Triple Sugar Iron Agar was originally proposed by Sulkin and Willett (1) and modified by Hajna (2) for identifying Enterobacteriaceae. This medium complies with the recommendation of APHA, for the examination of meat and food products (3), for the examination of milk and dairy products (4) and for microbial limit test for confirming the presence of Salmonellae (5, 6) and in the identification of gram-negative bacilli (6, 7).
Casein enzymic hydrolysate, peptic digest of animal tissue, yeast extract and beef extract provide nitrogenous compounds, sulphur, trace elements and vitamin B complex etc. Sodium chloride maintains osmotic equilibrium. Lactose, sucrose and dextrose are the fermentable carbohydrates. Sodium thiosulphate and ferrous ions make H2S indicator system. Phenol red is the pH indicator. Organisms that ferment glucose produce a variety of acids, turning the colour of the medium from red to yellow.
More amount 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.
Storage and Shelf-life:
Store below 30°C in tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label.
1. Sulkin E.S. and Willett J.C., 1940, J. Lab. Clin. Med., 25:649.
2. Hajna A.A., 1945, J. Bacteriol, 49:516.
3. Downes F. P. and Ito K., (Eds.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th Ed., APHA, Washington, D.C.
4. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
5. Finegold S. M. and Baron E. J., 1986, Bailey and Scotts Diagnostic Microbiology, 7th Ed., The C.V. Mosby Co., St. Louis.
6. Eaton A. D., Clesceri L. S. and Greenberg A. W., (Eds.), 2005, Standard Methods for the Examination of Water and Wastewater, 21st Ed., APHA, Washington, D.C.
7. MacFaddin J., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.