Urease test

 Aim

To determine the abilty of microorganisms to degrade urea by the production of the enzyme urease.

Principle

Urea is a major organic waste product of protein digestion in most vertebrates and is excreted in the urine.  Some microorganisms have the ability to produce the enzyme urease.  The urease is ahydrolytic enzyme which attacks the carbon and nitrogen bond amide compounds (eg: urea) with the liberation of ammonia as shown below:

Urea + 2H₂O      urease        Carbondioxide + water + ammonia

Urease test is performed by growing the test organisms on urea agar medium containing the pH indicator phenol red (pH 6.8).  During incubation, microorganisms possessing urease will produce ammonia that raises the pH of the medium.  As the pH becomes higher, the phenol red changes from a yellow color to a red or deep pink color.  Failure of the development of  a deep pink color due to lack of ammonia production is evidence of the absence of urease production by the microorganisms.

Requirements

24 hr nutrient agar cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Urea broth, Bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1.  Using sterile techniques inoculate each organism into appropriately labeled tubes by means of streak       inoculation.  One uninoculated tube kept as control.

2.  Incubate all inoculated tubes at 37^o C for 24-48 hrs.

 

Observations

Tubes inoculated with Staphylococcus showed color change of the medium from yellow to red indicating a positive result.  Tubes inoculated with  Streptococcus, Pseudomonas, Bacillus and E.coli  maintained the yellow color of the medium, indicating a negative result.

 Result 

Staphylococcus is urease positive where as Streptococcus,Pseudomonas, Bacillus and E.coli, are urease negative.