Introduction of Urease Test / Urea hydrolization Test / Urea Test
The urease test is important for certain bacteria and fungi that possess the enzyme urease 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 Urease 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 CO2and 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
Requirements for Urease Test
Following are the requirements for urea hydrolyzation test-
Christensen’s urea agar
Inoculated wire/ wooden stick
Test tube rack
Procedure of Urease Test
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
Leave the cap on loosely and incubate the tube at 37° C in ambient air for 18-24 hours.
Observe any change of color in the inoculated medium.
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.
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
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 urease test is considered the presence of Helicobacter pylori. Commercially available urease agar kits are also available.
B. Rapid Urease testis 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.
The test will help differentiate among gram-negative rods that grow well on MacConkey agar (MAC) and are likely members of the Enterobacteriaceae.
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.
Urease-positive, oxidase-positive, gram-negative coccobacilli that are isolated from the urinary tract may be Oligella ureolytica.
It helps to identify further any significant numbers of urea-positive corynebacteria from respiratory or urine specimens.
Urea-positive, oxidase-positive, curved rods from gastric specimens are identified as Helicobacter pylori.
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.
Jean F. Mac Faddin Biochemical tests for Identification of Medical bacteria
Bailey’s and Scott’s Diagnostic Microbiology-13th Edition
Mackie and McCartney Practical Medical Microbiology-14th Edition