USE OF TRIPLE SUGAR IRON AGAR (AS PER BIS)

TRIPLE SUGAR IRON AGAR (AS PER BIS) for confirmation of gram negative enteric bacilli on behalf of dextrose, lactose and sucrose fermentation and H2S production.

Composition 

Ingredients	Gms/Ltr
Peptone	20.000
Yeast extract	3.000
Meat extract	3.000
Lactose	10.000
Sucrose	10.000
Dextrose	1.000
Sodium chloride	5.000
Ferrous sulphate, heptahydrate	0.200
Sodium thiosulphate, pentahydrate	0.300
Phenol red	0.024
Agar	12.000

* 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

Suspend 64.32 grams in 1000 ml refined water. Gently heat to boiling with gently swirling to dissolve the medium completely. Sterilize by autoclaving at 15 psi pressure (121°C) for 15 mins. 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 was originally proposed by Sulkin and Willet and modified by Hajna for identifying Enterobacteriaceae. ISO Committee and BIS has recommended a slight modification for the identification of Salmonellae. BIS has recommended the medium for detection of Escherichia coli and Vibrios.

Triple Sugar Iron Agar is recommended for confirmation of gram negative bacilli on basis of dextrose, lactose and sucrose fermentation and H2S production. The ingredients included in the medium such as peptone, yeast extract, meat 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 carbohydrate 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.

Interpretation

Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.

Test Strains	ATCC	Inoculum (cfu/ml)	Growth	Butt/Slants/Gas/H2S
Salmonella Paratyphi A	9150	103	Luxuriant	Yellow /Red/+/-
Salmonella Typhi	6539	103	Luxuriant	Yellow /Red/-/+
Salmonella Typhimurium	14028	103	Luxuriant	Yellow /Red/+/+
Vibrio cholerae	15748	103	Luxuriant	Yellow /Red/-/-

Reference

1. Sulkin E.S. and Willett J.C., 1940, J. Lab. Clin. Med.

2. Hajna A.A., 1945, J. Bacteriol, 49:516.

3. Finegold and Baron, 1986, Bailey and Scotts Diagnostic Microbiology, 7th ed., The C.V. Mosby      Co., St. Louis.

4. Greenberg A. E., Trussell R. R. and Clesceri L. S. (Eds.), 1985, Standard Methods for the Examination of Water and Wastewater, 16th ed., APHA, Washington, D.C.

5. MacFaddin J., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.