Urease Test- Principle, Media, Procedure and Results

 

Purpose

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

Principle

Christensen in 1946 first developed Urea agar for the differentiation of enteric bacilli. All amides are easily hydrolyzed with the release of ammonia and carbon dioxide. Urease is an enzyme possessed by many species of microorganisms that can hydrolyze urea. The ammonia reacts in solution to form ammonium carbonate ((NH4)2CO3), resulting in alkalinisation and an increase in the pH of the medium. Two units of ammonia are formed with resulting alkalinity in the presence of enzyme and the increased pH is detected by a pH indicator.  The test organisms are cultured in Christensen’s Urea broth/agar.  The medium contains the pH indicator phenol red which under acid conditions (pH 6.8) is yellow.  In alkaline condition (pH 8.4) the indicator turns the medium pink.

Media and Reagents

Christensen’s urea agar is the two media most commonly used in clinical laboratories for the detection of urease activity.

Composition
Urea	20 gm
Sodium chloride	5 gm
Monopotassium phosphate	2 g
Peptone	1 g
Glucose	1 g
Phenol red	0.012 g
Distilled water	1 L
Agar	15 g
Final pH	6.8

Quality Control

Positively and negatively reacting control organisms should be run with each new batch of medium. The following organisms are suggested:

 Positive control: Proteus species

Positive control (weak): Klebsiella species

Negative control: Escherichia coli

Test Procedure

Inoculate Christensen’s urea agar slant with a colony of the test organism over the surface of the agar slant is streaked with loop.

Incubate inoculated slope at 37 °C for 18–24 hours

Examine the medium after overnight and look for any change in colour.

Results and Interpretation

Positive: Red- pink (Urease produced)

Negative:  No red – pink medium (No Urease produced)

Organisms that hydrolyze urea rapidly may produce positive reactions within 1 or 2 hours; less active species may require 3 or more days. The reactions are as follows:

Rapid urea hydrolyzers (Proteus species): red color throughout medium

Slow urea hydrolyzers (Klebsiella species): red color initially in slant only, gradually converting the entire tube

No urea hydrolysis: medium remains original yellow color.

Urease Test

Reference

Koneman’s Color Atlas and Text book of Diagnistic Microbiology.

Bailey and Scott’s Diagnostic Microbiology.

Mackie and McCartney Practical Medical Microbiology.

BBL Manual of Products and Laboratory Procedures. 5th Ed. Cockeysville, MD: BioQuest, 1968:154.

Christensen WB. Urea decomposition as a means of differentiating Proteus and paracolon cultures fromeach other and from Salmonella and Shigella types. J Bacteriol 1946; 52:461–466.

MacFaddin JF. Biochemical Tests for Identification of Medical Bacteria. 2nd Ed. Baltimore, MD: Williams & Wilkins, 1980:298–308.

Stuart CA, Van Stratum E, Rustigian R. Further studies on urease production by Proteus and related organisms. J Bacteriol 1945;49:437–444.