Urease test | Principle | Protocol | Results

☰ Content :

Ⅰ. Overview

Urease test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria.

Reoch, in 1875, proposed to test the alkaline fermentation of urea and the production of ammonia by bacteria to determine an organism's ability to produce urease, alkalinization causes a purplish-red coloration of the medium in presence of phenol red

- It can be used to detect the presence of Helicobacter pylori

- This test can also be used to differentiate between yeasts : Candida albicans (negative urease) and Cryptococcus neoformans (rapid production of urease).

Ⅱ. Principle

● Hydrolysis of urea by urease , a constitutively expressed enzyme, produces ammonia and CO2. The formation of ammonia alkalizes the medium and the change in pH is detected by the change in color from phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1.


the reaction of the Urease test

Ⅲ. Procedure and results

Two types of media are commonly used to detect urease activity :

1- Solid medium : Christensen Urea Agar Slant

◉ Results : A positive result is indicated by the presence of a bright pink color (fuchsia) in the agar slant. A negative result if no color change in the agar slant

2- Liquid medium : Rustigian Stuart Urea Broth

◉ Results : A positive result is indicated by the presence of a bright pink color (fuchsia) in the broth. A negative result if no color change in the broth

Ⅳ- Helicobacter pylori case

Helicobacter pylori breaks up urea quickly, usually within 30 seconds. Its speed is a key distinguishing factor for H. pylori from other Helicobacter species.

Many special tests and media have been developed for the detection of H. pylori in the clinical laboratory.

1- Rapid urease test

◉ Results : Results can be interpreted in 1 minute up to 3 hours, although the CLO test may be suspended for up to 24 hours in some cases

2- Urea breath test

Ⅴ- Urease positive and negative test

◍ Positive and negative urease bacteria (9, 10, 11)

Bacteria Urease positive Variable urease Negative urease
Enterobacteriaceae Proteus , Klebsiella (lent) / Salmonella, Citrobacter, Escherichia coli, Edwardsiella, Shigella, Providencia, Enterobacter, Hafnia alvei, Serratia marcescens et liquefaciens
Yersinia spp. Y.enterocolitica, Y.pseudotuberculosis / Y.pestis
Vibrionaceae / / Aeromonas, Plesiomonas, Vibrio
Haemophilus influenzae biotypes : I II III IV / biotypes : V VI VII VIII
Bordetella spp. B. parapertussis(24 h), B. bronchiseptica(24 h) / B. pertussis, B. trematum, B. holmesii
Brucella spp. B. suis , B. canis (fast) B. melitensis B. abortus (plus long ou peut être négatif) /
Bartonella spp. / / Bartonella spp.
Helicobacter species H. pylori, H. heilmannii / H. cinaedi, H. fennelliae
Ureaplasma urealyticum Ureaplasma urealyticum / /
Streptococcus spp S. vestibularis (Group Salivarius) / Group Mitis, Group Anginosus, Group Mutans

Ⅵ- Limits and technical tips

- Urea broth is a highly buffered medium requiring large amounts of ammonia to raise the pH above 8.0, resulting in a color change.

- Urea media if exposed to light may develop peroxide, which may interfere with the urease test. Urea is also known to undergo self-hydrolysis; it is therefore advisable to store the media in the refrigerator at 4-8 ° C.


  1. Public health England : Identification of Enterobacteriaceae
  2. Public health England : Urease test
  3. Biorad manuel : UREA INDOLE
  4. American society for microbiology : Urease Test Protocol
  5. ASM MicrobeLibrery
  6. M Peeters: a diagnostic tool in the management of Helicobacter pylori-related gastrointestinal diseases
  7. Senthilkumar Sankararaman; Leila Moosavi : Urea Breath Test
  8. Color Atlas of Medical Bacteriology (ASM Books) 3rd Edition