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.

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

Result Mohammed Laqqan E. Coli: only citrate negative Klebsilla: only non motile. Citrobacter: only H2S positive Protus: only ureas Positive Salmonella: H2S positive and Motile. Mohammed Laqqan

Urease Test Some bacteria are able to produce an enzyme called urease that attacks the nitrogen and carbon bond in amide compounds such as urea, forming the end products ammonia, CO2, and water. Urease activity (the urease test) is detected by growing bacteria in medium containing urea and using a pH indicator such as phenol red. When urea is hydrolyzed, ammonia accumulates in the medium and makes it alkaline. This increase in pH causes the indicator to change from orange-red to deep pink and is a positive test for urea hydrolysis. This test is particularly useful in distinguishing the genus Proteus from other enteric bacteria

How to Perform Test: Inoculate Urea broth or urea slant agar with inoculating loop. Property it tests for: This test is done to determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease. Reading Results: Urea slant is a yellow color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.

Limitations of the procedure Some bacteria have a delayed urease reaction that may require an incubation period longer than 48 hours.

Urease-Producing Enterobacteriaceae Proteus Klebsiella pneumoniae Enterobacter cloacae Yersinia enterocolitica

End of lecture