Composition

*Dehydrated powder, hygroscopic in nature, store in a dry place, in tightly-sealed containers below 25°C and protect from direct Sunlight.

Instructions for Use

Dissolve 65.0g in 1000ml distilled water. Gently heat to boil with gentle swirling and dissolve the medium completely. Distribute into test tubes. Sterilize by autoclaving at 10 psi (115°C) for 15 minutes. Allow the medium to cool at room temperature for preparing the slants.

Appearance: Pinkish – red colour, clear to slightly opalescent gel in slant position in screw cap tubes

pH (at 25°C): 7.4 ± 0.2

Principle

TRIPLE SUGAR IRON AGAR is used for confirmation of gram negative enteric bacilli on basis of  dextrose, lactose and sucrose fermentation and H2S production.

The ingredients included in the medium such as, Peptic digest of animal tissue, Casein enzymatic hydrolysate, Beef extract and Yeast extract provide the nitrogen, carbon, and vitamins required for organism growth.

Triple Sugar Iron Agar consists of three carbohydrates, Dextrose, Lactose and Sucrose. When the carbohydrates are fermented, acid production is detected by the Phenol Red pH indicator. Sodium thiosulphate is reduced to hydrogen sulphide, and hydrogen sulphide reacts with an iron salt yielding the typical black iron sulphide. Ferric ammonium citrate is the hydrogen sulphide (H2S) indicator.

Sodium chloride maintains the osmotic balance of the medium. Agar is used as a solidifying agent. Inoculating the microorganisms on the slants prepared in test tubes for the amount of 103 cfu/ml, observed on incubation period at 37°C after 24 hours has shown good growth in all bacteria tested only with the gas production (H2S) in Proteus vulgaris and Salmonella typhimurium.

Microbiological parameters (Growth promotion test)

Cultural characteristics observed after inoculation (103CFU/ml) and incubation at 37°C for 18 – 24 hours.

• Colour changes to red due to alkalinity.
• Colour changes to yellow due to acid production.
• Colour changes to yellow and gas is produced.
• Blackening due to H2S production.
• + = positive reaction
  • – = negative reaction

References

1. Kligler, I. J. A simple medium for the differentiation of members of the typhoid-paratyphoid group. Am. J. Public Health 7:1042-1044. (1917).
2. Kligler, I. J. Modifications of culture media used in the isolation and differentiation of typhoid, dysentery, and allied bacilli. J. Exp. Med. 28:319-322. (1918).
3. MacFaddin, J. F. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams & Wilkins, Baltimore, MD. (1985).
4. Marshall, R. T. (ed.). Standard methods for the examination of dairy products, 16^th ed. American Public Health Association, Washington, D.C. (1992).
5. P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (eds.). Manual of clinical microbiology, 6^th ed. American Society for Microbiology, Washington, D. C. (1995).
6. Padron, A. P. and W. B. Dockstader. Selective medium for hydrogen sulfide production. Appl. Microbiol. 23:1107. (1972).
7. Russell, F. F. The isolation of typhoid bacilli from urine and feces with the description of a new double sugar tube medium. J. Med. Res. 25:217. (1911).
8. Sulkin, S. E., and J. C. Willett. A triple sugar-Ferric ammonium citrate medium for use in identification of enteric organisms. J. Lab. Clin. Med. 25:649-653. (1940).
9. US Food and Drug Administration. Bacteriological analytical manual, 8^th ed. AOAC International, Gaithersburg, M.D. (1995).