Agar Medium M (Triple Sugar, Iron Agar)

Principle And Interpretation

Triple Sugar Iron Agar, cited as Agar Medium M, is recommended for identification and differentiation of Enterobacteria by European Pharmacopoeia, 2008 (1). It was originally proposed by Sulkin and Willett (2) and modified by Hajna (3) for identifying Enterobacteriaceae.

Peptones (casein and beef), yeast extract and beef extract provide nitrogenous compounds, sulphur, trace elements and vitamin B complex etc. Sodium chloride maintains osmotic equilibrium. Lactose (monohydrate), sucrose and Glucose (monohydrate) in the medium are the fermentable carbohydrates. Sodium thiosulphate and ferric ions make H2S indicator system. Sodium thiosulphates are also inactivators of halogens and can minimize its toxicity in the testing sample, if any during microbial limit tests. Phenol red is the pH indicator.

Organisms that ferment glucose monohydrate produce a variety of acids, turning the colour of the medium from red to yellow. More amounts of acids are liberated in butt (fermentation) than in the slant (respiration). Growing bacteria also form alkaline products from the oxidative decarboxylation of peptone and these alkaline products neutralize the large amounts of acid present in the butt. Thus the appearance of an alkaline (red) slant and an acid (yellow) butt after incubation indicates that the organism is a glucose fermenter but is unable to ferment lactose and/or sucrose. Bacteria that ferment lactose or sucrose (or both), in addition to glucose, produce large amounts of acid enables no reversion of pH in that region and thus bacteria exhibit an acid slant and acid butt. Gas production (CO2) is detected by the presence of cracks or bubbles in the medium, when the accumulated gas escapes. Thiosulphate is reduced to hydrogen sulphide by several species of bacteria and H2S combines with ferric ions of ferric salts to produce the insoluble black precipitate of ferrous sulphide. Reduction of thiosulphate proceeds only in an acid environment and blackening usually occurs in the butt of the tube.

Triple Sugar Iron Agar should be used in parallel with Urea Agar / Broth (M112/M111) to distinguish between Salmonella and Proteus species. The reactions can be summarized as follows:

Alkaline slant / acid butt - only glucose fermented

Acid slant/ acid butt - glucose and sucrose fermented or glucose and lactose fermented or all the three sugars, glucose, lactose and sucrose fermented.

Bubbles or cracks present - gas production

Black precipitate present - H2S gas production

Some members of the Enterobacteriaceae and H2S producing Salmonella may not be H2S positive on TSI Agar. Some bacteria may show H2S production on Kligler Iron Agar but not on TSI Agar. This can happen because utilization of sucrose in TSI Agar suppresses the enzymic pathway that result in H2S production.