BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA

Oxidation-Fermentation test

This test is done to determine the oxidative or fermentative metabolism of carbohydrate resulting in production of various organic acids as end product. Some bacteria are capable of metabolizing carbohydrates (as exhibited by acid production) only under aerobic conditions, while others produce acid both aerobically and anaerobically. Most medical bacteria are facultative anaerobes.

The test organism was stabbed into the bottom of two sets of tubes with Hugh and Leifson's media, bromothymol blue being the pH indicator. The inoculated medium in one of the tubes was covered with a 10 mm deep layer of sterile paraffin oil. The tubes were then incubated at 37°C for 24 hours. After incubation the tubes were examined for carbohydrate utilization as shown by acid production.

Fermentative organism utilizes the carbohydrate in both the open and sealed tubes as shown by a change in colour of the medium from green to yellow. Oxidative organisms, however, are able to use the carbohydrate only in the open tube.

Indole Production test

This test detects the ability of the organism to produce an enzyme: ‘tryptophanase’ which oxidizes tryptophan to form indolic metabolites: indole, skatole (methyl indole) and indoleacetic acid.

A smooth bacterial colony was stabbed on SIM (Sulphide Indole Motility) medium by a sterile stab wire and the inoculated media was incubated at 37°C for 24 hours. After 24 hours incubation, 0.5 ml of Kovac's reagent was added. Appearance of red color on the top of media indicates indole positive. Indole if present combines with the aldehyde present in the reagent to give a red color in the alcohol layer. The color reaction is based on the presence of the pyrrole structure present in indole.

Methyl Red test

This test is performed to test the ability of an organism to produce sufficient acid from the fermentation of glucose to give a red color with the indicator methyl red (denotes changes in degree of acidity by color reactions over a pH range of 4.4-6.0).

A pure colony of the test organism was inoculated into 2 ml of MRVP medium and was incubated at 37°C for 24 hours. After incubation, about 5 drops of methyl red reagent was added and mixed well. The positive test was indicated by the development of bright red color, indicating acidity.

Voges Proskauer (VP) test

This test is employed to detect the production of acetyl methyl carbinol (a neutral end product) or its reduction product 2, 3-butanidiol during fermentation of carbohydrates. 

A pure colony of the test organism was inoculated into 2 ml of MRVP medium and was incubated at 37°C for 24 hours. After incubation, about 5 drops of Barritt's reagent was added and shaken well for maximum aeration and kept for 15 minutes, positive test is indicated by the development of pink red colour.

Citrate Utilization test

This test is performed to detect whether an organism utilizes citrate as a sole source of carbon for metabolism with resulting alkalinity. Organisms capable of utilizing citrate as its sole carbon source also utilizes the ammonium salts present in the medium as its sole nitrogen source, the ammonium salts are broken down to ammonia with resulting alkalinity.

A loopful of test organism was streaked on the slant area of Simmon's Citrate Agar medium and incubated at 37°C for 24 hours. A positive test was indicated by the growth of organism and change of media by green to blue, due to alkaline reaction. The pH indicator bromothymol blue has a pH range of 6.0-7.6, i.e. above pH 7.6; a blue color develops due to alkalinity of the medium.

Motility test

The motility media used for motility test are semisolid, making motility interpretations macroscopic. Motile organisms migrate from the stabline and diffuse into the medium causing turbidity. Whereas non-motile bacteria show the growth along the stabline, and the surrounding media remains colorless and clear.

Triple Sugar Iron (TSI) Agar

The TSI agar is used to determine the ability of an organism to utilize specific carbohydrate incorporated in the medium (glucose, sucrose and lactose in concentrations of 0.1%, 1.0% and 1.0% respectively), with or without the production of gas (indicated by cracks in the media as well as an air gap at the bottom of the tube) along with determination of possible hydrogen sulfide production (detected by production of black color in the medium).

The test organism was streaked and stabbed on the surface of TSI and incubated at 37°C for 24 hours. Acid production limited only to the butt region of the tube is indicative of glucose utilization, while acid production in slant and butt indicates sucrose or lactose fermentation. Phenol red is the pH indicator which gives yellow reaction at acidic pH, and red reaction to indicate an alkaline surrounding.

Urea Hydrolysis test

This test demonstrates the urease activity present in certain bacteria which decomposes urea, releasing ammonia and carbon dioxide. Ammonia thus produced changes the color of indicator incorporated in the medium.

The test organism was inoculated in a medium containing urea and the indicator phenol red. The inoculated medium was incubated at 37°C overnight. Positive organism shows pink red color due to the breakdown of urea to ammonia. With the release of ammonia the medium becomes alkaline as shown by a change in colour of the indicator to pink.