Urease Test- Principle, Procedure and Result



To determine the ability of the organism to hydrolyse urea by the action of urease enzyme.


Urea is a nitrogen-containing compound that is produced during decarboxylation of the amino acid arginine in the urea cycle. Urea is a major organic waste product of protein digestion in most vertebrate and is excreted in urine. Some bacteria have the ability to produce an enzyme urease as part of its metabolism to break down urea. The urease is a hydrolytic enzyme which attacks the carbon and nitrogen bond with the liberation of ammonia and carbon dioxide. It is a useful diagnostic test for identifying bacteria, especially to distinguish members of the genus Proteus from Gram-negative pathogens. Proteus vulgaris is an important and fast producer of urease.

Urease test is performed by growing test organism on urea agar slant or urea broth with phenol red as an indicator with pH6.8. During the incubation period, the organism capable of producing urease enzyme hydrolyse urea and produce ammonia that raises the pH level. As the pH increases, the phenol red changes from yellowish orange as the initial colour of medium to deep pink. Failure of development of a pink colouration due to no ammonia production is evidence of an inability of the organism to produce urease enzyme.


Enzymatic digest of gelatin (1 g), dextrose (1 g), NaCl (5 g), KH2PO4 (2 g), urea (20 g), phenol red (0.012 g), per 1000 mL, pH 6.8.


  1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate test organism on urea broth containing phenol red as the indicator.
  2. Incubate the urea agar slant or urea broth at 37°C for 24-48 hours.
  3. Examine the development of pink colour.
  4. In case of unknown result incubate the tube for 7 days to check for slow urease production.


Positive: Deep pink colouration, Light Orange, Magenta

Negative: No Color change, Yellowish orange colouration



  1. Some organisms rapidly split urea (Proteus spp, H. pylori), while others react slowly.
  2. Urea Agar should not be used to determine the quantitative rate of urease activity, as organisms vary in their capability and rate of hydrolysis.
  3. Prolonged incubation may cause an alkaline reaction in the medium and give the false positive result.

Quality Control

Positive: Proteus vulgaris (ATCC13315)

Weak positive: Klebsiella pneumoniae (ATCC13883)

Negative: Escherichia coli (ATCC25922)

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