For differentiation of gram-negative bacteria on the basis of citrate utilization. SIMMONS CITRATE AGAR(VEG.) 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 thismedium 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, bacteriathat 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.