BIOCHEMICAL TEST
Prof. Dr. Marlina, MS, Apt.
Laboratory Investigation of Microbial
infections :
 Laboratory Investigation of Microbial infections
Examining specimens to detect isolate and
identify pathogens: 1- Microscopy 2- Culture
techniques 3- Biochemical reactions 4Serological identification: 5- Molecular biology
techniques 6- Bacteriophage typing
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Identification of an Unknown Bacterium: :
 Identification of an Unknown Bacterium:
Dr.T.V.Rao MD 3 Microbiologists use biochemical
tests, noting a particular microbe's ability to
utilize or produce certain chemicals
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Biochemical tests help in Identification of
several Bacterial isolates :
 Biochemical tests help in Identification of several
Bacterial isolates EVERYTHING that a living
organism does is the result of the activity of an
ENZYME, the SUMMATION of the activities of
all an organism's enzymes equals its
BIOCHEMICAL FINGERPRINT.
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That is, an organism is the totality of its
enzymes, so by determining which enzymes are
present in an unknown organism one can
DESCRIBE & IDENTIFY that organism 4
Dr.T.V.Rao MD
BIOCHEMICAL REACTION
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Biochemical Reaction Use of substrates and sugars to
identify pathogens:
a- Sugar fermentation: Organisms ferment sugar
with production of acid only Organisms ferment sugar
with production of acid and gas Organisms do not
ferment sugar
b- Production of indole: Depends on production of
indole from amino acid tryptophan Indole is detected
by addition of Kovac’s reagent Appearance of red ring
on the surface e- H2S production: Depends on
production H2S from protein or polypeptides
Detection by using a strip of filter paper containing
lead acetate
BIOCHEMICAL REACTION (CONT.)
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c- Methyl red reaction (MR): Fermentation of
glucose with production of huge amount of acid
Lowering pH is detected by methyl red indicator
d- Voges proskaur’s reaction (VP): Production of
acetyl methyl carbinol from glucose fermentation
Acetyl methyl carbinol is detected by addition
KOH Color of medium turns pink (positive) e-
BIOCHEMICAL REACTION (CONT.) :
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f- Oxidase test: Some bacteria produce Oxidase
enzyme Detection by adding few drops of colorless
oxidase reagent Colonies turn deep purple in color
(positive)
g- Catalase test: Some bacteria produce catalase
enzyme Addition of H2O2 lead to production of gas
bubbles (O2 production)
h- Coagulase test: Some bacteria produce coagulase
enzyme Coagulase enzyme converts fibrinogen to
fibrin (plasma clot) Detected by slide or test tube
method
i- Urease test: Some bacteria produce urease enzyme
Urease enzyme hydrolyze urea with production of
NH3 Alklinity of media and change color of indicator
from yellow to pink
COMMON TESTS TO IDENTIFY
BACTERIAL ISOLATES
Common Tests To identify Bacterial isolates :
 Indole
 Methyl Red/Voges Proskauer
 Citrate H2S production
 Urea hydrolysis
 Motility
 Lactose fermentation
 Sucrose fermentation
 Glucose fermentation
 Gas production
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CATALASE TEST
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Catalase test . This test is used to identify
organisms that produce the enzyme, catalase.
This enzyme detoxifies hydrogen peroxide by
breaking it down into water and oxygen gas. The
bubbles resulting from production of oxygen gas
clearly indicate a catalase positive result .
Catalase test :
 Catalase test 'Ten vol.' H2O2, is run into a
capillary tube, followed by suspension. Gas is
usually evolved immediately and only tubes not
showing gas within 10 sec. Are sealed for longer
observation
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Oxygen is sometimes toxic.
 Small amounts of superoxide free radicals are
formed during the normal respiration of
organisms that use oxygen as the final electron
acceptor.
 Obligate anaerobes from some oxygen free
radicals that are toxic to the cell. Hence, if
bacteria wants to grow in oxygen environment,
enzymes like catalase and superoxidase
dismutase must be present for neutralization
of the toxic form of oxygen(oxygen radical)
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During normal aerobic respiration, hydrogen ions
are produced and have to be removed by bacterial
cell. The electron transport system (ETS) in
cellular respiration (a part of glycolysis) involves
these H+ ions and combines them with oxygen to
form water. Water is harmless. Energy is given
off and stored in the form of Adenosine
Triphosphate.
AEROBES-ANAEROBES
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During normal aerobic respiration, hydrogen ions
are produced and have to be removed by bacterial
cell. The electron transport system (ETS) in
cellular respiration (a part of glycolysis) involves
these H+ ions and combines them with oxygen to
form water. Water is harmless. Energy is given
off and stored in the form of Adenosine
Triphosphate.
What is toxic is Hydrogen Peroxide that is
formed by the cytochromes in ETS. Water being
harmless is not required to be removed by the
bacteria. So, what is harmful to bacteria cell that
requires it to be removed instantly??
 answer: H2O2.
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Functions of catalase
 Protects bacteria from toxic hydrogen
peroxide (H2O2) accumulation, which can occur
during aerobic metabolism. If hydrogen
peroxide accumulates, it becomes toxic to the
organism.
 Since Catalase breaks H2O2 down into water and
O2, the presence of oxygen can be characterized
by bubbles which indicates a (+) result.
 What bacteria could mostly likely be
detected?
 most aerobic organism make catalase.
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catalase +
OXIDASE TEST
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OXIDASE TEST
The Oxidase test (also known as the Cytochrome Oxidase
test) is used to look for oxidase enzymes produced by
certain bacteria. Oxidases catalyse electron transport
between substrates acting as electron donors in the
bacterium and tetramethyl-p-phenylenediamine OR
dimethyl-p-phenylenediamine - a redox dye present as the
hydrochloride or oxalate salt The dye is reduced to a deep
violet-blue colour in the presence of oxidase enzymes.
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Oxidase test :
Oxidase test The oxidase test is a test used in microbiology
to determine if a bacterium produces certain cytochrome c
oxidases. It uses disks impregnated with a reagent such as
N,N,N′,N′- tetramethyl-p-phenylenediamine (TMPD) or
N,N-Dimethyl-p-phenylenediamine (DMPD), which is also
a redox indicator.
FILTER STRIP METHOD
Filter strip method
 Soak strips of filter paper in a fresh dye
solution, drain and freeze dry. Strips should be
stored in an air-tight bottle and kept in a cool
dark environment. Strips prepared in this
manner will keep for several months, and have
a faint pastel-violet color. To use, take a strip
and soak in distilled water. Pick the colony to be
tested with a loop and rub onto moistened strip.
A color change within 10 seconds indicates a
positive reaction.
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Oxidase testing needs controls :
 Oxidase testing needs controls Positive control:
Pseudomonas aeruginosa Negative control
Enterobactericia E.coli. Klebsiella spp.
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HYDROGEN SULFIDE PRODUCTION
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HYDROGEN SULFIDE PRODUCTION
 Some
bacteria have the enzymatic
capability to degrade amino acids (cysteine,
cystine etc.) that contain sulfhydryl group (SH) producing hydrogen sulfide. Hydrogen
sulfide reacts with heavy metals such as
lead or iron forming a black precipitate.
You can use TSI medium (contains iron) or
prepare a nutritive agar with lead acetate
(1g Pb acetate to 100 ml nutritive agar).
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PROCEDURE and Reading result :
 Harvest a well isolated colony and inoculate a
TSI tube by stabbing the medium. Incubate at 37
°C, 24 hours. Reaction is positive if a black color
appears. Bacteria growing in TSI degrade amino
acids forming ferrous sulfide which blackens the
medium
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Hydrogen sulphide production :
 H2S production: Depends on production H2S
from protein or polypeptides Detection by using a
strip of filter paper containing lead acetate H2S
production. H2S production, either via cysteine
catabolism or thiosulfate reduction
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NITRATE MEDIUM – NITRATE REDUCTION
TEST
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Nitrate Medium – Nitrate reduction Test This is
a differential medium. It is used to determine if
an organism is capable of reducing nitrate (NO3-)
to nitrite (NO2-) or other nitrogenous compounds
via the action of the enzyme nitratase (also called
nitrate reductase). This test is important in the
identification of both Gram-positive and Gramnegative species.
Nitrate reduction Test :
 Nitrate reduction Test After incubation, these
tubes are first inspected for the presence of gas in
the Durham tube. In the case of non fermenters,
this is indicative of reduction of nitrate to
nitrogen gas. However, in many cases gas is
produced by fermentation and further testing is
necessary to determine if reduction of nitrate has
occurred.
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Nitrate reduction Test :
 The reduction of nitrate to nitrite was detected
with dimethyl-a-naphthylamin (Wallace &
Neave, 1927) and sulphanilic acid. The reaction
was rapid with all the species tested; at 30 min.
the results were consistent with the usual
cultural method.
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I M VI C TESTS
I M Vi C Tests
 I M Vi C is an acronym that stands for :indole ,
methyl red, Voges-Proskauer , and citrate .
 To obtain the results of these four tests
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three test tubes are inoculated:
 tryptone broth (indole test),
 methyl red –
 Voges Proskauer broth (MR-VP broth),
 and citrate test.
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INDOLE TEST
Indole Test How to Perform Test:
 Inoculate Tryptone broth with inoculating loop.
Property it tests for: This test is performed to help
differentiate species of the family
Enterobacteriaceae. It tests for the bacteria
species’ ability to produce indole. Bacteria use an
enzyme, tryptophanase to break down the amino
acid, tryptophan, which makes by-products, of
which, indole is one. Media and Reagents Used:
Tryptone broth contains tryptophan. Kovac’s
reagent—contains hydrochloric acid,
dimethylaminobenzaldehyde, and amyl alcohol—
yellow in color. Reading Results: Kovac’s reagent
reacts with indole and creates a red color at the
top part of the test tube. 22 Dr.T.V.Rao MD
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PRINCIPLES OF INDOLE TEST :
Principles of Indole Test
 The test organism is inoculated into tryptone
broth, a rich source of the amino acid tryptophan.
 Indole positive bacteria such as Escherichia coli
produce tryptophanase, an enzyme that cleaves
tryptophan, producing indole and other products.
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When Kovac's reagent (p-dimethylamino
benzaldehyde) is added to a broth with indole in
it, a dark pink colour develops.
 The indole test must be read by 48 hours of
incubation because the indole can be further
degraded if prolonged incubation occurs.
 The acidic pH produced by Escherichia coli limits
its growth.
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CONT:
Indole Test :
 Indole Test Indole is a product of the breakdown
of another amino acid, tryptophan by the enzyme
TRYPTOPHANASE.
 To test for indole Kovacs reagent is added to the
SIM medium following growth.
 If indole is present a red ring forms around the
surface of the medium.

Tryptone Broth after addition of Kovacs(+)
Indole test on left --- (-) Indole test on
right :
 Tryptone Broth after addition of Kovacs(+) Indole
test on left --- (-) Indole test on right
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METHYL RED/VOGES PROSKAUER (MR/VP)
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Methyl Red/Voges Proskauer (MR/VP)
How to Perform Tests: Inoculate 2 glucose broths with
inoculating loop.
After 48 hours of incubation, add a few drops of MR to
one tube, and VP reagents to the other tube.
Properties they test for:
Both tests are used to help differentiate species of the
family Enterobacteriaceae.
MR—tests for acid end products from glucose
fermentation.
VP—tests for acetoin production from glucose
fermentation.
Media and Reagents Used: Glucose Broth
Methyl Red
 indicator for acid Voges Proskauer reagents
 A: 5% Alpha-Naphthol, & ethanol,
 B: Potassium Hydroxide, & Deionized Water.
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METHYL RED (MR) AND VOGES-PROSKAUER (VP)
TESTS
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Methyl red (MR) and Voges-Proskauer (VP) tests
The methyl red (MR) and Voges-Proskauer (VP) tests
are read from a single inoculated tube of MR-VP
broth.
After 24-48 hours of incubation the MR-VP broth is
split into two tubes.
One tube is used for the MR test; the other is used for
the VP test.
MR-VP media contains glucose and peptone.
All enterics oxidize glucose for energy; however the
end products vary depending on bacterial enzymes.
Both the MR and VP tests are used to determine what
end products result when the test organism degrades
glucose.
MR/VP CONTINUED :
MR/VP continued Reading Results:
 MR— a + result is red (indicating pH below 6)
and a – result is yellow (indicating no acid
production)
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VP—A + result is red after VP reagents are
added (indicating the presence of acetoin) and a –
result is no color change.
Methyl Red: left – and right +
METHYL RED
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This test is used to determine which fermentation
pathway is used to utilize glucose.
In the mixed acid fermentation pathway, glucose is
fermented and produces several organic acids (lactic,
acetic, succinic, and formic acids).
The stable production of enough acid to overcome the
phosphate buffer will result in a pH of below 4.4.
If the pH indicator (methyl red) is added to an aliquot
of the culture broth and the pH is below 4.4, a red color
will appear (first picture, tube on the left
METHYL RED TEST
Methyl Red Test
 If the pH indicator (methyl red) is added to an
aliquot of the culture broth and the pH is below
4.4, a red color will appear (first picture, tube on
the left).
 If the MR turns yellow, the pH is above 6.0 and
the mixed acid fermentation pathway has not
been utilized (first picture, tube on the right).
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METHYLENE- BLUE REDUCTION
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Methylene- blue reduction
Standardized methylene blue in concentrations of 0.1
and 0.01 yo are mixed,with suspension and sealed.
Readings are made after 4 and 24 hr. at 37".
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Voges-Proskauer (VP) test :
The reagents used for the VP test are :
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Barritt's A (alpha-napthol) and Barritt's B (potassium
hydroxide).
When these reagents are added to a broth in which
acetyl methyl carbinol is present, they turn a pinkburgundy colour (a positive VP test).
This colour may take 20 to 30 minutes to develop. E.
coli does not produce acetyl methyl carbinol, but
Enterobacter and Klebsiella do.
CITRATE TEST
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:
Citrate Test How to Perform Test: Inoculate slant
with inoculating loop. Property it tests for: This test is
used to help differentiate species of the family
Enterobacteriaceae. It is selective for bacteria that
has the ability to consume citrate as its sole source of
carbon and ammonium as sole nitrogen source. Media
and Reagents Used: Simmon’s Citrate Agar contains
sodium citrate (carbon source), ammonium ion
(nitrogen source), & pH indicator—bromthymol blue.
Reading Results: A + result is blue (meaning the
bacteria metabolised citrate and produced an acid end
product) and a – result remains green 34 Dr.T.V.Rao
MD
SIMMON'S CITRATE AGAR :

Simmon's citrate agar Uninoculated Simmon's
citrate agar has a pH of 6.9, so it is an
intermediate green color. Growth of bacteria in
the media leads to development of a Prussian
blue color (positive citrate). Enterobacter and
Klebsiella are citrate positive while E.coli is
negative. 35 Dr.T.V.Rao MD
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Citrate Test :
 Citrate Test Left positive and right negative. 36
Dr.T.V.Rao MD
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Urea Hydrolysis :
Urea Hydrolysis How to Perform Test: Inoculate Urea
broth with inoculating loop. Property it tests for: This
test is done to determine a bacteria’s ability to
hydrolyze urea to make ammonia using the enzyme
urease. Media and Reagents Used: Urea broth
contains a yeast extract, monopotassium phosphate,
disodium phosphate, urea, and phenol red indicator.
Reading Results: Urea broth is a yellow-orange color.
The enzyme urease will be used to hydrolyze urea to
make ammonia. If ammonia is made, the broth turns
a bright pink color, and is positive. If test is negative,
broth has no color change and no ammonia is made.
37 Dr.T.V.Rao MD
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Urease test :
Urease test
This test is used to identify bacteria capable of hydrolyzing
urea using the enzyme urease.
It is commonly used to distinguish the genus Proteus from
other enteric bacteria.
The hydrolysis of urea forms the weak base, ammonia, as one
of its products.
This weak base raises the pH of the media above 8.4 and the
pH indicator, phenol red, turns from yellow to pin.
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Urease Test :
Urease Test
Urea is broken down by the enzyme UREASE into carbon
dioxide and ammonia.
Ammonia turns the medium alkaline; that is it raises the pH
to above 7.0.
In this test the bacteria are inoculated into urea broth which
contains the pH indicator (phenol red) which changes from
yellow to red/pink as the pH increases (Atlas pg. 79).
After 24 to 48 hours of incubation the tubes are observed for a
color change indicative of urea digestion.
UREASE TEST
Urease test
 This test is used to identify bacteria capable of
hydrolyzing urea using the enzyme urease.
 It is commonly used to distinguish the genus
Proteus from other enteric bacteria.
 The hydrolysis of urea forms the weak base,
ammonia, as one of its products.
 This weak base raises the pH of the media above
8.4 and the pH indicator, phenol red, turns from
yellow to pink
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GLUCOSE FERMENTATION & GAS
PRODUCTION
Glucose Fermentation & Gas Production How to
Perform Test:
 Inoculate broth with inoculating loop. Property it
tests for:
 This test is done to help differnetiate species of
the family Enterobacteriaceae.
 This tests for the bacteria’s ability to ferment
glucose and produce gas and/or an acid endproduct.
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MEDIA AND REAGENTS USED
Glucose broth contains beef extract, gelatine
peptone, and glucose.
 A phenol red indicator is added to indicate an
acid enproduct.
 A Durham tube is added to indicate gas
production.
 Results A positive result for acid is yellow after
indicator is added (indicating glucose
fermentation) A positive result for gas is a bubble
in the Durham tube.
 A completely negative result has no color change
or reddish color and no bubble
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GLUCOSE BROTH WITH DURHAM TUBES
Glucose broth with Durham tubes
 This is a differential medium.
 It tests an organism's ability to ferment the sugar
glucose as well as its ability to convert the end
product of glycolysis, pyruvic acid into gaseous
byproducts.
 This is a test commonly used when trying to
identify Gram-negative enteric bacteria, all of
which are glucose fermenters but only some of
which produce gas.

Carbohydrate Fermentation tubesLeft is
(+) -- Middle indicates (+) with gas -- Right
is a (-) test :
 Carbohydrate Fermentation tubesLeft is (+) -Middle indicates (+) with gas -- Right is a (-)
test Dr.T.V.Rao MD 43
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Sugar Fermentation Tests :
 Sugar Fermentation Tests Tube
 1: Negative acid /Negative gas
 Tube 2A: Must incubate longer (ambiguous
result)
 Tube 2B: Positive acid /Negative gas
 Tube 3A: Positive acid/ Positive gas

SUCROSE FERMENTATION
Sucrose Fermentation How to Perform Test:
 Inoculate sucrose broth with inoculating loop.
Property it tests for:
 This test is done to help differentiate species of the
family Enterobacteriaceae.
 This tests for the bacteria’s ability to ferment
sucrose and production of acid end-product
 Media and Reagents Used: Sucrose broth contains
beef extract, gelatin peptone, and sucrose.
 Phenol red indicator is added to indicate an acid
end-product.
 Results A positive result is yellow after indicator is
added (indicating sucrose fermentation)
 A negative result has no color change or is reddish.
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LACTOSE FERMENTATION
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Lactose Fermentation How to Perform Test:
Inoculate lactose broth with inoculating loop.
Property it tests for:
This tests for the bacteria’s ability to ferment lactose.
Media and Reagents Used:
Lactose broth contains beef extract, gelatin peptone,
and lactose.
A phenol red indicator is added to indicate acid
production from fermentation.
Results A positive result is yellow after indicator is
added (indicating lactose fermentation)
A negative result will have no color change or will be
redish.
MOTILITY TESTING
Motility Testing Motility agar is a differential
medium used to determine whether an organism
is equipped with flagella and thus capable of
swimming away from a stab mark.
 The results of motility agar are often difficult to
interpret.
 Generally, if the entire tube is turbid, this
indicates that the bacteria have moved away from
the stab mark (are motile).
 The organisms in the two tubes pictured on the
right are motile
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COAGULASE TEST
Coagulase Test How to Perform Test:
 Inoculate rabbit plasma with one single colony.
 Break up colony and stir until blended in plasma.
Incubate at 37 degrees C for 24 hours.
 Property it tests for: This tests for the bacteria’s
ability to clot blood plasma using the enzyme
coagulase.
 If the organism has coagulase it will clump rabbit
plasma.
 Media and Reagents: This media contains rabbit
plasma dissolved in buffer .
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COAGULASE TEST
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Coagulase test Coagulase is an enzyme that clots
blood plasma by catalyzing the conversion of a soluble
protein (fibrinogen) to an insoluble protein (fibrin).
This test is performed on Gram-positive, catalase
positive species to identify the coagulase positive
Staphylococcus aureus.
Coagulase is a virulence factor of S. aureus.
The formation of clot around an infection caused by
this bacteria likely protects it from phagocytosis.
Coagulase Results :
Coagulase Results Reading Results:
If the organism is has coagulase it will clump the
plasma.
If the organism does not have coagulase it will not
clump the plasma.
TRIPLE SUGAR IRON AGAR
Triple Sugar Iron Agar Bacteria that ferment any
of the three sugars in the medium will produce by
products .
 These byproducts are usually acids, which will
change the color of the red pH-sensitive dye
(phenol red) to a yellow color.
 Position of the color change distinguishes the acid
production associated with glucose fermentation
from the acidic byproducts of lactose or sucrose
fermentation.
 Many bacteria that can ferment sugars in the
anaerobic butt of the tube are enterobacteria.
