Biochemical tests

Citrate Utilization test Determine ability of an organism to use citrate as sole carbon source. Simmon's citrate agar: sodium citrate as sole carbon source and ammonium ion as the sole nitrogen source. Bromthymol blue (pH indicator), turns from green at neutral pH (6.9) to blue at pH higher than 7.6 (basic or alkaline). Organisms that metabolize citrate utilize the ammonium salts releasing ammonia and increasing the pH of the medium . Bromthymol blue is present in the medium as the indicator dye. Mohammed Laqqan

How to Perform Test: Inoculate slant with inoculating loop. Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon. Media and Reagents Used: Simmon’s Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator—bromthymol blue. Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an alkaline end product) and a – result remains green Mohammed Laqqan

Result Mohammed Laqqan

Tryptophan hydrolysis (Indole Production) The ability to degrade amino acids to identifiable end products is often used to differentiate among bacteria. Tryptophan, for example, is hydrolyzed to indole, pyruvic acid and ammonia by tryptophanase. The pyruvic acid can be further metabolized to produce large amounts of energy. The ammonia is available for use in synthesis of new amino acids. Indole can be detected by reaction with Kovac's reagent (para-dimethylaminobenzaldehyde in alcohol) to produce a red color. Mohammed Laqqan

How to Perform Test Property it tests for: Media and Reagents Used: Inoculate Tryptone broth or SIM media with inoculating loop. Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species’ ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one. Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color. Reading Results: After incubating the bacteria for at least 48 hours, Kovac’s reagent is added to the media to detect if indole has been made by the bacteria. The development of a red/pink layer (RED RING) on top of the media is a positive result (the bacteria can breakdown tryptophan to form indole). Failure to see a red layer is a negative result (indole was not formed from tryptophan). Mohammed Laqqan

Result Mohammed Laqqan

End of lecture Mohammed Laqqan