v Principle:-

The test organism is cultured in a medium containing sodium citrate, an ammonium salt and a bromothymol blue indicator. The organisms use citrate (the only source of carbon) and ammonia (the only source of nitrogen). The citrate utilization is followed by an alkaline reaction (change of the color from light green to blue) and growth in the medium is indicated by the appearance of turbidity.

    v Requirements

          Test tube (15 x 125 mm),   

             Simmon’s citrate medium, 

             Formula and preparation

 1.      Potassium dihydrogen phosphate                    : 1.0 g

2.      Sodium ammonium phosphate                          : 1.5 g

3.      Magnesium sulfate                                               : 0.2 g

4.      Sodium citrate                                                        : 2.5 g

5.      Bromothymol blue                                                 :0.016 g

6.      Agar                                                                          :15.0g

7.      Distilled water                                                           1000ml

The ingredients 1 to 6 are dissolved in about 900 ml of distilled water and after diluting to one liter, pH of this medium is adjusted to 6.7-6.9.

    v Procedure

           1.      Inoculate 3-4 ml of sterile Simmon’s citrate medium with a broth culture of test organisms.

              2.      Incubate at 35°C up to 4 days.

    v Results

          1.       Turbidity and blue color: Positive test.

             2.     No growth (no turbidity and persistence of original color): Negative test.

    v Quality control

           Use following microorganisms to confirm the reliability of reagents:

             Positive control: Klebsiella pneumonia

 Negative control: Escherichia coli