General Microbiology Laboratory Biochemical tests Citrate Utilization test

General Microbiology Laboratory Biochemical tests

Citrate Utilization test v Determine ability of an organism to use citrate as sole carbon source. v Simmon's citrate agar: sodium citrate as sole carbon source and ammonium ion as the sole nitrogen source. v Bromthymol blue (p. H indicator), turns from green at neutral p. H (6. 9) to blue at p. H higher than 7. 6 (basic or alkaline). v Organisms that metabolize citrate utilize the ammonium salts releasing ammonia and increasing the p. H of the medium. Bromthymol blue is present in the medium as the indicator dye. Mohammed Laqqan

v How to Perform Test: Inoculate slant with inoculating loop. v 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. v Media and Reagents Used: Simmon’s Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & p. H indicator—bromthymol blue. v 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) v 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. v 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. v 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 v Inoculate Tryptone broth or SIM media with inoculating loop. v Property it tests for: Ø This test is performed to help differentiate species of the family Enterobacteriaceae. Ø It tests for the bacteria species’ ability to produce indole. Ø Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one. v Media and Reagents Used: Ø Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color. v Reading Results: Ø 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