Kligler Iron Agar

Principle And Interpretation

Kligler Iron Agar is a combination of the lead acetate medium described by Kligler (1,2) and Russels Double Sugar Agar (3) and is used as a differentiation medium for typhoid, dysentery and allied bacilli (4). Bailey and Lacey substituted phenol red for andrade indicator previously used as pH indicator (4). Kligler Iron Agar differentiates lactose fermenters from the non-fermenters. It differentiates Salmonella Typhi from other Salmonellae and also Salmonella Paratyphi A from Salmonella Scottmuelleri and Salmonella Enteritidis (5). Fermentation of dextrose results in production of acid, which turns the indicator from red to yellow. Since there is little sugar i.e. dextrose, acid production is very limited and therefore a reoxidation of the indicator is produced on the surface of the medium, and the indicator remains red. However, when lactose is fermented, the large amount of acid produced, avoids reoxidation and therefore the entire medium turns yellow. Kligler Iron Agar, in addition to peptone, HM peptone B and yeast extract, contains lactose and glucose (dextrose), which enables the differentiation of species of enteric bacilli. Phenol red is the pH indicator, which exhibits a colour change in response to acid produced during the fermentation of sugars. The combination of ferrous sulphate and sodium thiosulphate enables the detection of hydrogen sulfide production, which is evidenced by a black color either throughout the butt, or in a ring formation near the top of the butt. Lactose non-fermenters (e.g.Salmonella and Shigella) initially produce a yellow slant due to acid produced by the fermentation of the small amount of glucose (dextrose). When glucose (dextrose) supply is exhausted in the aerobic environment of the slant, the reaction reverts to alkaline (red slant) due to oxidation of the acids produced. The reversion does not occur in the anaerobic environment of the butt, which therefore remains acidic (yellow butt). Lactose fermenters produce yellow slants and butts because of lactose fermentation. The high amount of acids thus produced helps to maintain an acidic pH under aerobic conditions. Tubes showing original colour of the medium indicates the fermentation of neither glucose (dextrose) nor lactose. Gas production (aerogenic reaction) is detected as individual bubbles or by splitting or displacement of the agar by the formation of cracks in the butt of the medium. Pure cultures of suspected organisms from plating media such as MacConkey Agar (M081), Bismuth Sulphite Agar (M027) or Deoxycholate Citrate Agar (M065), SS Agar (M108) etc. are inoculated on Kligler Iron Agar for identification.