Urease Test

Theoretical Background / Context

• Urea is a waste product excreted in urine by animals. 
• Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia through the urease reaction.
• The microbiology urease test is useful in identifying the genera Proteus, Providentia, and Morganella, which liberate this enzyme.

• Urease, which is produced by some microorganisms, 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 urease 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 a deep pink. 
• This is a positive reaction for the presence of urease in the urease test.
• Failure of the 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.

Principle of Work

• Urea is a nitrogen-containing compound that is produced during the 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 can produce an enzyme urease as part of their metabolism to break down urea into ammonia and carbon dioxide through the urea hydrolysis test.  
• 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 to their efficiency in carrying out this process in the urease enzyme activity experiment.
• 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 through the urease enzyme assay.
• Many enterics can hydrolyze urea, but only a few can degrade it rapidly.
• These are commonly referred to as "rapid urease-positive" organisms. Members of the genus Proteus have the ability to hydrolyze urea rapidly.