IV. Data & Results: Test
Catalase test
Pictograph of Expected Results
Positive and Negative results Bubbling indicates a positive test.
Specimen
Pictograph of Lab Results
Interpretation
From the Tube (Staphylococcus aureus)
No bubbling indicates a negative test.
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.
Coagulase test
Formation of a clot is taken as positive
Throat swab (Streptococcus pyogenes)
Negative no present of enzyme catalase.
From the teeth Streptococcusmutan
Negative no present of enzyme catalase.
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. There is no agglutination of the plasma, remained as a smooth suspension. There is no agglutination of the plasma, remained as a smooth suspension. There is no agglutination of the plasma, remained as a smooth suspension.
Rectal swab (Escherichia coli)
Throat swab (Streptococcus pyogenes)
From the teeth Streptococcusmutan
IMViC TEST
Indole test
A positive result has a red layer at the top. A negative result has a yellow or brown layer.
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
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. 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.
VogesProskauer test Appearance of red color is taken as a positive test.
Citrate test( Simmon citrated slant
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.
1. Staphylococcus aureus
A purssian blue color indicates that the organism used citrate as its source of carbon.
If the organism has the ability to utilize citrate, the medium changes its color from green to blue.
2. Staphlococcus epidermidis
3. Escherichia coli(urine)
4. Escheria coli(Rectal swab)
5. Streptococcus mutans
6. Streptococcus pyogenes
For Staphylococcus epidermidis it remained green resulting that it doesnt use citrate as its source of carbon. For the result of Escherichia coliUrine sample, gave a positive result which means that it utilizes citrate, For Escherichia coli-Rectal swab it remained green resulting that it doesnt use citrate as its source of carbon. For Streptococcus mutan it remained green resulting that it doesnt use citrate as its source of carbon. For Streptococcus pyogenes it
remained green resulting that it doesnt use citrate as its 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.
P ropionibacterium
spp.
Leuconostoc spp.
Kurthia spp.
Streptococcus spp.
Rhodococcus spp.
Erysipelothrix spp.
Arthrobacter spp.
Gardnerella spp. Lactobacillus spp.
y y y
y y
y
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 m ixture of hydrogen peroxide and bacteria. No bubbles is a negative result and means that the bacteria on the slide (or petri dish) could not pro duce 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 s lide, coagulase test whereas free bound is detected by the tube coagulase test. Bound coagulase absorbs fibrinogen from the plasma and alters it s o 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 tes t 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 P roteus mirabilis (indole negative) from all other P roteus 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 y our 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 re sult 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.
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 uti lize 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 med iums 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 A lpha-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 mediums color f rom green to blue. 2.
What
microorganisms give positive and negative results for each? TEST USING LABORATORY SPECIMEN
Catalase test
POSITIVE Staphylococcus aureus (tube specimen) Escherichia coli (Urine sample)
NEGATIVE Streptococcus pyogene Streptococcus mutans Escherichia coli (Rectal swab) Staphylococcus epidermidis(earlobe)
OTHER (+) MICROORGANISM
OTHER (-) MICROOGANISM
Micrococcus spp. Staphylococcus spp. Listeria spp. P ropionibacterium spp. Kurthia spp. Rhodococcus spp.
Enterococcus spp. Gemella spp. Lactococcus spp. Leuconostoc spp. Streptococcus spp. Erysipelothrix spp.
Arthrobacter spp.
Coagulase test
Staphylococcus aureus (tube specimen)
Streptococcus pyogene Streptococcus mutans Escherichia coli (Rectal swab and Urine sample) Staphylococcus epidermidis(earlobe)
Escherichia coli (Rectal swab and Urine sample)
Staphylococcus aureus (tube specimen) Streptococcus pyogene Streptococcus mutans Staphylococcus epidermidis(earlobe)
Other Proteus spp.
Escherichia coli (Rectal swab and Urine sample)
Staphylococcus aureus (tube specimen) Staphylococcus epidermidis(earlobe) Streptococcus pyogene Streptococcus mutans
Salmonella spp., Proteus spp.,
Voges-Proskauer test
none
Streptococcus pyogene Streptococcus mutans Escherichia 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 pyogene Streptococcus mutans Escherichia coli (Rectal
Klebsiella pneumonia, Enterobacter spp.
IMViC Test Indole test
Methyl Red test
Gardnerella spp. Lactobacillus spp.
S.intermedius, S. lugdensis, S chleiferi, S. hyicus
Klebsiella pneumonia Proteus mirabilis
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 a n 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
