrant of the
Bismuth Sulfite Agar plate and streak for isolation.
 This
will permit the development of discrete
colonies.
 Incubate
plates at 35°C.
 Examine at 24 hours and again at 48 hours for
colonies resembling Salmonella spp.
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
Expected results

The typical discrete S. Typhi surface colony is black
and surrounded by a black or brownish-black zone
which may be several times the size of the colony.

By reflected light, preferably daylight, this zone
exhibits a distinctly characteristic metallic sheen.

Plates heavily seeded with S. Typhi may not show
this reaction except near the margin of the mass
inoculation.
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 In
these heavy growth areas, this organism
frequently appears as small light green colonies.
 This
fact emphasizes the importance of inoculating
plates so that some areas are sparsely populated
with discrete S. Typhi colonies.
 Other
strains of Salmonella produce black to
green colonies with little or no darkening of the
surrounding medium.
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 Heat
with frequent agitation and boil for 1 minute
to completely dissolve the powder.
DO
 Use
NOT AUTOCLAVE.
the medium the same day it is prepared.
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Intended Use
 Brilliant Green Agar is a highly selective medium for
the isolation of Salmonella other than S. Typhi from
feces and other materials.
Principles of the Procedure
 Brilliant green dye inhibits gram-positive bacteria and a
majority of gram-negative bacilli.

Phenol red serves as a pH indicator and yields a yellow
color as a result of acid production in the fermentation of
the lactose and/or sucrose in the medium.
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Procedure

A less selective medium and a nonselective medium
should also be streaked to increase the chance of
recovery when the population of gram-negative
organisms is low and to provide an indication of other
organisms present in the specimen.

Incubate plates, protected from light, at 35 ± 2°C for 1824 hours.

If negative after 24 hours, reincubate an additional 24
hours.
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Salmonella on BPLS Agar. The colonies
are red because the bacterium does not
ferment lactose or sucrose.
Dr. S. S. Khoramrooz
Escherichia coli on BPLS Agar.
The colonies are yellow due to the
low pH which is caused by the
production of acid during
fermentation of lactose and/or
sucrose.
39
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Intended Use
 Decarboxylase media are used in the biochemical
differentiation of gram-negative enteric bacilli based on the
production:



Arginine dihydrolase
Lysine decarboxylase
Ornithine decarboxylase

Decarboxylase Medium Base, with added arginine, lysine or
ornithine is used for the same purpose.

Lysine Decarboxylase Broth is used for differentiating
microorganisms based on lysine decarboxylation.
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Summary and Explanation

Moeller introduced the decarboxylase media for detecting
the production of lysine and ornithine decarboxylase and
arginine dihydrolase.

These media are a useful adjunct to other biochemical
tests for the speciation and identification of the
Enterobacteriaceae and other gram-negative bacilli.

The production of OD is particularly useful for
differentiating Klebsiella and Enterobacter species.

Klebsiella species are non-motile and, except for K.
ornithinolytica, do not produce ornithine decarboxylase,
while most Enterobacter species are motile and, except for
E. agglomerans, usually produce this enzyme.
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Principles of the Procedure

Pyridoxal is an enzyme co-factor for the amino acid
decarboxylase.

Dextrose is a fermentable carbohydrate.

Bromcresol purple and cresol red are pH indicators.

The amino acids lysine, ornithine or arginine are added
to the basal medium at a concentration of 10.0 g/L to
detect the production of the enzyme specific for these
substrates.
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 When
the medium is inoculated with a bacterium
that is able to ferment dextrose, acids are produced
that lower the pH of the medium and change the
color of the indicator from purple to yellow.
 The
acidic condition also stimulates decarboxylase
activity.
 If
the organism produces the appropriate enzyme,
the amino acid in the medium is degraded, yielding
a corresponding amine.
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 Decarboxylation
 while
of lysine yields cadaverine.
decarboxylation of ornithine yields putrescine.
 Arginine
is first hydrolyzed to form ornithine, which
is then decarboxylated to form putrescine.
 The
production of these amines elevates the pH of
the medium, changing the color of the indicator from
yellow to purple or violet.
 If
the organism does not produce the appropriate
enzyme, the medium remains acidic (yellow).
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
Each isolate to be tested must also be inoculated into
a tube of the basal medium that does not contain the
amino acid.

If this tube becomes alkaline, the test is invalid.

To obtain the appropriate reactions, the inoculated
tubes must be protected from air with a layer of sterile
mineral oil.

Exposure to air may cause alkalinization at the surface
of the medium, which could cause a decarboxylasenegative organism to appear positive.
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
Expected Results

Compare the color of tubes of media containing the specific amino
acids with the color of control tubes of basal media (without
amino acid) that have been inoculated with the same isolate.

If inoculated control tubes show an alkaline reaction, the test is
invalid; i.e.,

Improperly performed or the test organisms

Degrade the peptone sufficiently to produce an alkaline reaction in the
absence of a specific amino acid.

The medium becomes purple to violet if the reaction is positive
(alkaline).

A yellow color indicates a negative test; i.e., the organism does
not produce the appropriate enzyme.
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Intended Use

Eosin Methylene Blue Agar, Levine is a slightly selective
and differential plating medium for the isolation of gramnegative enteric bacteria.

Principles of the Procedure

The eosin Y and methylene blue dyes in Levine EMB
Agar render the medium slightly selective in that they
inhibit gram- positive bacteria to a limited degree.
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 These
dyes also play a role in differentiating
between lactose fermenters and lactose
nonfermenters due to the presence or absence of
dye uptake in the bacterial colonies.
 Coliforms,
as lactose-fermenting organisms, are
visualized as blue-black colonies, whereas colonies
of Salmonella and Shigella, as lactose
nonfermenters, appear colorless, transparent or
amber.
 Some
gram-positive bacteria, such as fecal
streptococci, staphylococci and yeasts, will grow
on this medium and usually form pinpoint
colonies.
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 Expected
Results
 Typical colonial morphology on Eosin Methylene
Blue Agar, Levine is as follows:
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 Intended
Use
 Hektoen
Enteric (HE) Agar is a moderately selective
medium used in qualitative procedures for the
isolation and cultivation of gram-negative enteric
microorganisms, especially Shigella, from a variety
of clinical and nonclinical specimens.
Dr. S. S. Khoramrooz
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Principles of the Procedure
 The
selective nature of Hektoen Enteric Agar is
due to the incorporation of bile salts in the
formulation.
 These
substances inhibit gram-positive organisms
but also can be toxic for some gram-negative
strains.
Dr. S. S. Khoramrooz
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
This medium contains three carbohydrates, lactose, sucrose
(saccharose) and salicin, for optimal differentiation of
enteric pathogens

The lactose concentration is higher than in many other
media used for enterics in order to aid in the visualization
of enteric pathogens and minimize the problem of delayed
lactose fermentation.

Ferric ammonium citrate and sodium thiosulfate in the
medium enable the detection of hydrogen sulfide
production.

The indicator system, consisting of acid fuchsin and
bromthymol blue, has a lower toxicity than that of many
other enteric media, resulting in improved recovery of
enteric pathogens.
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Procedure
 A nonselective medium should also be streaked to
increase the chance of recovery when the
population of gram-negative organisms is low and
to provide an indication of other organisms present
in the specimen.
 Incubate plates, protected from light, at 35 ± 2°C
for 18-24 hours.
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DO NOT AUTOCLAVE.
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Salmonella mixed with normal fecal flora.
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Intended Use
 CLED Agar is used for the isolation, enumeration
and presumptive identification of microorganisms
from urine.
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Principles of the Procedure
 Lactose
is included to provide an energy source for
organisms capable of utilizing it by a fermentative
mechanism.
cystine permits the growth of “dwarf colony”
coliforms.
 The
 Bromthymol
blue is used as a pH indicator to
differentiate lactose fermenters from lactose
nonfermenters.
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 Organisms
that ferment lactose will lower the pH
and change the color of the medium from green to
yellow.
 Electrolyte
sources are reduced in order to restrict
the swarming of Proteus species.
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 Bacteriuria
is determined by inoculating the surface
of an agar medium using 0.1 mL of a 102 dilution of
the urine sample or using a calibrated loop (0.001
mL) of the undiluted sample.
 Current
guidelines are that for a single isolate a
density of >105 CFU/mL indicates infection, <104
CFU/mL indicates urethral or vaginal contamination,
and between 104 and 105 CFU/mL needs to be
evaluated based on clinical information.
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Procedure
 Inoculate the medium as soon as possible after the
specimen is received in the laboratory.
 It
is recommended that quantitative methods be used
for culturing urine specimens.
 Incubate
at 35 ― 2•
‹C for 24-48 hours.
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Expected Results
 Count the number of colonies on the plate or dipstick.

Multiply by an appropriate number to convert the count
to CFU per mL of sample.

Contaminant bacteria usually appear in low numbers
which vary in colonial morphology.

Urinary pathogens will usually yield high counts having
uniform colonial morphology and should be subcultured
directly to routine media for identification and
susceptibility testing.
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 Typical
colonial morphology on CLED Agar is as
follows:
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E. coli on CLED
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Intended Use
 Lauryl Tryptose Broth and Lauryl Sulfate Broth,
which are also known as Lauryl Sulfate Tryptose
(LST) Broth, are used for the detection of coliform
organisms in materials of sanitary importance.
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 Lactose
provides a source of fermentable
carbohydrate for coliform organisms.
 The
fermentation of lactose with gas formation is a
presumptive test for coliforms.
 Sodium
lauryl sulfate inhibits organisms other than
coliforms.
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 This
medium is used for the detection of
coliforms in foods and dairy products.

It is now the medium of choice for use in the
presumptive phase of the Standard Total Coliform
Multiple-Tube (MPN) Test for the microbiological
examination of water.
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Expected Results
 After incubation of the tubes with loosened caps at 35 ±
0.5°C for 24 hours, examine for turbidity and for gas
production in the Durham fermentation tubes.

If no gas has formed and been trapped in the inverted tube,
reincubate and reexamine after 48 hours.

Turbidity of the medium accompanied by formation of gas
in any amount in the Durham tubes within 48 hours is a
positive presumptive test for the presence of coliforms in
the sample.

The result should be confirmed by additional standard
testing.
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Intended Use
 Lysine Iron Agar is used for the differentiation of enteric
organisms based on their ability to decarboxylate or
deaminate lysine and to form hydrogen sulfide.
Principles of the Procedure
 Dextrose serves as a source of fermentable carbohydrate.

The pH indicator, bromcresol purple, is changed to a
yellow color at or below pH 5.2 and is purple at or above
pH 6.8.
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 Ferric
ammonium citrate and sodium thiosulfate
are indicators of hydrogen sulfide formation.
 Lysine
is the substrate for use in detecting the
enzymes, lysine decarboxylase and lysine
deaminase.
 Cultures
of enteric bacilli that produce hydrogen
sulfide cause blackening of the medium due to
the production of ferrous sulfides.
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 Those
that produce lysine decarboxylase produce
an alkaline reaction (purple color) or neutral
reaction in the butt of the medium.
 Organisms
that deaminate the lysine cause the
development of a red slant over an acid butt.
 Gas
may be formed but its formation is often
irregular or suppressed.
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Procedure
 Using an inoculating needle, stab the butt twice
then streak the slant with growth from a pure
culture.
 Incubate
tubes with loosened caps for 18-48 hours
at 35 ± 2°C in an aerobic atmosphere.
 Triple
Sugar Iron Agar slants should be inoculated
in parallel unless results from this medium have
already been obtained to distinguish coliforms
from Shigella, for example.
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Expected Results
 Lysine
decarboxylation is detected in the butt by
an alkaline (purple) reaction.
 Lysine
deamination is detected by a red slant.
 Hydrogen
sulfide production is detected by the
formation of a black precipitate.
 A negative
reaction (purple slant and yellow
butt) indicates fermentation of dextrose only.
Dr. S. S. Khoramrooz
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 Hydrogen
sulfide may not be detected in this
medium by organisms that are negative for lysine
decarboxylase activity since acid production in
the butt may suppress its formation.
 For
this reason H2S-producing Proteus species do
not blacken this medium
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Intended Use
 MR-VP Medium and MR-VP Broth (Methyl RedVoges Proskauer Medium/Broth, also known as
Buffered Peptone- Glucose Broth) are used for the
differentiation of bacteria by means of the methyl
red and Voges-Proskauer reactions.
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Principles of the Procedure

Methyl red-positive organisms produce high levels of acid
during fermentation of dextrose, overcome the phosphate buffer
system and produce a red color upon the addition of the methyl
red pH indicator.

In the Voges-Proskauer test, the red color produced by the
addition of potassium hydroxide to cultures of certain microbial
species is due to the ability of the organisms to produce a
neutral end product, acetoin (acetylmethylcarbinol), from
the fermentation of dextrose.

The acetoin is oxidized in the presence of oxygen and alkali
to produce a red color.

This is a positive Voges-Proskauer reaction.
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Procedure
 Using a light inoculum, inoculate tubes of MR-VP media
with 18- to 24-hour pure cultures.

Incubate tubes aerobically at 35 ― 2•
‹C for a minimum of
48 hours but preferably for 5 days.

Prepare the methyl red indicator:



0.1 g of methyl red in 300 mL of 95% ethyl alcohol.
Add sufficient purified water to make 500 mL.
After the appropriate incubation period, aseptically remove
aliquots (1 mL for the VP test) of the medium and conduct
the following tests:
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1. Methyl Red Test:
 Add 5 drops of methyl red indicator to an aliquot of the
broth.
 Interpret the color result immediately.
2. Voges-Proskauer Test:
Empty the contents (15 drops) from the reagent A
dropper
 5 drops from the reagent B dropper into 1 mL of broth
culture.


Shake well after the addition of each reagent to aerate the
sample.
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Expected Results
 1. Methyl Red Test
 a. Positive – red color at surface of the medium.
 b. Negative – yellow color at surface of the medium.
2. Voges-Proskauer Test
 A positive reaction is indicated by the development of a
distinct red color which occurs within 5 minutes.

Certain species within Enterobacteriaceae genera may react
differently or give variable results.

Consult appropriate texts for reactions of specific species.
Dr. S. S. Khoramrooz
94
Limitations of the Procedure
1. Results of the MR and VP tests need to be used in
conjunction with other biochemical tests to
differentiate genus and species within the
Enterobacteriaceae.
2. A precipitate may form in the potassium
hydroxide reagent solution.

This precipitate has not been shown to reduce the
effectiveness of the reagent.
Dr. S. S. Khoramrooz
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3. Most members of the family Enterobacteriaceae give
either a positive MR test or a positive VP test.
However, certain organisms such as Hafnia alvei and
Proteus mirabilis may give a positive result for both
tests.
4. Incubation time for the Methyl Red test cannot be
shortened by increasing the dextrose concentration in
the medium or by heavily inoculating the broth.
5. Incubate MR-negative tests for more than 48 hours and
test again.
Dr. S. S. Khoramrooz
96
6. Read the VP test at 48 hours. Increased incubation may
produce acid conditions in the broth that will interfere with
reading the results.
7. VP reagents must be added in the order and the amounts
specified or a weak-positive or false-negative reaction may
occur.

A weak-positive reaction may be masked by a copper-like color
which may form due to the reaction of KOH and α-naphthol.
8. Read the VP test within 1 hour of adding the reagents. The
KOH and α-naphthol may react to form a copper-like color,
causing a potential false-positive interpretation.
9. Due to the possible presence of acetoin, diacetyl or related
substances in certain raw materials, the use of media low in
these substances (such as MR-VP media) is recommended for
this test.
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
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Intended Use
 MacConkey agars are slightly selective and differential
plating media mainly used for the detection and isolation
of gram-negative organisms from clinical, dairy, food,
water, pharmaceutical, cosmetic, and other industrial
sources.

MacConkey Agar is used for isolating and differentiating
lactose-fermenting from lactose-nonfermenting gramnegative enteric bacilli.

MacConkey Agar Base is used with added carbohydrate
in differentiating coliforms based on fermentation
reactions.
Dr. S. S. Khoramrooz
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
MacConkey Agar without Crystal Violet is used for
isolating and differentiating enteric microorganisms
while permitting growth of staphylococci and
enterococci.

The medium can be used also to separate
Mycobacterium fortuitum and M. chelonae from other
rapidly growing mycobacteria.

MacConkey Agar without Crystal Violet or Salt and
MacConkey Agar without Salt are used for isolating
and differentiating gram-negative bacilli while
suppressing the swarming of most Proteus species.
Dr. S. S. Khoramrooz
100
Principles of the Procedure
 Lactose
is a fermentable carbohydrate.
 When
lactose is fermented, a local pH drop around
the colony cause a color change in the pH
indicator (neutral red) and bile precipitation.
Dr. S. S. Khoramrooz
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 Bile
salts, bile salts no. 3, oxgall and crystal
violet are selective agents that inhibit growth of
gram-positive organisms.
 Magnesium
sulfate is a source of divalent cations.
Agar is the solidifying agent.
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
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Expected Results
 Lactose-fermenting organisms grow as pink to
brick-red colonies with or without a zone of
precipitated bile.
 Lactose-nonfermenting
organisms grow as
colorless or clear colonies.
 Swarming
by Proteus spp. is reduced on
MacConkey agars without salt.
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
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Limitations of the Procedure
1. Although MacConkey media are selective
primarily for gram-negative enteric bacilli,
biochemical and, if indicated, serological testing
using pure cultures are recommended for complete
identification.
2. Incubation of MacConkey Agar plates under
increased CO2 has been reported to reduce the
growth and recovery of a number of strains of
gram-negative bacilli.
Dr. S. S. Khoramrooz
106
Intended Use

Malonate Broth is used for differentiating Enterobacter from
Escherichia based on malonate utilization.
Principles of the Procedure



Malonate Broth contains ammonium sulfate, which is the sole
source of nitrogen in the medium;
Sodium malonate is the sole source of carbon.
Increased alkalinity resulting from malonate utilization
causes the indicator, bromthymol blue, to change color from
green to blue.
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Dr. S. S. Khoramrooz
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Procedure
1. Inoculate tubes with a loopful of test organism.
2. Incubate at 35 ― 2•
‹C for 18-48 hours.
3. Examine tubes for a change in the color of the
medium from green to blue.
Expected Results
 Malonate utilization is indicated by a change in the
color of the medium from green to blue:
 Positive: Blue
 Negative: Green
Dr. S. S. Khoramrooz
109
Dr. S. S. Khoramrooz
110
Intended Use

OF (Oxidation Fermentation) media are used for the
determination of oxidative and fermentative
metabolism of carbohydrates by gram-negative rods on
the basis of acid reaction in either the open or closed
system.
Summary and Explanation
 OF Medium was developed by Hugh and Leifson who
described the taxonomic significance of fermentative
versus oxidative metabolism of carbohydrates by
gram-negative bacteria.
Dr. S. S. Khoramrooz
111

They showed that when an organism is inoculated into two tubes of
OF Basal Medium containing a carbohydrate and the medium in one
of the tubes is covered with melted petrolatum prior to incubation,
the patterns of metabolism are of differential significance.

Oxidative organisms only produce an acid reaction in the open
tube with little or no growth and no acid formation in the covered
tube.

Fermentative organisms will produce an acid reaction in both types
of tubes.

Changes in the covered agar are considered to be due to true
fermentation, while changes in the open tubes are due to oxidative
utilization of the carbohydrate present.

If the carbohydrate is not utilized by either method, there is no acid
production in either tube.
Dr. S. S. Khoramrooz
112
Principles of the Procedure
 Dextrose is the most important carbohydrate for
use in OF Basal Medium; however, certain
organisms may metabolize other carbohydrates
even if they are unable to utilize dextrose.
 Prepared
tubed media containing arabinose,
dextrose, dulcitol, fructose, galactose, lactose,
maltose, mannose, raffinose, rhamnose, salicin,
sorbitol, sucrose and xylose are provided.
Dr. S. S. Khoramrooz
113
Dr. S. S. Khoramrooz
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Procedure

Inoculate a pair of OF tubes of each carbohydrate used
with each organism being tested.

The tubes should be stabbed to approximately 1/4 inch
from the bottom using an inoculating needle and a light
inoculum.

Overlay one tube of each pair with sterile mineral oil.

Incubate tubes at 35 ± 2°C in an aerobic atmosphere for 48
hours.

Do not discard as negative until after 4 days of incubation.
Dr. S. S. Khoramrooz
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 Expected
Results
 Record results as acid (A) or alkaline/no change (–).
 Also
record whether or not the organism is motile
as evidenced by the appearance of growth away
from the line of inoculation.
 Typical
reaction patterns are as follows.
Dr. S. S. Khoramrooz
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Limitations of the Procedure
1. The acid reaction produced by oxidative organisms is
apparent at the surface and gradually spreads
throughout the medium.
If the oxidation is weak or slow, however, an initial
alkaline reaction at the surface of the open tube may
persist for several days and eventually convert to an
acid reaction.
2. If an organism is unable to grow on OF Basal Medium,
Cowan recommends adding either 2% serum or 0.1%
yeast extract to each carbohydrate tube.
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
118
Intended Use
 SIM
Medium is used to differentiate enteric bacilli
on the basis of sulfide production,indole
formation and motility.
Dr. S. S. Khoramrooz
119
Summary and Explanation
 Hydrogen
sulfide production, indole formation
and motility are distinguishing characteristics
which aid in the identification of the
Enterobacteriaceae, especially Salmonella and
Shigella.
 SIM
Medium, therefore ,is useful in the process of
identification of enteric pathogens.
Dr. S. S. Khoramrooz
120
Principles of the Procedure

Sodium thiosulfate and ferrous ammonium sulfate are
indicators of hydrogen sulfide production.

The ferrous ammonium sulfate reacts with H2S gas to
produce ferrous sulfide,a black precipitate.

The casein peptone is rich in tryptophan,which is attacked
by certain microorganisms resulting in the production of
indole.

The indole is detected by the addition of chemical
reagents following the incubation period.
Dr. S. S. Khoramrooz
121
 Motility
detection is possible due to the semisolid
nature of the medium.
 Growth
radiating out from the central stab line
indicates that the test organism is motile.
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
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Procedure
 Loosen caps, boil and cool before use.
 Using
growth from a pure culture, stab an
inoculating needle two-thirds of the distance to
the bottom in the center of the tube.
 Incubate
tubes with loosened caps for18-24 hours
at 35±2°C in anaerobic atmosphere.
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
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Intended Use
 SS Agar and Salmonella Shigella Agar are
moderately selective and differential media for the
isolation of pathogenic enteric bacilli, especially
those belonging to the genus Salmonella.
 This
formulation is not recommended for the
primary isolation of Shigella.
Dr. S. S. Khoramrooz
126
Principles of the Procedure

SS Agar and Salmonella Shigella Agar are designated as
moderately selective media based upon the degree of
inhibition of gram-positive microorganisms that they inhibit
due to their content of bile salts, brilliant green and
citrates.

Differentiation of enteric organisms is achieved by the
incorporation of lactose in the medium.

Organisms that ferment lactose produce acid which, in the
presence of the neutral red indicator, results in the
formation of red colonies.

Lactose nonfermenters form colorless colonies.
Dr. S. S. Khoramrooz
127
 The
latter group contains the majority of the
intestinal pathogens, including Salmonella and
Shigella.
 The
sodium thiosulfate and ferric citrate enable
the detection of hydrogen sulfide production as
evidenced by colonies with black centers.
Dr. S. S. Khoramrooz
128
Procedure

A nonselective medium should also be streaked to
increase the chance of recovery when the population
of gram-negative organisms is low and to provide an
indication of other organisms present in the
specimen.

Incubate plates, protected from light, at 35 ± 2°C for
18-24 hours.

If negative after 24 hours, reincubate an additional 24
hours.
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
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Expected Results
 Typical colonial morphology on Salmonella
Shigella Agar is as follows:
Dr. S. S. Khoramrooz
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Dr. S. S. Khoramrooz
132
Limitation of the Procedure
 Due to the relatively high level of selectivity, some
Shigella strains may not grow on SS Agar and
Salmonella Shigella Agar and, therefore, these
media are not recommended for the primary
isolation of Shigella.
 Media
recommended for the isolation of Shigella
are Hektoen Enteric and XLD agars.
Dr. S. S. Khoramrooz
133
Intended Use
 Selenite Broth (Selenite-F Broth) is used as an
enrichment medium for the isolation of Salmonella
from feces, urine, water, foods and other materials of
sanitary importance.
Principles of the Procedure
 The peptone provides essential nitrogenous and carbon
compounds.

The lactose in the medium serves to maintain a
uniform pH.
Dr. S. S. Khoramrooz
134

When selenite is reduced by the growth of bacteria,
alkali is produced, and such increase in pH would lessen
the toxicity of the selenite and result in overgrowth of
extraneous bacteria.

The acid produced by lactose fermentation serves to
maintain a neutral or slightly decreased pH.

The function of the phosphate is two-fold; it serves to
maintain a stable pH and lessens the toxicity of the
selenite, thus increasing the capacity of the medium.

Sodium selenite inhibits many species of grampositive
and gram-negative bacteria including enterococci and
coliforms.
Dr. S. S. Khoramrooz
135
Dr. S. S. Khoramrooz
136
Dr. S. S. Khoramrooz
137
Procedure

For feces and other solid materials, suspend 1-2 g of the
specimen in the broth (approximately 10-15% by
volume) and emulsify with an inoculating needle, if
necessary.

Incubate tubes with loosened caps at 35 ± 2°C for up to
24 hours.

Subcultures should be made after 12-18 hours of
incubation, if possible.

Coliforms will tend to overgrow the pathogens if
incubated longer than 24 hours.
Dr. S. S. Khoramrooz
138
Expected Results
 After incubation, there should be an increase in the
number of pathogens that the medium is designed
to select for and enrich.
 Subculture
onto appropriate selective and
differential media (e.g., MacConkey Agar,
Hektoen Enteric Agar, XLD Agar, XLT4 Agar,
CHROMagar™ Salmonella) to isolate
pathogens for identification.
Dr. S. S. Khoramrooz
139
Limitation of the Procedure
 Enrichment broths should not be used as the sole
isolation medium.
 They
are to be used in conjunction with selective
and nonselective plating media to increase the
probability of isolating pathogens, especially when
they may be present in small numbers.
Dr. S. S. Khoramrooz
140
Intended Use
 Simmons Citrate Agar is used for the differentiation of
gram negative bacteria on the basis of citrate utilization.
Principles of the Procedure
 Organisms able to utilize ammonium dihydrogen
phosphate and sodium citrate as the sole sources of
nitrogen and carbon, respectively, will grow on this
medium and produce an alkaline reaction as evidenced
by a change in the color of the bromthymol blue
indicator from green (neutral) to blue (alkaline).
Dr. S. S. Khoramrooz
141
Procedure

Inoculate slants with growth from a pure culture using a light
inoculum.

Incubate all tubes for 4 days at 35 ± 2°C in an aerobic
atmosphere.
Expected Results

A positive reaction is indicated by growth with an intense blue
color in the slant.

A negative reaction is evidenced by no growth to trace growth
with no change in color (medium remains dark green).
Dr. S. S. Khoramrooz
143
Dr. S. S. Khoramrooz
144
Intended Use

Triple Sugar Iron Agar (TSI Agar) is used for the
differentiation of gram-negative enteric bacilli based
on carbohydrate fermentation and the production of
hydrogen sulfide.
Principles of the Procedure

TSI Agar contains three sugars (dextrose, lactose and
sucrose),

Phenol red for detecting carbohydrate fermentation
Dr. S. S. Khoramrooz
145

Ferrous ammonium sulfate for detection of hydrogen sulfide
production (indicated by blackening in the butt of the tube).

Carbohydrate fermentation is indicated by the production of gas
and a change in the color of the pH indicator from red to yellow.

To facilitate the detection of organisms that only ferment
dextrose, the dextrose concentration is one-tenth the concentration
of lactose or sucrose.

The small amount of acid produced in the slant of the tube during
dextrose fermentation oxidizes rapidly, causing the medium to
remain red or revert to an alkaline pH.

In contrast, the acid reaction (yellow) is maintained in the butt of the
tube because it is under lower oxygen tension.
Dr. S. S. Khoramrooz
146
 After
depletion of the limited dextrose, organisms
able to do so will begin to utilize the lactose or
sucrose.
 To
enhance the alkaline condition of the slant, free
exchange of air must be permitted by closing the
tube cap loosely.
 If
the tube is tightly closed, an acid reaction
(caused solely by dextrose fermentation) will also
involve the slant.
Dr. S. S. Khoramrooz
147
Dr. S. S. Khoramrooz
148
Procedure
 To
inoculate, carefully touch only the center of an
isolated colony on an enteric plated medium with a
cool, sterile needle, stab into the medium in the butt
of the tube, and then streak back and forth along the
surface of the slant.
 Several
colonies from each primary plate should be
studied separately, since mixed infections may
occur.
Dr. S. S. Khoramrooz
149
 Incubate
with caps loosened at 35°C and examine
after 18-24 hours for carbohydrate fermentation,
gas production and hydrogen sulfide production.
 Any
combination of these reactions may be
observed.
 Do
not incubate longer than 24 hours because
the acid reaction in the slant of lactose and sucrose
fermenters may revert to an alkaline reaction.
Dr. S. S. Khoramrooz
150
Dr. S. S. Khoramrooz
151
Expected Results

Carbohydrate fermentation is indicated by a yellow coloration
of the medium.

If the medium in the butt of the tube becomes yellow (acidic),
but the medium in the slant becomes red (alkaline), the
organism being tested only ferments dextrose (glucose).

A yellow (acidic) color in the slant and butt indicates that
the organism being tested ferments dextrose, lactose and/or
sucrose.

A red (alkaline) color in the slant and butt indicates that the
organism being tested is a nonfermenter.
Dr. S. S. Khoramrooz
152
 Hydrogen
sulfide production results in a black
precipitate in the butt of the tube.
 Gas
production is indicated by splitting and
cracking of the medium.
Dr. S. S. Khoramrooz
153
1. Hydrogen sulfide production may be evident on Kligler Iron
Agar but negative on Triple Sugar Iron Agar.
Studies by Bulmash and Fulton showed that the utilization of
sucrose could suppress the enzymatic mechanisms responsible
for H2S production.
Padron and Dockstader8 found that not all H2S-positive Salmonella
are positive on TSI.
2. Sucrose is added to TSI to eliminate some sucrose-fermenting
lactose-nonfermenters such as Proteus and Citrobacter spp.
3. Further biochemical tests and serological typing must be
performed for definite identification and confirmation of
organisms.
Dr. S. S. Khoramrooz
154
4. Do not use an inoculating loop to inoculate a tube of
Triple Sugar Iron Agar.
While stabbing the butt, mechanical splitting of the medium
occurs, causing a false positive result for gas production.
5. A pure culture is essential when inoculating Triple Sugar
Iron Agar.
If inoculated with a mixed culture, irregular observations may
occur.
6. Tubes should be incubated with caps loosened.
This allows a free exchange of air, which is necessary to
enhance the alkaline condition on the slant.
Dr. S. S. Khoramrooz
155
Intended Use
 Urea Agar and Urease Test Broth are used for the
differentiation of organisms, especially the
Enterobacteriaceae, on the basis of urease
production.
Dr. S. S. Khoramrooz
156
Principles of the Procedure

The urea medium of Rustigian and Stuart is particularly
suited for the differentiation of Proteus species from other
gram negative enteric bacilli capable of utilizing urea.


Unable to do so in Urease Test Broth because of limited
nutrients and the high buffering capacity of the medium.
To provide a medium with greater utility, Urea Agar was
devised by Christensen with peptone and dextrose
included and reduced buffer content to promote more
rapid growth of many of the Enterobacteriaceae and permit
a reduction in incubation time.
Dr. S. S. Khoramrooz
157
 The
complete Urea Agar contains 15.0 g/L of agar
in addition to the ingredients in the base
medium.
 When
organisms utilize urea, ammonia is formed
during incubation which makes the reaction of
these media alkaline, producing a red-pink color.
 Consequently,
urease production may be detected
by the change in the phenol red indicator.
Dr. S. S. Khoramrooz
158
Urease medium
Dr. S. S. Khoramrooz
160
Dehydrated Product
 BBL™ Urea Agar Base
1. Dissolve 29 g of the powder in 100 mL of purified water.
Mix thoroughly. Sterilize by filtration.
2. Suspend 15 g of agar in 900 mL of purified water.
3. Autoclave at 121°C for 15 minutes.
4. Cool to 50°C and add 100 mL of the sterile Urea Aga Base.
5. Mix thoroughly and dispense aseptically in sterile tubes.
6. Cool tubed medium in a slanted position so that deep
butts are formed.
7. Do not remelt the complete medium.
8. Test samples of the finished product for performance using
stable, typical control cultures.
Dr. S. S. Khoramrooz
161
Procedure
 Using
a heavy inoculum (2 loopfuls) of growth from
an 18- to 24-hour pure culture (TSI Agar or other
suitable medium), inoculate the broth or agar
(streaking back and forth over the entire slant
surface).
Dr. S. S. Khoramrooz
162

Do not stab the butt since it serves as a color control.

For broth, shake tubes gently to suspend the bacteria.

Incubate tubes with loosened caps at 35 ― 2•
‹C in an
incubator or water bath.

Observe reactions after 2, 4, 6, 18, 24 and 48 hours.

For agar, continue to check every day for a total of 6
days; even longer incubation periods may be necessary.
Dr. S. S. Khoramrooz
163
 The
production of urease is indicated by an intense
pink-red (red-violet) color on the slant or
throughout the broth.
 The
color may penetrate into the agar (butt); the
extent of the color indicates the rate of urea
hydrolysis.
Dr. S. S. Khoramrooz
164
 A negative
reaction is no color change.
 The
agar medium remains pale yellow to buff; the
broth remains yellowish orange.
Dr. S. S. Khoramrooz
165
Urea Agar Base
1. The alkaline reaction produced in this medium after prolonged
incubation may not be caused by urease activity.
False positive reactions may occur due to the utilization of
peptones (especially in slant agar by Pseudomonas aeruginosa,
for example) or other proteins which raise the pH due to protein
hydrolysis and the release of excessive amino acid residues.
To eliminate possible protein hydrolysis, perform a control test
with the same test medium without urea.
2. Do not heat or reheat the medium because urea decomposes very
easily.
Dr. S. S. Khoramrooz
166
3. Urea Agar detects rapid urease activity of only the urease
positive Proteus species.
For results to be valid for the detection of Proteus, the results
must be read within the first 2-6 hours after incubation.
Urease-positive Enterobacter, Citrobacter or Klebsiella, in
contrast, hydrolyze urea much more slowly, showing
only slight penetration of the alkaline reaction into the butt
of the medium in 6 hours and requiring 3-5 days to
change the reaction of the entire butt.
Dr. S. S. Khoramrooz
167
Urea Broth
1. To rule out false positives due to protein
hydrolysis (as opposed to urea hydrolysis) that
may occur in the medium after prolonged
incubation, perform a control test with the same
test medium without urea.
2. Do not heat or reheat the medium because urea
decomposes very easily.
Dr. S. S. Khoramrooz
168
3. The high buffering system in this medium masks urease activity
in organisms that are delayed positive.
This medium is therefore recommended for the detection of urease
activity in all Proteus spp., Providencia rettgeri and ureasepositive Providencia stuartii.
M. morganii slowly hydrolyzes urea and may require approximately
a 36 hour incubation for a strong urease-positive reaction to
occur.
If in doubt as to a result, compare with an uninoculated tube or
incubate for an additional 24 hours.
4. Variations in the size of the inoculum can affect the time required
to reach positive (alkaline, pH 8.1) results.
Dr. S. S. Khoramrooz
169
Intended Use
 XL (Xylose Lysine) Agar Base is used for the
isolation and differentiation of enteric pathogens
and, when supplemented with appropriate
additives, as a base for selective enteric media.
 XLD Agar
is the complete Xylose Lysine
Desoxycholate Agar, a moderately selective
medium recommended for isolation and
differentiation of enteric pathogens, especially
Shigella species.
Dr. S. S. Khoramrooz
170
Principles of the Procedure

Xylose is incorporated into the medium because it is
fermented by practically all enterics except for the
shigellae.

This property enables the differentiation of Shigella species.

Lysine is included to enable the Salmonella group to be
differentiated from the nonpathogens.

Without lysine, salmonellae rapidly would ferment the
xylose and be indistinguishable from nonpathogenic
species.
Dr. S. S. Khoramrooz
171

After the salmonellae exhaust the supply of xylose, the
lysine is attacked via the enzyme lysine decarboxylase,
with reversion to an alkaline pH, which mimics the
Shigella reaction.

To prevent similar reversion by lysine-positive coliforms,
lactose and sucrose (saccharose) are added to produce
acid in excess.

Degradation of xylose, lactose and sucrose generates acid
products, which in the presence of the pH indicator
phenol red, causes a color change in the medium from
red to yellow.
Dr. S. S. Khoramrooz
172
 To
add to the differentiating ability of the
formulation, an H2S indicator system, consisting
of sodium thiosulfate and ferric ammonium citrate,
is included for the visualization of the hydrogen
sulfide produced, resulting in the formation of
colonies with black centers.
 The
nonpathogenic H2S producers do not
decarboxylate lysine; therefore, the acid reaction
produced by them prevents the blackening of the
colonies.
Dr. S. S. Khoramrooz
173
 XLD Agar
is both a selective and differential
medium.
 It
utilizes sodium desoxycholate as the selective
agent and, therefore, it is inhibitory to grampositive microorganisms.
Dr. S. S. Khoramrooz
174
Dr. S. S. Khoramrooz
175
Dr. S. S. Khoramrooz
176

Expected Results

Degradation of xylose, lactose and sucrose generates acid
products, causing a color change in the medium from red to
yellow.

Hydrogen sulfide production under alkaline conditions
causes colonies to develop black centers.

This reaction is inhibited by the acid conditions that
accompany carbohydrate fermentation.

Lysine decarboxylation in the absence of lactose and
sucrose fermentation causes reversion to an alkaline
condition and the color of the medium changes back to red.

Typical colonial morphology and reactions on XLD Agar are
as follows:
Dr. S. S. Khoramrooz
177
Dr. S. S. Khoramrooz
178
Dr. S. S. Khoramrooz
179
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Limitations of the Procedure
1. Red, false-positive colonies may occur with some
Proteus and Pseudomonas species.
2. Incubation in excess of 48 hours may lead to falsepositive results.
3. S. Paratyphi A, S. Choleraesuis, S. pullorum and S.
gallinarum may form red colonies without black
centers, thus resembling Shigella species.
4. Some Proteus strains will give black-centered colonies
on XLD Agar.
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Intended Use
 Transport
Medium Amies and Transport Medium
(Stuart, Toshach and Patsula) are used for collecting,
transporting and preserving microbiological
specimens.
 Cary
and Blair Transport Medium is used for
collecting, transporting and preserving microbiological
specimens, particularly those containing Vibrio
cholerae.
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Summary and Explanation

Transport media are chemically defined, semisolid,
nonnutritive, phosphate buffered media that provide a
reduced environment.

Transport media are formulated to maintain the viability
of microorganisms without significant increase in
growth.

In 1948, Moffett, Young and Stuart described a medium
for transporting gonococcal specimens to the laboratory.
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
The ability of Stuart’s medium to maintain the
viability of gonococci during transport led other
researchers to explore its use with a variety of
specimens.
 This
medium is currently recommended for throat,
vaginal and wound samples.
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
In 1964, Cary and Blair modified Stuart’s medium by
substituting inorganic phosphates for glycerophosphate
and raising the pH to 8.4.

The modified medium was effective in maintaining the
viability of Salmonella and Shigella in fecal samples.

Due to its high pH, Cary and Blair Transport Medium is
also effective in maintaining the viability of Vibrio
cultures for up to four weeks.

Cary and Blair Transport Medium is currently
recommended for fecal and rectal samples.
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 Transport
Medium Amies is recommended for
throat, vaginal and wound samples.
 Amies
media are especially suited for specimens
containing Neisseria gonorrhoeae.
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Procedure
1. Obtain specimen with sterile swab. Insert specimen swab(s)
into the upper third of the medium in the transport container.
2. Cut with sterile scissors or break-off the protruding portion of
the swab stick. Tightly screw the lid on the bottle or vial.
3. Label the bottle or vial and send to the laboratory with
minimum delay.
Specimens may be refrigerated until ready for shipment.
4. Submit to laboratory within 24 hours for culture and
analysis.
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Expected Results

Survival of bacteria in a transport medium depends on
many factors including the type and concentration of
bacteria in the specimen, the formulation of the
transport medium, the temperature and duration of
transport and inoculation to appropriate culture media
within 24 hours.

Optimal growth and typical morphology can only be
expected following direct inoculation and appropriate
cultivation.
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THE END
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