TSI Agar: Foundation, Preparation And Uses

The TSI agar or triple sugar iron agar is a solid medium which serves as a biochemical test to guide the initial identification of Gram negative bacilli. It is based on showing the fermentation of the sugars present, and the production of hydrogen sulfide and gas.

Its composition and basis is very similar to the Kligler iron test, with the difference that the latter contains only glucose and lactose. Instead, as its name implies, triple sugar iron agar contains three fermentable carbohydrates: glucose, lactose, and sucrose.

TSI agar

Images of the TSI test with different microorganisms A. Escherichia coli, B. Shigella flexneri, C) Salmonella typhimurium, D. Pseudomonas aeruginosa. Source: Witmadrid [Public domain], from Wikimedia Commons

In addition, the TSI medium has four protein derivatives that make it a very nutritious agar: yeast extract, meat extract, peptone and proteose peptone. It also contains ferrous ammonium sulfate, sodium thiosulfate, sodium chloride, phenol red, and agar.

The inability of a microorganism to ferment the glucose present in the medium immediately excludes it from belonging to the Enterobacteriaceae Family. Hence this test is essential in deciding which identification route to take to determine the genus and species.

Each laboratory decides whether to work with TSI agar or with Kligler iron agar.

Article index

  • one


    • 1.1

      Sodium chloride and agar

    • 1.2

      PH indicator (phenol red)

    • 1.3

      Protein derivatives (yeast extract, meat extract, peptone and proteose peptone)

    • 1.4

      Fermentation of carbohydrates (glucose, lactose and sucrose)

    • 1.5

      Gas production

    • 1.6

      Sodium thiosulfate and ferrous ammonium sulfate (hydrogen sulfide production)

  • two


  • 3


  • 4


  • 5


  • 6



Each of the compounds fulfills a function within the medium.

Sodium chloride and agar

Sodium chloride is necessary to maintain the osmotic balance of the medium. While the agar gives the solid consistency.

PH indicator (phenol red)

The pH of the prepared medium is balanced at 7.3 and the pH indicator (phenol red) turns yellow below 6.8. This means that small amounts of acids produced by the fermentation of sugars will turn the medium from red-orange to yellow.

If fermentation does not occur there will be alkalinization of the medium through the use of peptones, turning from red-orange to strong red.

Protein derivatives (yeast extract, meat extract, peptone and proteose peptone)

When bacteria metabolize the proteins present in TSI agar, amines are produced that alkalize the medium (mainly at the bevel level), because the reaction requires oxygen. The amines turn the bezel bright red.

But this will depend on the ability of the bacteria to ferment carbohydrates or not.

Fermentation of carbohydrates (glucose, lactose and sucrose)

The study of the fermentation of sugars can give several images and each one is interpreted differently. The test interpretation divides microorganisms into 3 categories: glucose non-fermenters, lactose non-fermenters, and lactose / sucrose fermenters.

It should be noted that the amount of glucose in the medium is limited, while the concentration of lactose and sucrose is 10 times higher.

Bacteria of the Enterobacteriaceae Family and other glucose-fermenting microorganisms will begin to ferment this sugar as it is the simplest carbohydrate for energy.

On the other hand, lactose and sucrose are complex carbohydrates that must be broken down and converted into glucose in order for them to enter the Embden-Meyerhof cycle.

-Microorganisms not fermenting glucose

When the inoculated microorganism is not able to ferment glucose, much less will it be able to ferment other carbohydrates. Therefore, no acids are formed here, but there is formation of amines in the bevel by the use of peptones.

In this case, the bezel turns to a stronger red and the bottom of the tube may remain unchanged or it may also be alkalized, leaving the entire tube red.

Interpretation: K / K means alkaline bevel / alkaline or neutral bottom

In the image at the beginning of the article see the image of tube D.

This result indicates that the microorganism does not belong to the Enterobacteriaceae Family.

-Microorganisms not fermenting lactose / sucrose

If the bacteria are able to ferment glucose but not lactose or sucrose, the following will happen:

The bacteria will consume all the glucose present after approximately 6 to 8 hours, being able to acidify both the bevel and the block; that is, the agar will have completely turned yellow. But when glucose is depleted and lactose and sucrose cannot be used, the bacteria will begin the metabolism of proteins.

This reaction needs oxygen, therefore the degradation of peptones occurs on the surface (bevel). The amines produced alkalize the bezel turning from yellow to red. This reaction is evidenced after 18 to 24 hours of incubation.

Interpretation: K / A means alkaline bevel and acid wad.

In the image at the beginning of the article see the image of tube B.

-Lactose / sucrose fermenting microorganisms

Microorganisms capable of fermenting lactose and sucrose can obviously ferment glucose. After the minimum amount of glucose present in the medium is used up, the pyruvate formed begins to metabolize to form acids through the aerobic Krebs cycle, and in the period of 8 to 12 hours all the medium will be yellow.

If the bacteria is capable of breaking down the lactose or sucrose, acids will continue to be produced, and after 18 to 24 hours the entire tube – bevel and plug – will continue to yellow.

It should be noted that the use of glucose is carried out in two ways: one aerobically at the bevel of the tube, and the other anaerobically at the bottom of the tube.

Interpretation: A / A means acid bevel / acid bottom. It may or may not have gas.

In the image at the beginning of the article see the image of tube A.

Gas production

Some microorganisms are capable of producing gas during the fermentation of sugars. The gas is evidenced in the tube by the pressure it exerts within the agar. The pressure causes the formation of bubbles or the displacement of the agar. Sometimes the gas formation can fracture the medium.

It is important that when sowing the TSI medium, the puncture is made cleanly through the center of the agar until it reaches the bottom. If the puncture is diverted towards the walls of the tube, it can cause false positives in the production of the gas, since it will escape through the wrongly formed channel.

The gas production, as well as the reactions that occur in the agar bevel, need oxygen, therefore it is recommended that the tube be covered with a cotton plug, and if a Bakelite cap is used, it should not be completely tight.

Gas production is reported as positive (+) or negative (-).

TSI Medium Gas Formation

Gas formation patterns. Source: Scheme made by MSc. Marielsa Gil. Image source: VeeDunnFlickr.com/Y_tambe [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

Sodium thiosulfate and ferrous ammonium sulfate ( hydrogen sulfide production)

Bacteria capable of producing hydrogen sulfide (colorless gas) take up the sulfur from sodium thiosulfate present in the medium. Once H 2 S is formed, it reacts with ferrous ammonium sulfate, producing iron sulfide (clearly visible black precipitate).

H 2 S production is reported as positive (+) or negative (-).

In the image at the beginning of the article see the image of tube C.


Weigh 62.5 g of the dehydrated triple sugar iron agar (TSI) medium and dissolve in one liter of distilled water.

Heat until the agar is completely dissolved. Boil for a minute, stirring frequently. Distribute 4 ml of the medium into 13/100 test tubes with cotton caps.

Sterilize in an autoclave at 121 ° C for 15 minutes. Remove from the autoclave and let it rest at an angle. Care must be taken that both the base and the bezel have the same distance.

Store in a refrigerator 2-8 ° C. Let it warm up before sowing the bacterial strain.

The color of the dehydrated medium is light beige and the prepared medium is red-orange.

The final pH of the prepared medium is 7.3 ± 0.2.


The TSI test is widely used at the microbiology laboratory level. This test is essential to guide the type of test that must be applied to reach the identification of the genus and species. 
Its good execution and interpretation can save material and labor.

If the result is a TSI K / K and the cytochrome oxidase test is positive, it is known that tests should be used to identify non-fermenting Gram negative rods, such as Pseudomonas, Alcaligenes, Achromobacter, Burkholderia, among other genera. If it is oxidase negative, it is oriented towards the genera Acinetobacter, Stenotrophomonas, etc.

On the other hand, if a TSI A / A or K / A is obtained and the cytochrome oxidase test is negative, the more nitrates reduce to nitrites, we will be certain that it is a microorganism belonging to the Enterobacteriaceae Family. In this case, the identification route will focus on specific tests for this group of bacteria.

On the other hand, if a K / A or A / A image is obtained and the cytochrome oxidase test is positive, the additional tests to be assembled will be aimed at the identification of fermenting strains that do not belong to the Enterobacteriaceae Family, such as: Aeromonas, Plesiomonas, Vibrio and Pasteurella.

A TSI with hydrogen sulfide, oxidase negative, will guide the identification of the following genera of the Enterobacteriaceae Family: Proteus, Citrobacter, Edwardsiella , Leminorella, Pragia, Trabusiella or Salmonella.

A TSI with little or moderate hydrogen sulfide in the alkaline bevel with an alkaline background and a positive oxidase, will guide the use of tests for the identification of Gram-negative, non-fermenting H 2 S- producing rods , such as Shewanella putrefaciens .

Finally, the TSI can be used for the investigation of hydrogen sulfide production in Gram positive bacilli, especially when Erysipelothrix rhusiopathiae is suspected .


The TSI medium must be inoculated with pure colonies, isolated in primary or selective cultures. If the colony is taken from selective media that were seeded with samples with mixed flora, care must be taken to take only from the surface, since viable strains inhibited in that medium may exist in the lower part of the colony.

Therefore, the loop should never be cooled on selective medium and then the colony is taken and inoculated with TSI medium.

The seeding will be done with a straight loop or needle. A puncture will be made, taking care that it is through the center of the middle until reaching the bottom, and then the seeding is finished by inoculating the surface in a zigzag shape. Do not do two punctures.

Incubate at 37 ° C in aerobiosis for 18-24 hours. Interpret at this time, neither before nor after.


The TSI test should be read within 18 to 24 hours of incubation. A reading before this time may give a false positive for A / A fermentation. Meanwhile, a reading after this time can give rise to a false negative image of a non-fermenter, due to the consumption of peptones that alkalize the medium.


  1. Mac Faddin J. (2003). Biochemical tests for the identification of bacteria of clinical importance. 3rd ed. Editorial Panamericana. Buenos Aires. Argentina.
  2. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
  3. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
  4. “TSI agar.”  Wikipedia, The Free Encyclopedia . 10 Jul 2018, 08:09 UTC. 10 Feb 2019, 03:33 Available at: es.wikipedia.org
  5. Britannia Laboratories. TSI Agar (Triple sugar iron agar). 2015.Available at: britanialab.com
  6. BD Laboratories. Triple sugar iron agar (TSI Agar). 2003.Available at: bd.com

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