This test is used to assist in the differentiation of Entero- bacteria. Vibrio cholerae, K. pneumoniae and some strains of Enterobacter, ferment sugar, with the formation of ac- etoin which can be detected by chemical methods.

v  Principle:- The test organisms are cultured in a glucose phos- phate peptone water for 48 hours. Acetoin formed is converted to diacetyl. It is converted to a pink com- pound by the action of creatine

v Requirements:-

1)       Glucose phosphate peptone water

2)       40 g/dl sodium hydroxide

 v Procedure:-

1.  Inoculate about 2 ml of sterile  glucose phosphate peptone water with the test organisms.

2.   Incubate at 35°C for 48 hours.

3.  Add small amount (knife point) of creatine powder.

4. Add 3 ml of sodium hydroxidereagent and mix well.

5.  Keep at room temperature (25° ± 5°C) for one hour and observe the color.

 v Observations:-

1.       Pink red color : Positive test

2.       No pink red color: Negative test

 v Quality control:-

Use following microorganisms to confirm the reliability of reagents:

Positive control: Klebsiella aerogenes

Negative control: E. coli