BIOCHEMICAL TESTS - PROCEDURES

CITRATE UTILIZATION:

This test is one of several techniques used to assist in the identification of enterobacteria. The test is based on the ability of an organism to use citrate as its only source of carbon and ammonia as its only source of nitrogen.

PRINCIPLE:

The test organism is cultured in a medium which contains sodium citrate, an ammonium salt, and the indicator bromo-thymol blue. Growth in the medium is shown by turbidity and a change in color of the indicator from light green to blue, due to the alkaline reaction, following citrate utilization.

REQUIREMENTS:

Koser’s citrate medium:

Formula and preparation:

Oxoid dehydrated medium

Grams per litre

Sodium ammonium phosphate                                                       1.5

Potassium dihydrogen phosphate                                                  1.0

Magnesium sulphate                                                                         0.2

Sodium citrate                                                                                   2.5

Bromothymol blue                                                                             0.016

The medium is used at a concentration of 0.52 grams in every 100ml of distilled water.   Prepare the medium as per the instructions in the kit. Distribute in 3ml amounts in small screw cap bottles or tubes. Sterilize by autoclaving (with caps loosened) at 121oC for 15 minutes .When cool, tighten the container tops.

Date the medium and give it a batch number.

QUALITY CONTROL:

PH of medium: This should be within the range pH 6.6-7.0 at room temperature.

Performance: Test the medium by inoculating it with bacterial species of known                                                 positive and negative citrate activity

Storage: Store in a cool dark place.

Shelf life: Up to 2 years providing there is no change in the volume or appearance of the medium to suggest contamination or an alteration of pH.

INOCULATION:

Use a straight wire to inoculate the medium to prevent any carry over of nutrients from the test culture .A light inoculum must be used, and the bottle caps should be loosened during incubation.

METHOD:

1.         Using a sterile straight wire, inoculate 3-4ml of sterile Koser’s citrate medium with a broth culture of the test organism. Care must be taken not to contaminate the medium with carbon particles, such as from a frequently flamed wire.

2.         Incubate the inoculated broth at 35-37oC for up to 4 days, checking daily for growth.

RESULTS:

Turbidity and blue color………….Positive test (Citrate utilized)

No Growth……………………….Negative test (Citrate not utilized)

CONTROLS:

Positive citrate control:                                     Klebsiella pneumoniae.

Negative citrate control:                                   Escherichia coli


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