Fish Microbiology and Quality Assurance

4. Citrate utilization test

The test is used to determine the ability of bacteria to utilize citrate as a sole source of carbon and energy, and ammonium as a sole source of nitrogen. In the absence of fermentable glucose or lactose, some microorganisms use citrate as carbon source. Pyruvic acid and carbon dioxide formed during the process of citrate utilization turns the medium alkaline. The increase in pH is due to the reaction of carbon dioxide with sodium resulting in alkaline sodium carbonate. Under alkaline condition the bromothymol blue indicator in the medium changes to green to deep Prussian blue colour.

Procedure:

The Simmon’s citrate agar slant is inoculated with the test organism and incubated at 37⁰C for 24- 48 h. The positive reaction is indicated by blue colour (alkaline) and the negative result by yellowish green colour (acidic). If it is negative, there will be no growth in the slant itself.

Simmon’s citrate medium or Citrate agar

Sodium chloride : 5.0 g
Magnesium sulphate : 0.2 g
Ammonium dihydrogen phosphate: 1.0 g
Dipotassium hydrogen phosphate: 1.0 g
Sodium citrate : 2.0 g
Agar : 15.0 g
Distilled water : 1000.0 ml
Bromothymol blue : 0.1 g
pH : 7.0 ± 0.2

Dissolve all the ingredients by boiling, distribute to test tubes and stterilize at 121⁰C for 15 minutes. Place the tubes in sloped condition to make slants. The agar slant should be in green colour.