5 Jan 2012

UREASE TEST


UREASE TEST

Testing for urease enzyme activity is important in differentiating enterobacteria. Proteus strains are strong urease producers. Y. Enterocolitica also shows urease activity (weekly at 35-37C). Salmonellae and shigellae do not produce urease.

PRINCIPLE

The test organism is cultured in a medium which contains urea and the indicator phenol red. If the strain is urease-producing, the enzyme will break down the urea (by hydrolysis) to give ammonia and carbon dioxide. With the release of ammonia, the medium becomes alkaline as shown by a change in color of the indicator to red-pink. The method described is that which uses the combined motility indole urea (MIU) medium.

REQUIREMENTS:

Motility indole urea (MIU) medium:
MIU semi solid medium is used to differentiate enterobacteria species by their motility, urease and indole reactions.
Formula and Preparation
To make 1 litre of MIU base medium
Tryptone………………………………………………………… 30g
(Or pancreatic digest of casein)
Potassium dihydrogen Phosphate……………………………        1g
Sodium Chloride………………………………………………… 5g
Agar……………………………………………………………….4g
Phenol red 2.5g/l (0.25%)………………………………………   2ml
Distilled water……………………………………………………1 liter

            Mix the dry ingredients in the water and heat to 100oC to dissolve the chemicals (place the flask in a container of boiling water).
            Allow to cool to 50-55oC and then add the phenol red solution. Mix well.
            Dispense in 95ml amounts in screw-cap bottles. Sterilize by autoclaving (with caps           loosened) at 121oC for 15 minutes. When the medium has cooled, tighten the bottle    caps.
METHOD:
1.         Using a sterile straight wire, inoculate a tube of sterile MIU medium with a smooth colony of the test organism. When inoculating KIA medium at the same time, inoculate the KIA tube first and then stab the MIU medium.
2.         Place an indole paper strip in the neck of the MIU tube above the medium. Stopper the tube and incubate at 35-37o C overnight.
3.         Examine for urease production by looking for a red-pink color in the medium.

RESULTS:

Red-pink color…………………Positive test(Urease produced)
No red-pink color………………Negative test (No urease produced)


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