Triple Sugar Iron Agar Test Procedure

In this triple sugar iron agar test procedure post we have briefly explained triple sugar iron agar test principle, objectives, requirements, triple sugar iron agar test procedure, uses and limitations.

Triple Sugar Iron Agar Test

Triple sugar iron agar test was introduced by Sulkin and Willett for detecting Enterobacteriaceae, which Hanja adapted. This medium complies with the Indian Pharmacopoeia’s recommendation for the identification of Gram-negative bacteria.

Lactose, sucrose, a tiny amount of glucose (dextrose), ferrous sulphate, and the pH indicator phenol red make up triple sugar iron agar (TSIA), a differential media. It’s utilised to distinguish between enterics that can decrease sulphur and ferment carbohydrates.

The test is carried out on an agar slant of a special medium containing multiple sugars, a pH-sensitive dye (phenol red), 1 percent lactose, 1 percent sucrose, 0.1 percent glucose, sodium thiosulfate, and ferrous sulphate or ferrous ammonium sulphate, as well as sodium thiosulfate and ferrous sulphate or ferrous ammonium sulphate.

When all of these materials are combined and allowed to solidify at an angle, the outcome is a slanted agar test tube.

The slanted shape of this medium creates a variety of surfaces that are either exposed to oxygen-containing air to variable degrees (an aerobic environment) or not exposed to air (an anaerobic environment), allowing for the study of organisms’ fermentation processes.


Triple sugar iron agar test is used to discriminate between the several Enterobacteriaceae groups or genera, which are all gram – negative bacilli capable of fermenting glucose with the generation of acid, and other gram negative intestinal bacilli.

The differences in carbohydrate fermentation processes and hydrogen sulphide production by the distinct types of intestinal microbes provide the basis for this classification. The presence of gas and a visible colour change in the pH indicator, phenol red, indicate carbohydrate fermentation.

The production of hydrogen sulphide in the medium is indicated by the formation of a black precipitate that will blacken the medium in the bottom of the tube.

Triple sugar iron agar test agar comprises three fermentative sugars, lactose and sucrose in 1% concentrations, and glucose in 0.1 percent concentration, to aid in the monitoring of carbohydrate usage patterns.

The pH decreases as acid builds up during fermentation. The acid-base indicator Phenol red is used to detect carbohydrate fermentation, which is indicated by a change in the colour of the carbohydrate medium from orange to yellow when acids are present.

When peptone is oxidatively decarboxylated, alkaline compounds are formed, and the pH rises. The change in colour of the medium from orange red to deep red indicates this.

The presence of sodium thiosulfate and ferrous ammonium sulphate in the medium detects hydrogen sulphide generation, which is shown by the black colour in the tube’s butt.

The creation of gas and a shift in the colour of the pH indicator from red to yellow indicate carbohydrate fermentation. The glucose content is one-tenth that of lactose or sucrose to make it easier to discover microbes that only digest glucose. During glucose fermentation, the small quantity of acid produced in the slant of the tube oxidises quickly, causing the medium to remain orange red or return to an alkaline pH. Because of the reduced oxygen tension in the butt of the tube, the acid reaction (yellow) is sustained.

After the restricted glucose supply has been depleted, organisms that are capable of doing so will proceed to use lactose or sucrose. By lightly shutting the tube cap, free interchange of air can be allowed to improve the alkaline condition of the slant. If the tube is tightly closed, the slant will be involved in an acid reaction (produced primarily by glucose fermentation).


Ingredients Gms / Litre Beef extract – 3.000, Peptone – 20.000, Yeast extract- 3.000, Lactose – 10.000, Sucrose – 10.000, Dextrose monohydrate – 1.000, Ferrous sulphate – 0.200, Sodium chloride – 5.000, Sodium thiosulphate – 0.300, Phenol red – 0.024, Agar – 12.000.

Media Preparation

Add 3.0 gram of Beef extract, 3 gram of yeast extract, 15 gram of peptone, 5 grams of protease peptone, 10.0 grams of lactose, 10.0 gram of saccharose, 1.0 gram of glucose, 0.2 gram of ferrous sulphate, 5.0 gram of sodium chloride, 0.3 gram of sodium thiosulphate, 0.024 gram of phenol red and 12 gram of agar and make the mixture up to 1000ml with distilled water.

The Peptone mixture and the Beef and Yeast extracts provide the nutrients essential for growth. Sodium chloride maintains the osmotic balance of the medium. The Bacteriological agar is the solidifying agent.


1. Triple sugar-iron agar slants

2. 24 broth culture

3. Bunsen burner

4. Inoculating needle

5. Test tubes

6. Marking pen

Triple Sugar Iron Agar Test Procedure

1. Sterilize the inoculating needle in the bunsen burner’s blue flame until red hot, then set aside to cool.

2. Take the Trypticase soy broth tube holding the 24-48 hour culture from the rack, remove the cap, and burn the tube’s neck.

3. With the needle, take the organism’s culture from the TSB (tryptic soy broth) tube using aseptic procedure.

4. Replace the tube in the test tube rack after re-flaming the tube’s neck. Remove the cap from a sterile Triple sugar iron agar test slant tube from the rack and burn the tube’s neck.

5. Insert the needle holding the pure culture into the medium up to the Triple sugar iron agar test tube’s butt, then streak the needle back and forth across the slant’s surface.

6. Flame the Triple sugar iron agar test tube’s neck once more, cap it, and place it in the test tube rack. Incubate at 37⁰C for 18 to 24 hours.


Only dextrose fermentation causes an alkaline/acid (red slant/yellow butt) response in Triple sugar iron agar test.

The fermentation of dextrose, lactose, and/or sucrose is indicated by an acid/acid (yellow slant/yellow butt) response.

An alkaline/alkaline reaction (red slant, red butt): Absence of carbohydrate fermentation leads to the absence of carbohydrate fermentation.

The medium is blackened: When H2 is present, this happens. Production of natural gas: The presence of bubbles or fissures in the agar indicates the presence of gas (formation of CO2and H2).

TSI test
TSI test


1. Triple sugar iron agar test is mostly used to distinguish Enterobacteriaceae bacteria from other gram-negative rods.

2. Triple sugar iron agar test can also be used to distinguish between Enterobacteriaceae based on their sugar fermentation processes.


1. Some organisms may produce hydrogen sulphide on Kligler Iron Agar, but not on Triple sugar iron agar test Agar, since the sucrose in Triple sugar iron agar test agar blocks the enzyme process that results in H2S formation. H2S-producing Salmonella and several Enterobacteriaceae members may not be H2S-positive on Triple sugar iron agar test Agar.

2. On Triple sugar iron agar test Agar, H2S-producing organisms may develop so much ferrous sulphide, a black precipitate, that the acidity produced in the butt is totally concealed.

3. Sucrose is added to Triple sugar iron agar test to eliminate some sucrose-fermenting non-lactose fermenters, such as Proteus and Citrobacter spp.

4. Some sucrose-fermenting non-lactose fermenters, including as Proteus and Citrobacter spp., are eliminated by adding sucrose to Triple sugar iron agar test agar.

5. For precise identification and confirmation of organisms, additional biochemical tests and serological typing must be undertaken.

Further Readings