Fish Microbiology and Quality Assurance

5. Urease test

This test is used to determine the ability of microorganisms to degrade urea in to ammonia and carbon dioxide by means of the enzyme, urease. This hydrolytic enzyme attacks nitrogen and carbon bond in amide compounds such as urea and forms the alkaline end product ammonia which can be detected by change in pH.

Procedure
The Christencen’s medium or urea agar slants containing the pH indicator phenol red is inoculated with young culture of test organism, incubated for 24 hr at 37⁰C. A positive reaction is indicated by slants turning purplish pink or red colour due to increase in pH. The uninoculated slants serving as control remain yellow in colour.

Christencen’s urease medium

Peptone : 1.0 g
Sodium chloride : 5.0 g
Glucose : 1.0 g
Potassium dihydrogen phosphate: 2.0 g
Agar : 15.0 g
Distilled water : 900.0 ml
Phenol red : 0.012 g
pH : 6.8 ± 0.1

Heat to dissolve the ingredients and sterilize at 115⁰C for 15 minutes. Mix 100 ml of filter-sterilized urea solution with to the sterile medium. Distribute it in sterile test tubes (approximately 3 ml each) and place the tubes in sloped condition for making slants.

Urea solution

Urea - 20.0 g
Distilled water - 100.0 ml

Dissolve urea in distilled water and filter-sterilize it.