Urease Test Principle and Procedure

In this urease test principle and procedure post we have briefly explained urease test principle, objectives, requirements, urease test in microbiology procedure, uses and limitations.

Urease Test Principle and Procedure

Urease test in microbiology is a biochemical test that detects bacteria producing ammonia as a result of alkaline fermentation of urea. The urease test in microbiology determines which organisms can hydrolyze urea and create ammonia and carbon dioxide.

Urease activity is one of the most essential traits for distinguishing Proteus species from non-lactose-fermenting Enterobacteriaceae members. To identify urease activity in a range of bacteria, Christensen’s urea agar is utilised. Stuart’s urea broth is primarily used to distinguish Proteus species.

Urease production is detected on Christensen’s urea agar by a vivid pink hue on the slant that may extend into the butt after 1-6 hours of incubation. If the organism is urease negative, the culture media will remain yellowish. Urease production is indicated by a vivid pink color throughout Stuart’s urea broth.


The urease test in microbiology determines which organisms can hydrolyze urea and create ammonia and carbon dioxide. It’s mostly utilized to tell the difference between urease-positive Proteeae and other Enterobacteriaceae.


Urea is a nitrogen-containing chemical created through the urea cycle’s decarboxylation of the amino acid arginine. Because urea is very soluble in water, it is an effective mechanism for the human body to expel excess nitrogen.

The excess urea is subsequently excreted from the body as a component of urine via the kidneys. As part of their metabolism, some bacteria can create the enzyme urease, which breaks down urea into ammonia and carbon dioxide.

While many enteric bacteria may hydrolyze urea as part of their metabolism, representatives of the genus Proteus are known as fast urease producers because of their effectiveness at doing so. As a result, based on their ability to rapidly hydrolyze urea, this experiment is beneficial in identifying individuals of Proteus, a urinary tract infection, from other enterics.

Although many intestinal bacteria can hydrolyze urea, only a few can breakdown it quickly. These organisms are usually referred to as “rapid urease-positive.” Proteus genus members have the ability to rapidly hydrolyze urea.



Urease generation can be detected using both urea agar slants and broth media. The Urea Agar Base (Christensen agar) is included in the agar media, and the urea broth is included in the broth.


1.0 gm

Peptic digest of animal tissue

1.5 gm

Sodium chloride

5.0 gm

Monopotassium phosphate

2.0 gm

Phenol red

0.012 gm


15.0 gm



1. Filter sterilizes the ingredients after dissolving them in 100 mL of distilled water (0.45-mm pore size). Boil the agar in 900 mL of distilled water to completely dissolve it.

2. Autoclave for 15 minutes at 121°C and 15 psi. Reduce the temperature of the agar to 50 to 55 degrees Celsius. To the chilled agar solution, add 100 mL of filter-sterilized urea base and carefully mix.

3. Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes until solidified during cooling.


1. Inoculate a urea agar slant with 1 to 2 drops from an overnight brain-heart infusion broth culture or stain the surface with a piece of a well-isolated colony.

2. Incubate the tube at 35°-37°C in ambient air for 48 hours to 7 days with the cap loosely on. For up to 7 days, keep an eye out for the development of a pink color in urease test in microbiology.

Urease Test

Urease Test in Microbiology


Proteus spp., Cryptococcus spp., Corynebacterium spp., Helicobacter pylori, etc., produce a vivid magenta to bright pink color in 15 minutes to 24 hours, indicating a positive test.

There is no color shift in a negative test in urease test in microbiology. Escherichia coli, Shigella sonnei, Salmonella sonnei, etc.


1. Some organisms should produce quick results since they breakdown urea quickly, while others may take longer.

2. The alkaline reaction observed in overnight testing using a medium containing peptone could be attributable to peptone hydrolysis rather than urease.

3. Urea is photosensitive and may be auto-hydrolyzed. As a result, the urease test in microbiology medium must be kept between 2 to 8°C in the dark. If the medium is not buffered, the urease test in microbiology is less sensitive.


1. The urease test in microbiology is used to discover organisms that can hydrolyze urea and create ammonia and carbon dioxide.

2. The urease test in microbiology is particularly useful for determining whether a Proteus species is related to another member of the Enterobacteriaceae family.

3. The urease test in microbiology also distinguishes Proteus from bacteria that do not digest lactose. To detect the presence of H. pylori, this test is performed as a fast test on gastric biopsy samples.

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