To determine the ability of microorganisms to degrade urea by means of the enzyme urease.
Urea is a nitrogen containing compound that is produced during decarboxylation of the amino acid arginine in the urea cycle. Urea is highly soluble in water and is therefore an efficient way for the human body to discharge excess nitrogen. This excess urea is then taken out of the body through the kidneys as a component of urine. Some bacteria have the ability to produce an enzyme urease as part of its metabolism to break down urea to ammonia and carbon dioxide.
While many enteric bacteria have the ability to hydrolyze urea as part of their metabolism, members of the genus Proteus are considered rapid urease producers due their efficiency in carrying out this process. Therefore, this experiment is useful in distinguishing members of Proteus, a urinary tract pathogen, from other enterics based on their ability to rapidly hydrolyze urea. Many enterics can hydrolyze urea but only a few can degrade it rapidly. These are commonly referred as "rapid urease-positive" organisms. Members of the genus Proteus have the ability to hydrolyze urea rapidly.
Rapid Urease Test:
Also known as the CLO test (Campylobacter-like organism test), is a rapid test for diagnosis of Helicobacter pylori. The basis of the test is the ability of H. pylori to secrete the urease enzyme, which catalyzes the conversion of urea to ammonia and bicarbonate.
Urea is waste product excreted in urine by animals. Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia. The urease test is useful in identifying the genera Proteus, Providentia, and Morganella, which liberate this enzyme.
Urease, which is produced by some micro organisms, is an enzyme that is especially helpful in the identification of Proteus vulgaris, although other organisms may produce urease, their action on the substrate urea tends to be slower than that seen with Proteus species. Therefore this test serves to rapidly distinguish members of this genus from other lactose non fermenting enteric microorganisms.
Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compounds such as urea and forms the alkaline end product ammonia. The presence of urease is detectable when the organisms are grown in a urea broth medium containing the pH indicator phenol red. As the substrate urea is split into its products, the presence of ammonia creates an alkaline environment that causes the phenol red to turn to deep pink. This is a positive reaction for the presence of urease. Failure of deep pink color to develop is evidence of a negative reaction.
Urea is unstable and is broken down at 15 psi or pressure. It cannot be added to the medium for autoclaving and is therefore filter sterilized and added to the medium after autoclaving.
Examine the urea slants inoculated with Proteus vulgaris and compare them to the unknown. If your unknown is negative, continue to incubate the slant for 7 days to check for slow urease production.
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Amrita CREATE (Center for Research in Advanced Technologies for Education),
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Amrita University, India 2009 - 2015
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