Triple Sugar Iron Agar (TSI) ISO

Peptone and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly the B-group. The three carbohydrates (glucose, sucrose and lactose) are the fermentable carbohydrates providing carbon and energy. When these are fermented, the acid production is indicated by the phenol red indicator, the color changing to yellow for acid production and to red for alkalinization. Sodium thiosulfate is reduced to hydrogen sulfide, which reacts with the iron salt to give the black iron sulfide. The ferric ammonium citrate is an H2S indicator. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Bacteriological agar is the solidifying agent.

The glucose concentration in the medium is one-tenth the concentration of lactose or sucrose in order to facilitate the detection of organisms that only ferment glucose. The fermentation of glucose produces a small amount of acid in the slant of the tube, which is rapidly oxidized, and the medium remains red or reverts to an alkaline pH. On the other hand, the same acid reaction in the butt of the tube keeps the acid pH (yellow) due to the lower oxygen tension.

When all glucose is used, organisms able to ferment lactose or glucose will begin to utilize them. In order to enhance the free exchange of air in the slant of the tube, the tube cap must be closed loosely.

The addition of 1% sucrose in the TSI Agar allows differentiating between Proteus and Salmonella. The fermentation of the sucrose by Proteus turns the color of the phenol red indicator in the slant from red to yellow. Dextrose-positive and lactose-negative members of the genus Salmonella all cause a reddening of the slant and acidify the depths of the agar tubes.

Preparation

Suspend 64.6 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into tubes and sterilize in autoclave at 121 °C for 15 minutes. Allow to cool in a slanted position in order to obtain butts of 1.5-2.0 cm depth.

Instructions for use

1. Streak the agar slant surface and stab the butt.
2. Incubate at 37 °C for 24 ± 3 h according to ISO 6579 and at 36 ± 2 °C for 24 ± 3 h according to ISO 19250.
3. Interpret the changes in the medium as follows:

Butt:

• Yellow: glucose positive.
• Red or unchanged: glucose negative.
• Black: formation of hydrogen sulphide.
• Bubbles or cracks: gas formation from glucose.

Slant surface:

• Yellow: lactose and/or sucrose positive.
• Red or unchanged: lactose and/or sucrose negative.

• ISO 6579 Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella spp.
• Standard Methods for the Examination of Dairy Products. APHA, 1972.
• Food and Drug Administration. Bacteriological Analytical Manual, 1976.
• Vanderzant, C. and D.F. Splittstoesser (ed) 1992. Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health Association, Washington D.C.
• European Pharmacopoeia. 4th Edition. 2002.
• ISO 19250 Water quality - Detection of Salmonella spp.