Simmons Citrate Agar is used to differentiate Gram-negative enteric bacilli based on Sodium citrate as a source of carbon and inorganic Ammonium salt as a source of nitrogen. It is recommended for the differentiation of coliforms isolated from water and clinical samples. This medium is used in the same manner as Koser Citrate Broth (Cat. CL-1200) for the utilization of citrate as one of the IMVIC reactions. Magnesium sulphate is a cofactor for various metabolic reactions. Sodium chloride maintains the osmotic balance. Dipotassium phosphate acts as a buffer system and bromothymol blue is a pH indicator. Ammonium dihydrogen phosphate is the sole source of nitrogen and sodium citrate is the sole source of carbon. Bacteriological agar is the solidifying agent.

Only those organisms capable of utilizing citrate as a source of carbon grow and produce a colour change from green to blue (alkaline), whilst when no citrate utilization takes place (negative test), the colour of the medium remains the same. Escherichia coli, alongside Shigella, Yersinia and Edwardsiella species, do not grow on the medium. Serratia and most Enterobacter, Citrobacter, Klebsiella, Proteus and Providencia species, except for Morganella morganii and Klebsiella rhinoscleromatis, utilize citrate and produce the typical blue coloration. Simmons Citrate Agar is also used to differentiate citrate-positive Salmonella enteritidis and members of Salmonella subgenus II, III and IV from the citrate-negative Salmonella typhi, Salmonella paratyphi A, Salmonella pullorum and Salmonella gallinarum. ISO 10273 recommends this medium for the confirmation of Yersinia enterocolitica. The medium remains green since Yersinia enterocolitica does not use citrate as the sole source of carbon.