Identification of an Unknown Bacterium:
Dr.T.V.Rao MD
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Microbiologists use biochemical tests, noting a particular
microbe's ability to utilize or produce certain
chemicals
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 BIOCHEMICALFINGERPRINT. 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
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Dr.T.V.Rao MD
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 Kovacs 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.)
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 proskaurs 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.)
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 mediaand change color of indicator from yellow to
pink
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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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
HYDROGEN SULFIDE PRODUCTION
Dr.T.V.Rao MD
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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).
PROCEDURE and Reading result
Dr.T.V.Rao MD
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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
Hydrogen sulphide production
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e- 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, produces a black precipitate in the
media.
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 Gram-negative
species.
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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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, three test tubes are inoculated: tryptone broth (indole
test), methyl red - Voges Proskauer broth (MR-VP broth), and
citrate test.
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Dr.T.V.Rao MD
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. Kovacs
reagentcontains hydrochloric acid, dimethylaminobenzaldehyde, and
amyl alcoholyellow in color.
Reading Results:Kovacs reagent reacts with indole and creates a red
color at the top part of the test tube.
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Dr.T.V.Rao MD
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. 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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Dr.T.V.Rao MD
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 formsaround the surface of the medium.
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Dr.T.V.Rao MD
Tryptone Broth after addition of Kovacs(+) Indole test on left
--- (-) Indole test on right
Dr.T.V.Rao MD
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Indole test reactions
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Dr.T.V.Rao MD
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.
MRtests for acid end products from glucose fermentation.
VPtests for acetoin production from glucose fermentation.
Media and Reagents Used:
Glucose Broth
Methyl Red indicator for acid
Voges Proskauer reagentsA: 5% Alpha-Naphthol, & ethanol, B:
Potassium Hydroxide, & Deionized Water.
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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
MR/VP continued
Reading Results:
MR a + result is red (indicating pH below 6) and a result is yellow
(indicating no acid production)
VPA + 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 +
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Dr.T.V.Rao MD
Methyl Red
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).
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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
Voges-Proskauer (VP) test
The reagents used for the VP test are 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 pink-burgundy 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.
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Dr.T.V.Rao MD
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:Simmons Citrate Agar contains sodium
citrate (carbon source), ammonium ion (nitrogen source), & pH
indicatorbromthymol blue.
Reading Results:
A + result is blue (meaning the bacteria metabolised citrate and
produced an acid end product) and a result remains green
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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
Citrate Test
Left positive and right negative.
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Dr.T.V.Rao MD
Urea Hydrolysis
How to Perform Test: Inoculate Urea broth with inoculating
loop.
Property it tests for: This test is done to determine a bacterias
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.
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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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
bacterias ability to ferment glucose and produce gas and/or an acid
end-product..
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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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
Carbohydrate Fermentation tubesLeft is (+) -- Middle indicates
(+) with gas -- Right is a (-) test
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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
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Dr.T.V.Rao MD
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Dr.T.V.Rao MD
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
bacterias 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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Dr.T.V.Rao MD
Lactose Fermentation
How to Perform Test: Inoculate lactose broth with inoculating
loop.
Property it tests for: This tests for the bacterias 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.
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Dr.T.V.Rao MD
Lactose fermenter and Non fermenter
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Dr.T.V.Rao MD
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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Dr.T.V.Rao MD
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 bacterias 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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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
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.
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Dr.T.V.Rao MD
Triple Sugar Iron Agar
Bacteria that ferment any of the three sugars in the medium will
produce byproducts 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.
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Dr.T.V.Rao MD
TSI slant
The TSI slant is a test tube that contains agar, a pH-sensitive dye
(phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as
sodium thiosulfate and ferrous sulfate or ferrous ammonium
sulfate.
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Dr.T.V.Rao MD
TSI - Reactions
Some bacteria utilize thiosulfate anion as a terminal electron
acceptor, reducing it to sulfide. If this occurs, the newly-formed
hydrogen sulfide (H2S) reacts with ferrous sulfate in the medium to
form ferrous sulfide, which is visible as a black precipitate.
Examples of sulfide-producing bacteria include Salmonella, Proteus,
Citrobacter and Edwardsiella species. The blackening of the medium
is almost always observed in the butt (bottom) of the medium.
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Dr.T.V.Rao MD
TSI - Reactions
All lactose fermenters result in yellow slant/yellow butt
(acid/acid reaction), whereas non-lactose fermenters may result in
pink/yellow or yellow/yellow (if sucrose is fermented). Blackening
of the butt due to H2S production may mask the acid reaction
(yellow) in the butt..
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Dr.T.V.Rao MD
Reading the results on TSI enables to identify the several
pathogens
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Dr.T.V.Rao MD
GELATIN LIQUEFACTION TEST
Dr.T.V.Rao MD
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The purpose of this test is to determine an organism's ability to
produce proteolytic-like enzymes and liquefy gelatin. This test is
used to differentiate between species in the genera Staphylococcus
and Clostridium as well as aid in the identification of other
species and genra.
GELATIN LIQUEFACTION TEST
Dr.T.V.Rao MD
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Gelatin is to large to enter a bacterial cell wall and thus
extracellular enzymesmust catabolize them into smaller components
before they can be utilized. Possession of these extracellular
gelatinases can aid in the differentiation of bacteria
INTERPRETING TEST RESULTS:
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POSITIVE TEST = medium liquefied.NEGATIVE TEST = medium remains
solid.
CAMP Test
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CAMP factor is a diffusible, heat-stable protein produced by group
B streptococci. This is a synergistic test between Staphylococcus
aureus and Streptococcus agalactiae. S. agalactiae produces CAMP
factor. S. aureus produces sphingomyelin C, which binds to red
blood cell membranes. The two bacteria are streaked at 90o angles
of one another. They do NOT touch
Dr.T.V.Rao MD
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Created by Dr.T.V.Rao MD for e learning by Microbiologists
Email
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