Composition

* Dehydrated powder, hygroscopic in nature, store, in a dry place in tightly- sealed containers below 25°C and protect from direct Sunlight.

Instructions for use

Dissolve 24.28gms in 1000ml of distilled water. Gently heat to boiling with gentle swirling and dissolve  the medium completely. Sterilize by autoclaving at 15psi (1210C) for 15 minutes. Cool to 50°C and pour in sterile Petri plates or into test tubes.

Appearance: Forest green colour, clear to slightly opalescent gel

pH: 6.9 ± 0.2

Principle

SIMMONS CITRATE AGAR is used for differentiation of gram-negative bacteria on the basis of citrate utilization. Citrate acts source of carbon and nitrogen in the medium. Ammonium dihydrogen phosphate provides nitrogen in the medium. Dipotassium phosphate acts as a buffer. Sodium chloride maintains the osmotic balance of the medium. Sodium citrate is the sole source of carbon in this medium. Magnesium sulphate is a cofactor for the metabolic process. Organisms that can utilize Ammonium dihydrogen phosphate and Sodium citrate as their sole sources of nitrogen and carbon will grow on this medium and produce a color change from green (neutral) to blue (alkaline). Agar is the solidifying agent. The alkaline reaction takes place when excess CO₂ is generated (Kreb’s cycle) as citrate is cleaved to form oxaloacetate. Oxaloacetate is decarboxylated to pyruvic acid and CO₂. The excess CO₂ combines with sodium and water that is already present in the medium to form sodium carbonate. In addition, bacteria that utilize citrate can extract nitrogen from the ammonium phosphate present in the medium, forming ammonia which combines with water to form NH₄OH. The formation of these end products produce an alkaline pH in the medium (greater than 7.6), resulting in a color change which is detected by Bromothymol blue. Colour change from green to blue is the indication of positive reaction.

Interpretation

Cultural characteristics observed after inoculating (103-105CFU/ml), on incubation at 350C for 24 – 48 hours.