Some bacteria split the urea molecule in two, releasing carbon dioxide and ammonia. This reaction, mediated by the enzyme urease, can be seen in the culture medium prepared with urea added as the substrate. Phenol red is also added as a pH indicator. When bacterial cells that produce urease are grown in this medium, urea is degraded, ammonia is released, and the pH become alkaline. This pH shift is detected by a change in the indicator color from orange-red to dark pink. PROCEDURE Step 1. Inoculate an isolated colony from your culture into one tube of Urease Broth. Step 2. Incubate tubes at 37C for 24 hours. Results. A positive test is observed by the production of a Pink (alkaline pH) phenol red end product. porast Urease test This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other entoric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid hydrolyzer of urea (center tube pictured here). The tube on the far right was inoculated with a reaso negative organism and the tube on the far left was uninoculated Urease 1. What is the enzyme we are testing for? Urease 2. What is the pH indicator? -phenol red 3. Give one example of an organism that would be positive and one that would be negative.

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