Hektoen Enteric Agar is a differential and selective medium that is used for isolating and differentiating enteric pathogens such as Salmonella and Shigella. Both cause a variety of serious human gastrointestinal diseases; and other Gram-negative Enterobacteriaceae. It is used particularly in foods where multi-steps are followed to isolate the pathogens of gastroenteritis.

The nutrients for growth are provided by the Meat Peptone and Yeast extract. The increased content of the Peptone and the three fermentable carbohydrates (Lactose, Sucrose, Salicin) as sources of carbon and energy reduce the inhibitory action of the Bile salts on Salmonella and Shigella spp. The lactose concentration in this medium is higher than in many other media used for enterics since this helps the visualization of enteric pathogens and minimizes the problem of delayed lactose fermentation. Bromothymol blue and Acid fuchsin are pH indicators. Sodium thiosulfate provides Sulphur, and Ferric ammonium citrate is the indicator for H2S production.

H2S positive colonies are black-centred and sodium chloride maintains the osmotic balance. The normal ISO 21567 recommends the Hektoen Agar as a selective solid media for the enumeration of Shigella spp. Although suppressed, partially inhibited E. coli and other organisms which use lactose, sucrose, and/or salicin with the production of acid, give colonies whose tones vary from yellow to orange to salmon. The Salmonella and Shigella are green or green blue. Proteus is not inhibited but produces a green-yellow colony when it grows. The colonies of Proteus and Salmonella may present a black centre and clear edges if they form iron sulphide because of H2S production.