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Bacteria are capable of performing several biochemical activities, one of which is fermentation of many molecules including carbohydrates, amino acids & organic acids. These properties could serve as tools of identification since they demonstrates some metabolic capability of the microbe. The set-up used in the laboratory to detect biochemical activity is with the use of Durham tubes inverted in test tubes containing different carbohydrates. The Durham tube would collect the gases such as Carbon Dioxide, Methane or Hydrogen that might be produced in the fermentation process. If gas is produced , a visible bubble will form clearly in the Durham tube. Some bacteria produce acid & gas as fermentation products and in some instances only one of the two is produced. Objective : 1. To understand the biochemical activity & metabolic capabilities of some closely related microbes. 2. To differentiate two groups of microorganisms based on their biochemical properties. (Escherichia coli & Enterobacter aerogenes) 3. To have an actual demonstration of the most common biochemical tests used in the microbiology laboratory.
Materials: 1. Broth culture of both E. coli & E. aerogenes 2. Erlich’s reagent 3. Clark & Lub’s media 4. Ether 5. Methyl red indicator 6. Simon Citrate media 7. 40 % KOH 8. Test tubes
A. Indole Test: The sulfur reduction test is useful in differentiating enteric organisms. The indole test is a component of the IMViC series of tests, which is used for differentiating theEnterobacteriaceae. The motility test is useful for testing a wide variety of organisms. As a whole, the SIM test is primarily useful for differentiating Salmonella and Shigella. SIM medium contains nutrients, iron, and sodium thiosulfate. One of the nutrients is peptone, which contains amino acids, including tryptophan. If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate. If there is any blackening of the medium, it indicates the reduction of sulfur and is a positive result. The sulfur and motility test results should be determined before you perform the indole test. Some bacteria possess the ability to produce the enzyme tryptophanase, which hydrolyzes tryptophan. The end products of this hydrolyzation are indole, pyruvic acid, and ammonia, by way of deamination.
Procedure : 1. Prepare 2 test tubes containing Specimen A ( E. coli) & Specimen B ( E. aerogenes ) in broth culture. 2. Add 1 ml of ether ( note: do not aspirate by mouth) to 48 hour broth culture of both specimen A & B 3. Shake well and allow to stand until ether rises to the surface. 4. Gently add about 0.5 ml of Ehrlich’s reagent drop by drop down the side of the test tube so that it forms a ring between the medium and the ether layer. The presence of indole is indicated by a cherry red or deep red color in the reagent layer at the top. Observation: After 48hrs. of incubation, We have observed that the test results shows change in color which is black with suspended particles that means a positive result for indole. B. Methyl Red Test Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC tests as the medium in which both the Methyl Red and Voges-Prosakuer tests can be performed. It is a simple broth that contains peptone, buffers, and dextrose or glucose.
Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways. Some of these pathways produce unstable acidic products which quickly convert to neutral compounds. Some organisms use the butylene glycol pathway, which produces neutral end products, including acetoin and 2,3-butanediol. Other organisms use the mixed acid pathway, which produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are stable and will remain acidic. The Methyl Red test involves adding the pH indicator methyl red to an inoculated tube of MR-VP broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red. Methyl red differs from Phenol red (which is used in the fermentation test and the MSA plates) in that it is yellow at pH 6.2 and above and red at pH 4.4 and below. Phenol red turns yellow below a pH of 6.8. If you get these two pH indicators confused, you will have a difficult time interpreting test results. The MR and VP tests are particularly useful in the identification of the Enterobacteriaceae. Procedure: 1. Prepare 2 tubes containing Clark & Lub’s broth 2. Inoculate specimen A & B into the tubes 3. Incubate the tubes for 48 hours at 37 degrees 4. After incubation, add 2 to 3 drops of Methyl Red. 5. Red Color would be an indication for the production of acid and yellow color would be indicative of alkaline or negative results Observation: After incubation, we put 3 drops of Methyl Red, and we notice the change in color, it turns out to be pinkish in color which is a positive result for Methyl Red test. C. Citrate Test Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicator bromthymol blue. This test is part of the IMViC tests and is helpful in differentiating theEnterobacteriaceae . Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport the citrate into the cell. These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium. At pH 7.5 or above, bromthymol blue
turns royal blue. At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media. If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. Some citrate negative organisms may grow weakly on the surface of the slant, but they will not produce a color change.
Procedure: 1. Prepare two tubes with Simon Citrate media. 2. Inoculate by streaking on the slant portion of the media. 3. Incubate the tubes at 37 degrees for 48 hours. 4. Blue color would be indicative of positive results and green color would be negative. Observation: We have observed blue color in this test and also presence of white-dotted spots on the surface of the slanted prepared media. D. Voges-Proskauer Test The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a copper color is present, the result is negative. The MR and VP tests are particularly useful in the identification of the Enterobacteriaceae.
Procedure: 1. Prepare 2 tubes of Clark & Lubs media 2. Inoculate specimen A & B into the tubes 3. Incubate the tubes at 37 degrees for 72 hours. 4. After incubation add an equal amount of 40% KOH and shake vigorously for 2-5 min. 5. Allow the tubes to stand and note the color reaction. 6. Production of an Eosin like color up to 2 hours would be positive. Color would depend on the oxidation of acetoin.
Observation: After dropping the reagent into the test tube, we notice that there is no change in color which gives us a negative result for this test.
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