TRIPLE SUGAR IRON AGAR (TSI) EUROPEAN PHARMACOPOEIA - preparation and uses

Sep 27, 2026

TRIPLE SUGAR IRON AGAR (TSI) (European Pharmacopoeia) is a differential culture medium for identifying and differentiating Enterobacteria based on carbohydrate fermentation and H2S production. The formula uses phenol red for pH-driven color change, thiosulfate and ferric ammonium citrate to indicate H2S formation, and bacteriological agar as the solidifying agent. Preparation specifies dissolving 64.6 g/L, autoclaving at 121°C for 15 minutes, and storing at 2–8°C. Uses include aiding identification from routine material samples and confirming Salmonella via characteristic deep and surface reactions.

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CategoryHealthcare

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Preparation

Uses

Microbiological test

Storage

Bibliography

TRIPLE SUGAR IRON AGAR (TSI) EUROPEAN PHARMACOPOEIA CAT Nº: 21046 For the identification and differentiation of Enterobacteria FORMULA IN g/l Phenol red indicator, being the colour changes yellow for acid production and red for alkalinisation. Sodium thiosulfate is reduced to Hydrogen sulfide, which reacts with the iron salt to give the black iron sulfide. Ferric ammonium citrate is a H2S indicator. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Bacteriological agar is the solidifying agent. The mode of action is similar to Kligler Iron Agar (Cat. 21042) which contains two sugars. The addition of 1% Sucrose in TSI Agar allows to differentiate between | Peptones (Beef & Casein) | 20.00 | Glucose Monohydrate | 1.00 | | --- | --- | --- | --- | | | | | | | Lactose
Monohydrate | 10.00 | Ferric Ammonium Citrate | 0.30 | | Sucrose | 10.00 | Sodium Thiosulfate | 0.30 | | --- | --- | --- | --- | | Sodium Chloride | 5.00 | Phenol Red | 0.025 | | --- | --- | --- | --- | | Beef Extract | 3.00 | Bacteriological Agar | 12.00 | | --- | --- | --- | --- | | Yeast Extract | 3.00 | | | --- | --- | --- | Final pH 7.4 ± 0.2 at 25ºC 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 121ºC for 15 minutes. Allow to cool in aslanted position in order to obtain butts of 1.5-2.0 cm. depth. The prepared medium should be stored at 2- 8°C. The color is red. The dehydrated medium should be homogeneous, freeflowing and pink in color. If there are any physical changes, discard the medium. USES TRIPLE SUGAR IRON AGAR (TSI) is a differential medium used to differentiate enteric Gram-negative Enterobacteria on the basis of carbohydrate fermentation and H2S production. It is used as an aid in the identification of pathogenic and saprophytic Enterobacteria isolated from routine bacteriological analysis of material samples such as faeces. This medium is used to initiate the identification of Enterobacteria in some FDA schemas. Peptone mixture and the Beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group. TSI contains three carbohydrates (Dextrose, Sucrose and Lactose) as sources of carbon and energy. When these are fermented the acid production is indicated by the Proteus and Salmonella. The fermentation of sucrose by Proteus turns the colour 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. The European Pharmacopoeia recommends Triple Sugar Iron (TSI) Agar as one of the media used for the confirmation of Salmonella. The Eur. Pharmacopoeia recommends transferring separately a few of the suspect colonies to TSI Agar using surface and deep inoculation. The presence of salmonellae is provisionally confirmed if in the deep inoculation, but not in the surface culture, there is a change of colour from red to yellow and, usually, a formation of gas, with or without production of Hydrogen sulphide in the agar. Precise confirmation may be carried out by the appropriate biochemical and serological tests. The product passes the test if colonies of the type described do not appear, or if the confirmatory biochemical and serological tests are negative. Inoculate with sample and incubate at 35±2 for 18-72 hours. MICROBIOLOGICAL TEST The following results were obtained in the performance of the medium from type cultures after incubation at a temperature of 35±2ºC and observed after 18-72 hours. BIBLIOGRAPHY | EP | | | --- | --- | Standard Methods for the Examination of Dairy Products. APHA, 1972. 1 DEBEN DIAGNOSTICS LTD [www.debendiagnostics.co.uk

FAQs

What is TRIPLE SUGAR IRON AGAR (TSI) used for?

TSI is a differential medium used to differentiate enteric Gram-negative Enterobacteria by carbohydrate fermentation patterns and H2S production, aiding identification from routine bacteriological analysis such as feces.

How does TSI indicate carbohydrate fermentation and H2S production?

Phenol red shows acid production as a yellow change and alkalinisation as red. Sodium thiosulfate is reduced to hydrogen sulfide, which reacts with iron salts to form black iron sulfide; ferric ammonium citrate functions as an H2S indicator.

What are the key steps and conditions in preparing the medium?

Suspend 64.6 g in 1 liter distilled water, heat with frequent agitation until dissolved, boil for one minute, dispense into tubes, and autoclave at 121°C for 15 minutes. Cool in a slanted position to form butts, then store at 2–8°C.

Sep 27, 2026

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