Triple Sugar Iron (TSI) Test

Objective:

            To differentiate the enterobacteriaceae members according to their ability to ferment lactose, sucrose and glucose sugars and production of the hydrogen sulphide.

Principle:

            The fermentation of sugars will help to distinguish enteric bacteria from other gram negative bacilli. The TSI agar contains 1% concentrations of lactose and sucrose, 0.1 % of glucose. The phenol red is an acid base indicator is incorporated in this medium to detect acid production from carbohydrate fermentation.

            Acidification of medium is caused by intestinal organisms to attacking the sugars and it changes the phenol red to yellow color. This medium also contains sodium thio sulfate, the organisms reduces the sulfur to form hydrogen suphide gas and it is react with ferrous sulfate which is present in the medium to give black precipitation.

-0.1% Glucose: If only glucose is fermented, only enough acid is produced to turn the butt yellow.  The slant will remain red.

-1.0% lactose/1.0% sucrose:  a large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose.

-Sodium thio sulfate : Substrate for Production of Hydrogen sulphide (H₂S)

-Iron: Ferrous sulfate: Indicator of H₂S formation

-Phenol red: Indicator of acidification (It is yellow in acidic condition and red under alkaline conditions).

Materials required:

24 Hours old bacterial cultures (E.coli ., Proteus sp. and Klebsiella sp.)

Triple sugar iron agar

Procedure:

- Prepare TSI medium and adjust the pH to 7.4

- Sterilize the medium and pour into sterile test tubes and make slants

- Inoculate TSI agar slants by first stabbing in center of the medium and streaking on the surface of the slant

- Incubate the tubes at 37°C for 18 - 24 hours

Result:

-E.coli and Klebsiella sp., - shows yellow (acid) slant/ acid (yellow) butt with gas production.

-Proteus sp., shows acid butt, alkaline (red) slant with hydrogen sulphide (black precipitation) and gas production.

E.coli  & Klebsiella sp.             -   A/A with gas

Proteus sp.,                                -  K/A with H2S production

Interpretation:

From the above results klebsiella sp. and E.coli both are able to ferment all the sugars present in the TSI agar and produces acid and gas production indicate by bubble formation. Proteus sp. is ferment the glucose only, doesn’t ferment the remaining sugars, this organism also reduced the sulfur to hydrogen sulphide gas and it is observed as blackening precipitation.

Composition of TSI Agar

Beef extract   3.0 g

Yeast extract  3.0 g,

Peptone     15 g,

Protease peptone  5 g,

Lactose 10.0 g

Sucrose 10.0 g

Glucose 1.0g

Ferrous sulphate   0.2 g

Sodium chloride   5.0 g

Sodium thiosulphate   0.3 g,

Phenol red    0.024 g

Agar   12 g

Distilled water    1000ml

some examples of Triple Sugar Iron (TSI) Agar Reactions: 

Name of the organisms	Slant	Butt	Gas	H2S
Escherichia, Klebsiella, Enterobacter	Acid (A)	Acid (A)	Pos (+)	Neg (-)
Shigella, Serratia	Alkaline (K)	Acid (A)	Neg (-)	Neg (- )
Salmonella, Proteus	Alkaline (K)	Acid (A)	Pos (+)	Pos (+)
Pseudomonas	Alkaline (K)	Alkaline (K)	Neg (-)	Neg (-)