Urease test: Principle, Procedure, interpretation and urease positive organisms

Medium used for urease test: Any urea medium, agar or broth (It can be a sole medium or part of panel like motility indole urease (MIU) test)
Indicator used in urease test: Phenol red
Color change:
  • Original: orange yellow color
  • Final color (in positive test): Bright pink
Urease test principle
Urea is a diamide of carbonic acid.  It is hydrolyzed with the release of ammonia and carbon dioxide.
Many organisms especially those that infect the urinary tract, have an urease enzyme which is able to split urea in the presence of water to release ammonia and carbon dioxide. The ammonia combines with carbon dioxide and water to form ammonium carbonate which turns the medium alkaline, turning the indicator phenol red from its original orange yellow color to bright pink.

Procedure for urease test

  1. The broth medium is inoculated with a loopful of a pure culture of the test organism; the surface of the agar slant is streaked with the test organism.
  2. Leave the cap on loosely and incubate the test tube at 35 °C in ambient air for 18 to 24 hours; unless specified for longer incubation.

Result and Interpretation 

Organisms that hydrolyze urea rapidly (e.g. Proteus spp) may produce positive reactions within 1 or 2 hours; less active species (e.g. Klebsiella spp) may require 3 or more days. In routine diagnostic laboratories the urease test result is read within 24 hours.

  • If organism produces urease enzyme, the color of the slant changes from light orange to magenta.
  • If organism do not produce urease the agar slant and butt remain light orange (medium retains original color).

    Urease Test Results

Diagnostic utility of Urease test

  1. Urease test is used for the presumptive evidence of the presence of Helicobacter pylori in tissue biopsy material. This is done by placing a portion of crushed tissue biopsy material directly into urease broth. A positive urease test is considered presence of Helicobacter pylori. Commercially available urease agar kits are also available.
  2. Rapid Urease test is can be used to differentiate between the yeasts, Candida albicans and Cryptococcus neoformans. A presumptive identification of C. neoformans may be based on rapid urease production, whereas Candida albicans do not.
  3. Urea breath test: A common noninvasive test to detect Helicobacter pylori also based on urease activity. This is highly sensitive and specific test.

Principle of Urea Breath Test:  

Patient ingests radioactively labeled (13C or 14C) Urea. If infection is present, the urease produced by Helicobacter pylori hydrolyzes the urea to form ammonia and labeled bicarbonate that is exhaled as CO2. The labeled CO2 is detected either by a scintillation counter or a special spectrometer.

Urease test is useful test: 
This test can be used as part of the identification of several genera and species of Enterobacteriaceae including Proteus and Klebsiella. It is also useful to identify Cryptococcus species, Brucella, Helicobacter pylori.
Name of urease positive organisms (Bacteria)
  1. Proteus spp
  2. Cryptococcus spp
  3. Corynebacterium spp
  4. Helicobacter pylori
  5. Brucella spp

Note: Mneomonics to remember urease positive organisms: PUNCH (Similar mneomonic for oxidase positive organism is PVNCH)

  • P: Proteus
  • U:Ureaplasma
  • N: Nocardia
  • C: Cryptococcus neoformans/Corynebacterium spp
  • H: Helicobacter pylori