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Urease Test- Principle, Media, Procedure and Result

Last updated: August 10, 2022 by Sagar Aryal

Urea Agar was developed by Christensen in 1946 for the differentiation of

enteric bacilli. The urease test is used to determine the ability of an organism to
split urea, through the production of the enzyme urease.

Principle of Urease Test

Urea is the product of decarboxylation of amino acids. Hydrolysis of urea
produces ammonia and CO2. The formation of ammonia alkalinizes the
medium, and the pH shift is detected by the color change of phenol red from
light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive
organisms turn the entire medium pink within 24 hours.

Weakly positive organisms may take several days, and negative organisms
produce no color change or yellow as a result of acid production.

Rapid Urease Test (RUT)

The rapid urease test (RUT) is a popular diagnostic test for diagnosis of
Helicobacter pylori. It is a rapid, cheap and simple test that detects the
presence of urease in or on the gastric mucosa. It is also known as the CLO test
(Campylobacter-like organism test). This test uses a procedure called gastric
endoscopy and biopsy to collect stomach lining cells.
The test is performed at the time of gastroscopy. A biopsy of mucosa is taken
from the antrum of the stomach, and is placed into a medium containing urea
and an indicator such as phenol red. The urease produced by H. pylori
hydrolyzes urea to ammonia, which raises the pH of the medium, and changes
the color of the specimen from yellow (NEGATIVE) to red (POSITIVE).

The test can also be used to provide an informal assessment of the accuracy of
the histopathology result and discrepancies should prompt a review of the
histopathology and discussions with the pathologist.

Urea Breath Test

Urea breath test is a common non-invasive test to detect Helicobacter pylori
also based on urease activity. This is highly sensitive and specific test. 
:
Patient ingests radioactively labeled Urea (either radioactive carbon-14 or
non-radioactive carbon-13). 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 (Carbon-14) and a isotope ratio mass spectrometry or by
mass correlation spectrometry (Carbon-13).

Uses of Urease Test

1. This test is used to differentiate organisms based on their ability to
hydrolyze urea with the enzyme urease.
2. This test can be used as part of the identification of several genera and
species of Enterobacteriaceae, including Proteus, Klebsiella, and some
Yersinia and Citrobacter species, as well as some
Corynebacterium species.
3. It is also useful to identify Cryptococcus spp., Brucella, Helicobacter
pylori, and many other bacteria that produce the urease enzyme.
4. Directly, this test is performed on gastric biopsy samples to detect the
presence of H. pylori.

Media used in Urease Test

Christensen’s Urea Agar
:
Composition

Ingredients per liter of deionized water:*

Composition

Urea 20.0 gm

Sodium Chloride 5.0 gm

Monopotassium Phosphate 2.0 gm

Peptone 1.0 gm

Dextrose 1.0 gm

Phenol Red 0.012 gm

Agar 15.0 gm

Final pH 6.7 +/- 0.2 at 25 degrees C.

Preparation

1. Dissolve the 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.
3. Autoclave at 121 degree C and 15 psi for 15 minutes.
4. Cool the agar to 50 to 55 degree C.
5. Aseptically add 100 ml of filter-sterilized urea base to the cooled agar
solution and mix thoroughly.
6. Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes
during cooling until solidified.
:
Procedure of Urease Test
1. Streak the surface of a urea agar slant with a portion of a well-isolated
colony or inoculate slant with 1 to 2 drops from an overnight brain-heart
infusion broth culture.
2. Leave the cap on loosely and incubate the tube at 35°-37°C in ambient
air for 48 hours to 7 days.
3. Examine for the development of a pink color for as long as 7 days.

Result Interpretation of Urease Test

negativeureatest positiveureatest

UreaseTest UreaseTest
Negative Positive

Positive Reaction: Development of an intense magenta to bright pink color in

15 min to 24 h.
Examples: Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicobacter
pylori, Yersinia spp, Brucella spp, etc.

Negative Reaction:  No color change.

:
Examples: Escherichia, Shigella, Salmonella, etc.

Quality Control of Urease Test

Positive: Proteus vulgaris (ATCC13315)
Weak positive: Klebsiella pneumoniae (ATCC13883)
Negative: Escherichia coli (ATCC25922)

Limitations of Urease Test

1. Some organisms rapidly split urea (Brucella and H. pylori), while others
react slowly.
2. It is recommended that biochemical and/or serological tests be
performed on colonies from pure culture for complete identification.
3. To facilitate growth and the urea hydrolysis reaction, do not use inoculum
from a broth suspension.
4. After prolonged incubation times a false-positive alkaline reaction may be
seen. To rule out this occurrence, check the test with a control (an
uninoculated tube of Urea Agar) along with the inoculated tube during
prolonged incubation.
5. Do not heat the Urea Agar Slants, as urea decomposes very readily when
heated.
6. To detect Proteus species, the Urea Agar, Slants must be examined within
6 hours of inoculation for a reaction.
7. Urea Agar should not be used to determine the quantitative rate of urease
activity, as organisms vary in their capability and rate of hydrolysis.
8. Failure to incubate this medium with loose caps may cause erroneous
results to occur.
9. Urea is light sensitive and can undergo autohydrolysis. Store at 2 to 8 C
in the dark.

References
:
 1. ASM Microbe Library: Urease Test Protocol
2. Bailey & Scott’s Diagnostic Microbiology 13th Edition: Urease Test
(Christensen’s Method)
3. Clinical Microbiology Procedures Handbook 2nd Edition: Urease Test
4. UK Standards for Microbiology Investigations: Urease Test
5. Virtual Labs at Amrita Vishwa Vidyapeetham
6. Takahiro Uotani and David Y. Graham: Diagnosis of Helicobacter pylori
using the rapid urease test
7. Bermejo F, et al.: Rapid urease test utility for Helicobacter pylori infection
diagnosis in gastric ulcer disease.
8. Austin Community College
9. Allina Health
10. MedicineNet, Inc
11. Microbe Online
12. Hardy Diagnostics
13. The Centre for Digestive Diseases
14. Wikipedia

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1 thought on “Urease Test- Principle, Media, Procedure and Result”

Wilhelmina.Debbie
March 6, 2020 at 9:06 PM

Carefully observe the winners, don’t always pay attention to the losers

Reply

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