Some bacteria produce urease , an enzyme that hydrolyzes urea , a common metabolic waste product of vertebrates that contains nitrogen and is excreted in the urine. Urease splits urea into ammonia and carbon dioxide, making the two products available for bacterial use. The test for urease production relies on the fact that the ammonia produced upon hydroysis is alkaline.

The test organism is inoculated into a urea broth that contains phenol red, a pH indicator, and has a pH of 6.8. At this pH phenol red is salmon color. However, when the pH rises above 8.1 phenol red turns a cerise (hot pink) color. Organisms that produce urease will turn cerise due to the ammonia produced upon hydrolysis of urea (a positive result). Organisms that are unable to synthesize urease will not produce ammonia and thus will not experience the subsequent rise in pH. Thus a negative test is indicated by the continuance of a salmon color in the urea broth. The urease test is useful for differentiating Salmonella , which is urea negative, from Proteus , which is urea positive.