IV.

Data & Results:

Test Pictograph of Positive and Specimen Pictograph of Lab Results Interpretation

Expected Results Negative
results
Catalase Bubbling From the Tube
test indicates a (Staphylococcus
positive test. aureus)

No bubbling Positive for

indicates a catalase test,
negative test. Staphylococcus
is present with
the enzyme
catalase

From the Earlobe Negative no

(Staphylococcus present of
epidermidis) enzyme catalase.
From Urine sample Positive for
(Escherichia coli) catalase test,
Staphylococcus
is present with
the enzyme
catalase

Rectal swab Negative no

(Escherichia coli) present of
enzyme catalase.
 Throat swab Negative no
(Streptococcus present of
pyogenes) enzyme catalase.

From the teeth Negative no

Streptococcusmutan present of
enzyme catalase.

Coagulase Formation of From the Tube There is the

test a clot is taken (Staphylococcus presence of a
as positive aureus) clump which
gives a positive
result.
 From the Earlobe There is no
(Staphylococcus agglutination of
epidermidis) the plasma,
remained as a
smooth
suspension.

From Urine sample There is no

(Escherichia coli) agglutination of
the plasma,
remained as a
smooth
suspension.
Rectal swab There is no
(Escherichia coli) agglutination of
the plasma,
remained as a
smooth
suspension.
Throat swab There is no
(Streptococcus agglutination of
pyogenes) the plasma,
remained as a
smooth
suspension.
From the teeth There is no
Streptococcusmutan agglutination of
the plasma,
remained as a
smooth
suspension.
IMViC TEST
Indole A positive 1. Staphylococcus The result for
test result has a aureus #1&2,5&6-a
red layer at 2. Staphlococcus yellow ring was
the top. A epidermidis observed at the
negative 3. Escherichia top layer
result has a coli(urine) resulting in a
yellow or 4. Escheria negative result.
brown layer. coli(Rectal For
swab) #3&4(Escherichia
5. Streptococcus coli from Urine &
mutans Rectal swab) a
6. Streptococcus red layer at the
pyogenes top indicates
that Escherichia
coli can produce
Indole from
amino acid
tryptophan using
the enzyme
tryptophanase.
Methyl 1. Staphylococcus The result of
red test aureus Methyl Red Test
2. Staphlococcus for #1,2,5,& 6 a
Development
epidermidis slightly yellow
of a red color
3. Escherichia color that is a
is taken as
coli(urine) negative result,
positive, and
4. Escheria and for #3&4 it
a yellow
coli(Rectal swab) gave a positive
color is to be
5. Streptococcus red color.
negative
mutans
6. Streptococcus
pyogenes
Voges- 1. Staphylococcus For VP test the 6
Proskauer aureus specimens
test 2. Staphlococcus results were all
epidermidis negative because
Appearance 3. Escherichia there is no
of red color coli(urine) presence of
is taken as a 4. Escheria acetoin.
positive test. coli(Rectal swab)
5. Streptococcus
mutans
6. Streptococcus
pyogenes
Citrate
test( Sim
mon If the
citrated organism has
slant the ability to
utilize
citrate, the
medium
changes its
color from
green to
blue.

1. Staphylococcus A purssian blue

aureus color indicates
that the
organism used
citrate as its
source of
carbon.
2. Staphlococcus For
epidermidis Staphylococcus
epidermidis it
remained green
resulting that it
doesn’t use
citrate as it’s
source of
carbon.
3. Escherichia For the result of
coli(urine) Escherichia coli-
Urine sample,
gave a positive
result which
means that it
utilizes citrate,
4. Escheria For Escherichia
coli(Rectal swab) coli-Rectal swab
it remained
green resulting
that it doesn’t
use citrate as it’s
source of
carbon.
5. Streptococcus For
mutans Streptococcus
mutan it
remained green
resulting that it
doesn’t use
citrate as it’s
source of
carbon.
6. Streptococcus For
pyogenes Streptococcus
pyogenes it
 remained green
resulting that it
doesn’t use
citrate as it’s
source of
carbon.

V. Discussion:

CATALASE TEST

Catalase (also known as peroxidase) is an enzyme that catalyses the breakdown of hydrogen peroxide to oxygen and water. Most higher organisms
produce catalase, but in bacteriology this test is usually used to differentiate staphylococci (Catalase positive) from streptococci (Catalase negative).

Chemical equation for the breakdown of hydrogen peroxide:

2H2O2 → 2H2O + O2

A summary of typical results seen with some commonly encountered Gram-positive organisms.

Catalase positive Catalase negative

Micrococcus spp. Enterococcus spp.

Staphylococcus spp. Gemella spp.

Listeria spp. Lactococcus spp.

Propionibacterium spp. Leuconostoc spp.

Kurthia spp. Streptococcus spp.

Rhodococcus spp. Erysipelothrix spp.

Arthrobacter spp. Gardnerella spp.

  Lactobacillus spp.

 This test identifies organisms that are capable of producing the enzyme catalase.
 Organisms that produce catalase can break down hydrogen peroxide into water and oxygen gas.
 When a drop of 3% hydrogen peroxide is added to a glass slide (or petri dish) that contains catalase positive bacteria on it, bubbles of
oxygen gas become clearly visible in the mixture of hydrogen peroxide and bacteria.
 No bubbles is a negative result and means that the bacteria on the slide (or petri dish) could not produce catalase.
 Controls must always be run, because hydrogen peroxide is unstable and its integrity must be confirmed in order to rule out a false negative
result.
 Streptococcus is catalase negative, whereas Staphylococcus is catalase positive.

COAGULASE TEST

Coagulase exists in two forms: “bound coagulase” or clumping factor which is bound to cell wall and for free coagulase “coagulase test’ which
liberated by the cell wall. Bound coagulase is detected by the slide, coagulase test whereas free bound is detected by the tube coagulase test.

Bound coagulase absorbs fibrinogen from the plasma and alters it so it precipitates on the staphylococci causing them to clump resulting in cell
agglutination. The tube coagulase test detects both bound and free coagulase test. Free coagulase reacts with a substance in plasma to form fibrin
clot.

Clumps that will not mix uniformly into coagulase plasma represent a positive slide coagulase test and are indicative of S. aureus. A negative
reaction is recorded when colonies mix smoothly into solution. Clumping in both the coagulase and control indicate that the organism
autoagglutinates and is unsuitable for the slide coagulase test. When auto-agglutination is observed, the tube coagulase test should be employed
as an alternative to the slide agglutination test.

IMViC TEST
IMViC 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.

The IMViC tests are used to differentiate the enterics (Family Enterobacteriaceae). These are the Indole test (tryptone broth), the Methyl Red and
Voges-Proskauer tests (MRVP broth) and the Citrate test (Citrate agar slants). For these IMViC tests use the enterics
E. coli and Enterobacter. Work in groups of 4-5 students.

The significance of these tests is that when testing drinking water for the presence of the sewage indicator E. coli, one must be able to rule out
Enterobacter aerogenes.
E. aerogenes is not always associated with sewage, and its presence in water would not necessarily indicate sewage contamination.

1. INDOLE TEST
Principle:
Some bacteria can produce Indole from amino acid tryptophan using the enzyme tryptophanase.

Description:
Tryptophan hydrolysis -Some bacteria split tryptophan into indole and pyruvic acid using the hydrolase called tryptophanase. Indole
can be detected with Kovac's reagent (Indole reagent). This test is very important in differentiating E. coli (indole positive) from some closely
related enteric bacteria. It also differentiates Proteus mirabilis (indole negative) from all other Proteus species (indole positive). Tryptone
broth is used for this test as it contains a large amount of tryptophan.

Interpretation:

After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Add a few
drops of Indole reagent to the broth culture (tryptone broth). DO NOT SHAKE THE TUBE. A positive result has a red layer at the top.
A negative result has a yellow or brown layer.

2. METHYL RED TEST

Principle:
This test detects the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Some bacteria
produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. Methyl red is a pH indicator,
which remains the same color at a pH of 4.4 or less.

Description:
 Mixed acid fermentation - Many gram-negative intestinal bacteria can be differentiated based on the products produced when they ferment the
glucose in MR-VP medium. Escherichia, Salmonella, and Proteus ferment glucose to produce lactic, acetic, succinic, and formic acids and CO 2, H2,
and ethanol. The large amounts of acids produced lowers the pH of the medium - Methyl red (a pH indicator) will turn red when added to the
medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas.

Interpretation:
After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Remove 1
ml of broth and place into a sterile tube before performing the methyl red test if you are going to use the same broth for the VP test. Add 3-4
drops of methyl red to the original broth. DO NOT SHAKE THE TUBE. A positive result has a distinct red layer at the top of the broth. A negative
result has a yellow layer.

3. VOGES-PROSKAUER TEST
Principle:
This test detects butylenes glycol procedures. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylenes
glycol. In these test two reagents , 40% KOH and Alpha-napthol are added to the test broth after incubation and exposed to atmospheric
oxygen. If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then reacts with guanidine components of
peptone, in the presence of alpha-napthol to produce red color. Role of alpha-napthol is that of a catalyst and a color intensifier.

Discription:

Organisms that are negative in the methyl red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products
do not lower the pH as much as acids do. Enterobacter, Serratia and some species of Bacillus produce these substances. There is no satisfactory
test for determining production of 2, 3 butanediol. A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent.

Interpretation:
After incubation: Read the VP test when you have good turbidity. A clear broth indicates that your organism did not grow and
cannot be tested. Barritt's reagent A (VP A) contains naphthol and Barritt's B (VP B) contains KOH. Test 1 ml of your culture from the MRVP
broth. If you have already conducted the methyl red test, you should have already placed 1 ml of untested broth in a sterile tube. If you haven’t
done this, do so now. Add the entire contents of the VP A reagent (15 drops) and 5 drops of the VP B reagent to the 1 ml of your broth culture.
SHAKE WELL. This reaction will take a few minutes before you will see a color change. SHAKE the tube every few minutes for best results. With
a positive reaction the medium will change to pink or red indicating that acetoin is present. With a negative reaction the broth will not change
color or will be copper colored. Wait at least 15 minutes for color to develop before calling the test negative.
4. CITRATE TEST ( SIMMON CITRATE SLANT)
Principles:

This test detects the ability of an organism to utilize citrate as the sole source of carbon and energy.
Bacteria are inoculated on a medium containing sodium citrate and a pH indicator bromothymol blue. The medium also contains inorganic
ammonium salts, which is utilized as sole source of nitrogen. Utilization of citrate involves the enzyme citritase, which breaks down citrate to
oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and CO2. Production of Na2CO3 as well as NH3 from utilization of
sodium citrate and ammonium salt respectively results in alkaline pH. This results in change of medium’s color from green to blue.

Description:
Simmon's citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called
bromthymol blue. The agar media changes from green to blue at an alkaline pH.

Interpretationton:
After incubation. A positive reaction is indicated by a slant with a Prussian blue color. A negative slant will have no growth of bacteria and
will remain green.

VI. Questions:
1. What are the principles involved in each tests?

Catalase test
This test determines the organisms that produce the enzyme catalase that breaks down hydrogen peroxide to water and oxygen.
The presence of catalase enzyme is detected using hydrogen peroxide.

Coagulase test
a. Slide Coagulase Test
This bound coagulase is also known as clumping factor. It cross-links the alpha and beta chain of fibrinogen in plasma to form fibrin
clot that deposits on the cell wall. As a result, individual coccus sticks to each other and clumping is observed.
b. Tube Coagulase Test
The free coagulase secreted by S. aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin.
This converts fibrinogen to fibrin resulting in clotting of plasma.
 IMViC Test

a. Indole test
Some bacteria can produce indole from amino acid tryptophan using enzyme tryptophanase.

b. Methyl Red test

This test detects the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Some
bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. Methyl red is
a pH indicator, which remains the same color at a pH of 4.4 or less.

c. Voges-Proskauer Test
This test detects butylenes glycol procedures. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylenes
glycol. In these test two reagents , 40% KOH and Alpha-napthol are added to the test broth after incubation and exposed to atmospheric
oxygen. If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then reacts with guanidine components of
peptone, in the presence of alpha-napthol to produce red color. Role of alpha-napthol is that of a catalyst and a color intensifier.

d. Citrate Test (Simmon Citrate Test)

This test detects the ability of an organism to utilize citrate as the sole source of carbon and energy. Bacteria are inoculated on a
medium containing sodium citrate and a pH indicator bromothymol blue. The medium also contains inorganic ammonium salts, which is
utilized as sole source of nitrogen. Utilization of citrate involves the enzyme citritase, which breaks down citrate to oxaloacetate and
acetate. Oxaloacetate is further broken down to pyruvate and CO2. Production of Na2CO3 as well as NH3 from utilization of sodium
citrate and ammonium salt respectively results in alkaline pH. This results in change of medium’s color from green to blue.

2. What microorganisms give positive and negative results for each?

TEST USING LABORATORY SPECIMEN OTHER (+) OTHER (-)
MICROORGANISM MICROOGANISM
POSITIVE NEGATIVE
Catalase test Staphylococcus aureus Streptococcus pyogene Micrococcus spp. Enterococcus spp.
(tube specimen) Streptococcus mutans Staphylococcus spp. Gemella spp.
Escherichia coli (Urine Escherichia coli (Rectal Listeria spp. Lactococcus spp.
sample) swab) Propionibacterium spp. Leuconostoc spp.
Staphylococcus Kurthia spp. Streptococcus spp.
epidermidis(earlobe) Rhodococcus spp. Erysipelothrix spp.
Arthrobacter spp. Gardnerella spp.
Lactobacillus spp.
Coagulase test Staphylococcus aureus Streptococcus pyogene S.intermedius, S.
 (tube specimen) Streptococcus mutans lugdensis, S chleiferi, S.
Escherichia coli (Rectal hyicus
swab and Urine sample)
Staphylococcus
epidermidis(earlobe)

IMViC Test
Indole test Escherichia coli (Rectal Staphylococcus aureus Other Proteus spp. Klebsiella pneumonia
swab and Urine sample) (tube specimen) Proteus mirabilis
Streptococcus pyogene
Streptococcus mutans
Staphylococcus
epidermidis(earlobe)

Methyl Red test Escherichia coli (Rectal Staphylococcus aureus Salmonella spp.,
swab and Urine sample) (tube specimen) Proteus spp.,
Staphylococcus
epidermidis(earlobe)
Streptococcus pyogene
Streptococcus mutans

Voges-Proskauer test none Streptococcus pyogene Klebsiella pneumonia,

Streptococcus mutans Enterobacter spp.
Escherichia coli (Rectal Klebsiella spp., Serratia
swab and Urine sample) and some of Bacillus spp.
Staphylococcus aureus
(tube specimen)
Staphylococcus
epidermidis(earlobe)

Citrate test none Streptococcus pyogene Klebsiella pneumonia,

Streptococcus mutans Enterobacter spp.
Escherichia coli (Rectal
swab and Urine sample)
Staphylococcus aureus
(tube specimen)
 Staphylococcus
epidermidis(earlobe)

VII. Conclusion:

Based on the experiment perfomed, we have observed and learned that; catalase test determines if the organism produces the enzyme
catalase that breaks down hydrogen peroxide from water to oxygen, and the positive result for catalase test is when there is a production of
bubbles. Coagulase test determines if the organism produce the enzyme coagulase that has the ability to clot blood plasma, if there is the presence
of agglutination or clumping it indicates a positive result. Indole test is used to test bacteria that can produce indole from amino acid
trytophanusing enzyme tryptophanase. Methyl red test used to detect the ability of an organism to produce and maintain stable acid end products
from glucose fermentation. Voges-Proskauer test used to detect butylenes glycol procedures. Citrate Utilization test detects the ability of an
organism to utilize citrate as the sole source of carbon and energy.

VIII. References:
http:/www.en.wikipedia.org/wiki/Staphylococcus_aureus
http://www.microrao.com/micronotes/ imvic. pdf