IV. Data & Results:

Test Pictograph of Expected Results

Positive and Negative results

Specimen Pictograph of Lab Results Interpretation

Catalase test

Bubbling indicates a positive test.

No bubbling indicates a negative test.

From the Tube(Staphylococcus aureus)

Positive for catalase test,

Staphylococcus is present with

the enzyme catalase

From the Earlobe(Staphylococcus epidermidis)

Negative no present of enzyme catalase.

From Urine sample(Escherichia coli)

Positive for catalase test, Staphylococcus is present with the enzyme catalase

Rectal swab(Escherichia coli)

Negative no present of enzyme catalase.

Throat swab(Streptococcus

pyogenes)

Negative no present of enzyme catalase.

From the teethStreptococcusmutan

Negative no present of enzyme catalase.

Coagulase test

Formation of a clot is taken as positive

From the Tube(Staphylococcus aureus)

There is the presence of a clump which gives a positive result.

From the Earlobe(Staphylococcus epidermidis)

There is no agglutination of the plasma, remained as a smooth suspension.

From Urine sample(Escherichia coli)

There is no agglutination of the plasma, remained as a smooth suspension.

Rectal swab(Escherichia coli)

There is no agglutination of the plasma, remained as a smooth suspension.

Throat swab(Streptococcus

pyogenes)

There is no agglutination of the plasma, remained as a smooth suspension.

From the teethStreptococcusmutan

There is no agglutination of the plasma, remained as a smooth suspension.

IMViC TEST

Indole test

A positive result has a red layer at the top. A negative result has a yellow or brown layer.

1. Staphylococcus aureus

2. Staphlococcus epidermidis

3. Escherichia coli(urine)

4. Escheria coli(Rectal swab)

5. Streptococcus mutans

6. Streptococcus pyogenes

The result for #1&2,5&6-a yellow ring was observed at the top layer resulting in a negative result. For #3&4(Escherichia coli from Urine & Rectal swab) a red layer at the top indicates that Escherichia coli can produce Indole from amino acid tryptophan using the enzyme tryptophanase.

Methyl red test

Development of a red color is taken as positive, and a yellow color is to be negative

1. Staphylococcus aureus

2. Staphlococcus epidermidis

3. Escherichia coli(urine)

4. Escheria coli(Rectal swab)

5. Streptococcus mutans

6. Streptococcus pyogenes

The result of Methyl Red Test for #1,2,5,& 6 a slightly yellow color that is a negative result, and for #3&4 it gave a positive red color.

Voges-Proskauer test

Appearance of red color is taken as a positive test.

1. Staphylococcus aureus

2. Staphlococcus epidermidis

3. Escherichia coli(urine)

4. Escheria coli(Rectal swab)

5. Streptococcus mutans

6. Streptococcus pyogenes

For VP test the 6 specimens results were all negative because there is no presence of acetoin.

Citrate test( Simmon citrated slant

If the organism has the ability to utilize citrate, the medium changes its color from green to blue.

1. Staphylococcus aureus

A purssian blue color indicates that the organism used citrate as its source of carbon.

2. Staphlococcus epidermidis

For Staphylococcus epidermidis it remained green resulting that it doesn’t use citrate as it’s source of carbon.

3. Escherichia coli(urine)

For the result of Escherichia coli- Urine sample, gave a positive result which means that it utilizes citrate,

4. Escheria coli(Rectal swab)

For Escherichia coli-Rectal swab it remained green resulting that it doesn’t use citrate as it’s source of carbon.

5. Streptococcus mutans

For Streptococcus mutan it remained green resulting that it doesn’t use citrate as it’s source of carbon.

6. Streptococcus pyogenes

For 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 TESTPrinciple:

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 TESTPrinciple:

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 CO2, 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 TESTPrinciple:

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 tooxaloacetate 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 testThis 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 testa. 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 TestThe 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 testSome bacteria can produce indole from amino acid tryptophan using enzyme tryptophanase.

b. Methyl Red testThis 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 TestThis 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 (+)

MICROORGANISMOTHER (-) MICROOGANISM

POSITIVE NEGATIVECatalase test Staphylococcus aureus

(tube specimen)Escherichia coli (Urine sample)

Streptococcus pyogeneStreptococcus mutansEscherichia coli (Rectal swab)Staphylococcus epidermidis(earlobe)

Micrococcus spp.Staphylococcus spp.Listeria spp.Propionibacterium spp.Kurthia spp.Rhodococcus spp.Arthrobacter spp.

Enterococcus spp. Gemella spp.Lactococcus spp.Leuconostoc spp.Streptococcus spp.Erysipelothrix spp.Gardnerella spp.Lactobacillus spp.

Coagulase test Staphylococcus aureus Streptococcus pyogene S.intermedius, S.

(tube specimen) Streptococcus mutansEscherichia coli (Rectal swab and Urine sample)Staphylococcus epidermidis(earlobe)

lugdensis, S chleiferi, S. hyicus

IMViC TestIndole test Escherichia coli (Rectal

swab and Urine sample)Staphylococcus aureus (tube specimen)Streptococcus pyogeneStreptococcus mutansStaphylococcus epidermidis(earlobe)

Other Proteus spp. Klebsiella pneumoniaProteus mirabilis

Methyl Red test Escherichia coli (Rectal swab and Urine sample)

Staphylococcus aureus (tube specimen)Staphylococcus epidermidis(earlobe)Streptococcus pyogeneStreptococcus mutans

Salmonella spp.,Proteus spp.,

Voges-Proskauer test none Streptococcus pyogeneStreptococcus mutansEscherichia coli (Rectal swab and Urine sample)Staphylococcus aureus (tube specimen)Staphylococcus epidermidis(earlobe)

Klebsiella pneumonia,Enterobacter spp. Klebsiella spp., Serratia and some of Bacillus spp.

Citrate test none Streptococcus pyogeneStreptococcus mutansEscherichia coli (Rectal swab and Urine sample)Staphylococcus aureus (tube specimen)

Klebsiella pneumonia,Enterobacter spp.

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_aureushttp://www.microrao.com/micronotes/ imvic. pdf