Urease Test: Principle, Procedure, Results

Last updated on June 21st, 2021

Urease is a constitutively expressed enzyme that hydrolyzes urea to carbon dioxide and ammonia. Many organisms especially those that infect the urinary tract, have a urease enzyme that is able to split urea in the presence of water to release ammonia and carbon dioxide.

Urease test principle

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.

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 a panel like motility indole urease (MIU) test. The composition and preparation of these media are given at the end of this blog post.

Microorganisms Tested

  • A. The urea test is part of the battery of tests to identify the following.
    • Gram-negative enteric pathogens, including Yersinia spp.
    • Fastidious Gram-negative rods—Brucella, H. pylori, and Pasteurella
    • Gram-positive rods—Corynebacterium and Rhodococcus spp.
    • Yeasts—Cryptococcus spp.
  • B. Directly, this test is performed on gastric biopsy samples to detect the presence of H. pylori

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.
Urea broth test
Urease test using urea broth.
Left: Positive, Right: Negative
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 does not produce urease the agar slant and butt remain light orange (medium retains original color).
Urease test
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 reaction 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 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.
  2. Urease test helps for the identification of Proteus species (urease positive) and to differentiate it from other non-lactose fermenting members of the Enterobacteriaceae family.
  3. 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 the presence of Helicobacter pylori. Commercially available urease agar kits are also available.
  4. Rapid urease test is can be used to differentiate between the yeasts, Candida albicans, and Cryptococcus neoformans. Presumptive identification of C. neoformans may be based on rapid urease production, whereas Candida albicans do not.
  5. Urea breath test: A common noninvasive test to detect Helicobacter pylori also based on urease activity. This is a highly sensitive and specific test.

Name of urease positive organisms

  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

Limitations

  • Some organisms rapidly split urea (Brucella and H. pylori), while others react slowly.
  • When performing overnight tests from medium that contains peptone, the alkaline reaction may be due not to urease but to hydrolysis of peptone.
  • Urea is light sensitive and can undergo autohydrolysis. Store at 2 to 8°C in the dark.
  • The test is less sensitive if the medium is not buffered.

Composition of Christensen’s urea agar

IngredientsGrams/liter
Peptone1 g
Dextrose1 g
Sodium chloride5 g
Potassium phosphate, monobasic2 g
Urea20 g
Phenol red0.012 g
Agar15 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 base 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

IngredientsGrams/liter
Yeast extract0.1 g
Potassium phosphate, monobasic9.1 g
Potassium phosphate, dibasic9.5 g
Urea20 g
Phenol red0.01 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).

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.

References and further reading 

  1. Benita Brink. 2010. Urease test protocol. American Society for Microbiology
  2. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814

About Acharya Tankeshwar 474 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.