Procedure
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1. Indole Production Principle: Indole is produced in tryptone broth by bacterial enzyme tryptophanase. Tryptone broth is rich in tryptophan which can be used as a source of carbon, nitrogen and energy. Tryptophan ® (Tryprophanase) ® Indole (Benzopyrole compound) Pyrole + weakly acid alcoholic solution of p-DMAB ® red violet colour To an 18 – 24 h culture in tryptone broth, 1 ml of Kovac’s reagent is added. Formation of a red ring at the surface of the medium is taken as positive reaction for indole production. Methyl Red Test: Glucose is provided as the sole source of carbon and energy and when it is utilized, it produces mixed acids which reduce the pH. When pH is less than 4.5, on addition of methyl red-pink colouration develops. Break down of glucose to acidic end products by the organism is tested by adding a few drops of methyl red reagent to 24 – 48 h- culture grown in MR – VP broth. Positive test is indicated by an orange to pink colouration. Voges – proskauer Test: The production of neutral products like acetyl methyl carbinol (Acetoin) from glucose is tested by inoculating the cultures to 5 ml of MRVP broth and incubating at 37oC for at least 48 h. After incubation, add VP reagent consisting of solution A (0.6 ml) and solution B (0.2ml) one by one. A pinch of creatine enhances the reaction. Positive test is indicated by port-wine colouration. Results are read after 2 hours. Citrate utilization test: The ability of microorganisms to utilize sodium citrate as the sole source of carbon has been used chiefly in the differentiation of gram negative bacilli. Prepare Simmon’s citrate agar slants. Inoculate the surface of the slant lightly with a straight wire. Incubate for a minimum period of 24 h at 37oC. Growth utilizing citrate will produce alkaline condition of the medium and the indicator bromothymol blue turns to Prussian blue at pH 9.0 from the original green colour at pH 6.8 – 6.9. |