Urease test: Principle, Procedure, Results and Uses

This post was most recently updated on September 24th, 2019

Medium used for urease test: Any urea medium, agar (Christensen’s Urea Agar) or broth (Stuart’s Urea Broth). Urease test medium can be a sole medium or part of panel like motility indole urease (MIU) test.
Indicator used in urease test: Phenol red

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.Principle-of-Urease-Test

Composition of Christensen’s Urea Agar

Ingredients Grams/liter
Peptone 1 g
Dextrose 1 g
Sodium chloride 5 g
Potassium phosphate, monobasic 2 g
Urea 20 g
Phenol red 0.012 g
Agar 15 to 20 g

Preparation of Christensen’s Urea Agar

  1. Dissolve the first six ingredients in 100 ml of distilled water and filter sterilize (0.45 mm pore size).
  2. Suspend the agar in 900 ml of distilled water, boil to dissolve completely, and autoclave at 121°C and 15 psi for 15 minutes
  3. Cool the agar to 50-55 °C
  4. Aseptically add 100 ml of filter-sterilized urea based to the cooled agar solution and mix thoroughly
  5. Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes during cooling until solidified (It is desirable to have a long slant and short butt).

Composition and preparation of Stuart’s Urea Broth

Ingredients Grams/liter
Yeast extract 0.1 g
Potassium phosphate, monobasic 9.1 g
Potassium phosphate, dibasic 9.5 g
Urea 20 g
Phenol red 0.01 g
  • Dissolve all ingredients in 1 liter of distilled water and filter sterilize (0.45-mm pore size).
  • Distribute 3 ml of prepared broth per sterile tube (13X100 mm).

Note: Prepared media (both Christensen’s Urea Agar and Stuart’s Urea Broth) will have a yellow-orange color. Once prepared, do not reheat the medium as the urea will decompose. Prepared media can be stored in the refrigerator at 4 to 8 °C until needed.

Procedure for urease test

For Christensen’s Urea Agar
  1. Streak the entire slant surface with a heavy inoculum from an 18-24 hour pure culture (Do not stab the butt as it will serve as a color control).
  2. Incubate tubes with loosened caps at 35°C.
  3. Observe the slant for a color change at 6 hours and 24 hours unless specified for longer incubation.
For Stuart’s Urea Broth
  1. Inoculate the broth with a heavy inoculum from an 18-24 hour pure culture
  2. Shake the tube gently to suspend the bacteria
  3. Incubate the tubes with loosened caps at 35°C.
  4. Observe the broth for a color change at 8, 12, 24 hours.

Result and Interpretation 

Organisms that hydrolyze urea rapidly (Proteus spp., Morganella morganii, and some Providencia stuartii strains) will produce strong positive reactions within 1 or 6 hours of incubation;  delayed positive organisms (e.g. Klebsiella spp and Enterobacter species ) will produce weak positive reactions in the slant in 6 hours of incubation which will be intense during further incubation. The culture medium will remain a yellowish color if the organism is urease negative e.g. Escherichia coli. 

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).
Urea agar test (a) uninoculated, (b) Proteus mirabilis (rapidly urease positive), (c) Klebsiella pneumoniae (delayed urease positive), (d) Escherichia coli (urease negative). (Benita A. Brink, Adams State College, Alamosa, CO)

If Stuart’s Urea Broth is used; rapidly urease positive organisms (Proteus spp., Morganella morganii) will produce a strong positive reactions within 8-24 hours of incubation but delayed positive organisms (e.g., Enterobacter) will not produce a positive reaction due to high buffering capacity of this medium.

urease test
Urease Test Results

Diagnostic utility of Urease test

  1. Urease test helps for the identification of Proteus species (urease positive) and to differenitate it from other non-lactose fermenting members of the Enterobacteriaceae family.
  2. 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.
  3. 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.
  4. 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. Proteeae (Proteus spp., Morganella morganii, and some Providencia stuartii strains)
  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

References and further reading 

  1. Benita Brink. 2010. Urease test protocol. American Society for Microbiology
About tankeshwar 390 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion, I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

15 Comments

  1. Hi, Thank you for your posts, they have been of great help to me. I am doing a journal review on the antimicrobial effects of Innula britannica against H. pylori. One of the methods tested was the inhibitory effect of I. britannica on urease activity, which was ultimately measured via a spectrophotometer. Which test is this, and is there a post that explains how it works? Thank you!

  2. My wife’s RUT test was done on date 23/10/2017.But it’s colour changed yellow to pink that means +be. What are the causes? Is she in danger? Pl. Mail me. .

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