Urea hydrolyzation Test: Principle, Procedure, Interpretation, Urease Positive Organisms, Limitations and Keynotes

Introduction of Urea Test / Urea hydrolization Test / Urease Test

Urea Test or Urea hydrolyzation Test or Urease Test are other names of the same test. The urease test is important for certain bacteria and fungi that possess this enzyme that hydrolyzes urea releasing ammonia into the medium. This produces a change in the pH of the medium that can be detected by the color change in the indicator dye. This test can be used to differentiate different groups of bacteria and fungi.

Principle of Urea hydrolyzation Test

This test is used to determine the ability of an organism to produce the enzyme urease, which hydrolyzes urea. Hydrolysis of urea produces two molecules of ammonia and CO₂. The ammonia combines with CO₂ and water to form ammonium carbonate which turns medium alkaline, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta at pH 8.4

Test Requirements for Urea hydrolyzation Test

Following are the requirements for urea hydrolyzation test-

• Christensen’s urea agar
• Test organism
• Inoculated wire/ wooden stick
• Bunsen burner
• Incubator
• Test tube rack

Test Procedure of Urea hydrolyzation 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 37° C in ambient air for 18-24  hours.
3. Observe any change of color in the inoculated medium.

Quality Control

• Positive control (PC): Proteus mirabilis (Urease positive bacteria)
• Negative Control (NC): Escherichia coli (Urease negative bacteria)
• Uninoculated (UN): An uninoculated medium is incubated along with the test to compare the color change.

Observation

Examine the medium after 4 hours and after overnight incubation. The test should not be considered negative till after 4 days of incubation.

Result and Interpretation of Urea hydrolyzation Test

• Organisms that hydrolyze urea rapidly (e.g. Proteus species) may produce positive reactions within 1 or 2 hours; less active species (e.g. Klebsiella species) may require up to 4 days. In routine diagnostic laboratories, the urease test result is read within 24 hours.
• If an organism produces a urease enzyme, the color of the slant changes from light orange to magenta/ bright pink/ red.
• If an organism does not produce urease, the agar slant and butt remain light orange (medium retains original color).

Diagnostic Utility of Urease Test

A. 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 the urea broth. A positive test is considered the presence of Helicobacter pylori. Commercially available urease agar kits are also available.

B. Rapid Urease tests can be used to differentiate between the yeasts i.e. Candida albicans and Cryptococcus neoformans. Presumptive identification of neoformans may be based on rapid urease production, whereas Candida albicans do not.

C. Urea breath test: A common noninvasive test to detect Helicobacter pylori also based on urease activity. This is a highly sensitive and specific test.

Principle of Urea Breath Test

The patient ingests radioactively labeled (13C or 14C) Urea in a capsule form. If the infection is present, the urease produced by Helicobacter pylori hydrolyzes the urea to form ammonia and labeled bicarbonate that is exhaled as CO₂. The labeledCO₂ is detected either by a scintillation counter or a special spectrometer for more details  go with  this link:

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, Trichophytonmentagrophyte, Brucella, Helicobacter pylori.

Urease producing bacteria

Strong or most rapid urease producers-

• Brucella species
• Helicobacter pylori

Rapid producers

• Proteus species
• Morgenella species

Slow  producers

• Klebsiella species
• Enterobacter species

 Urease producing fungi

• Cryptococcus neoformans
• Trichophyton  mentagrophytes

Keynotes on Urea hydrolyzation Test

1. The test will help differentiate among gram-negative rods that grow well on MacConkey agar (MAC) and are likely members of the Enterobacteriaceae.
2. Urease is positive, oxidase-positive, gram-negative coccobacilli that do not grow on MAC in 24 hours are presumptively identified as Brucella unless they are isolated from urine. In this condition, immediately transfer culture to a biosafety cabinet because an organism comes under risk group third.
3. Urease-positive, oxidase-positive, gram-negative coccobacilli that are isolated from the urinary tract may be Oligella ureolytica.
4.  It helps to identify further any significant numbers of urea-positive corynebacteria from respiratory or urine specimens.
5. Urea-positive, oxidase-positive, curved rods from gastric specimens are identified as Helicobacter pylori.

Limitations of Urea Test

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

Further Readings

1. Jean F. Mac Faddin Biochemical tests for Identification of Medical bacteria
2. Bailey’s and Scott’s Diagnostic Microbiology-13th Edition
3. Mackie and McCartney Practical Medical Microbiology-14th Edition
4. ASM Clinical Microbiology Procedures Handbook-2^nd Edition
5.  Monica Cheesbrough Distinct Laboratory Practice in Tropical Countries…2^nd Edition