Urease Test: Introduction, Principle, Procedure, Result-Interpretation, Urease Positive Organisms List, Limitations, and Keynotes

Introduction

The urease test is also known as the urea hydrolyzation test or urea test and it is important for certain bacteria and fungi that possess this enzyme which 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 assay can be used to differentiate different groups of bacteria and fungi.

Principle

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

Test Requirements

The following are the requirements for the urea hydrolyzation test-

• Christensen’s urea agar (test medium)
• Test organism
• Inoculating wire/ wooden stick
• Bunsen burner
• Incubator
• Test tube rack

Test Procedure

1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate the slant with 1 to 2 drops from an overnight brain heart infusion broth culture
2. Leave the cap or cotton plug 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.
4. Follow the above steps for control strains too excluding uninoculated medium which needs only incubation together.

Quality Control

• Positive control (PC): Proteus mirabilis (Urease positive)
• Negative Control (NC): Escherichia coli (Urease negative)
• 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

• 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).

Urease Producing Microbes List

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

1. Urease test will help differentiate among gram-negative rods that grow well on MacConkey agar (MAC) and are likely members of the Enterobacteriaceae.
2. Urease-positive, oxidase-positive, and 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 the culture to a biosafety cabinet (BSC) since this organism comes under the risk group third.
3. Urease-positive, oxidase-positive, and 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 hydrolyzation test-positive, oxidase-positive, curved rods from gastric specimens is identified as Helicobacter pylori.

Limitations

• Some organisms rapidly split urea (Brucella and H. pylori), while others react slowly.
• Urea is light sensitive and can undergo autohydrolysis and therefore 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 low sensitivity if the medium is not buffered.

Further Readings

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