Triple Sugar Iron (TSI) Agar- Composition, Principle, Preparation, Results, Uses

Most bacteria have the ability to ferment carbohydrates, particularly sugars. Among them, each bacterium can ferment only some of the sugars, while it cannot ferment the others. Thus, the sugars, which a bacterium can ferment, and the sugars, which it cannot is the characteristic of the bacteria and thus an important criterion for its identification. The Triple Sugar Iron (TSI) agar is a culture medium named for its ability to test a microorganism’s ability to ferment sugars and to produce hydrogen sulfide.

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Composition of Triple Sugar Iron (TSI) Agar

Pancreatic Digest of Casein15.0
Sodium Chloride5.0
Peptic Digest of Animal Tissue5.0
Yeast Extract3.0
Beef Extract3.0
Ferric Ammonium Citrate0.5
Sodium Thiosulfate0.3
Phenol Red0.024

Final pH (at 25°C): 7.3 +/- 0.2

Principle of Triple Sugar Iron (TSI) Agar

  1. The TSI agar is a special medium with multiple sugars constituting a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate.
  2. All of these ingredients when mixed together and allowed solidification at an angle resulting in an agar test tube at a slanted angle.
  3. The slanted shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying degrees (aerobic environment) or not exposed to air (an anaerobic environment) under which fermentation patterns of organisms are determined.
  4. The triple sugar- iron agar is designed to differentiate among organisms based on the differences in carbohydrate fermentation patterns and hydrogen sulfide production.
  5. Carbohydrate fermentation is indicated by the production of gas and a change in the color of the pH indicator from red to yellow.
  6. Due to the building of acid during fermentation, the pH falls. The acid-base indicator Phenol red is incorporated for detecting carbohydrate fermentation that is indicated by the change in color of the carbohydrate medium from orange-red to yellow in the presence of acids.
  7. In the case of oxidative decarboxylation of peptone, alkaline products are built and the pH rises. This is indicated by the change in color of the medium from orange-red to deep red.
  8. Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects the production of hydrogen sulfide and is indicated by the black color in the butt of the tube.
  9. To facilitate the detection of organisms that only ferment glucose, the glucose concentration is one-tenth the concentration of lactose or sucrose.
  10. The meagre amount of acid production in the slant of the tube during glucose fermentation oxidizes rapidly, causing the medium to remain orange-red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube since it is under lower oxygen tension.
  11. After depletion of the limited glucose, organisms able to do so will begin to utilize the lactose or sucrose.
  • An alkaline/acid (red slant/yellow butt) reaction: It is indicative of dextrose fermentation only.
  • An acid/acid (yellow slant/yellow butt) reaction: It indicates the fermentation of dextrose, lactose and/or sucrose.
  • An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate fermentation results.
  • Blackening of the medium: Occurs in the presence of H2S.
  • Gas production: Bubbles or cracks in the agar indicate the production of gas (formation of CO2 and O2)

Preparation and Method of Use

  1. Suspend 64.42 grams (the equivalent weight of dehydrated medium per liter) in 1000 ml purified distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Mix well and distribute into test tubes.
  4. Sterilize by maintaining at 10lbs pressure (115°C) for 30 minutes or as per validated cycle.
  5. Allow the medium to set in the sloped form with a butt about 2.5cm long.
  6. With a straight inoculation needle, touch the top of a well-isolated colony.
  7. Inoculate TSI by first stabbing through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant.
  8. Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 18 to 24 hours.
  9. Examine the reaction of the medium.

 Result Interpretation on Triple Sugar Iron (TSI) Agar

Triple Sugar Iron (TSI) Agar

Image Source: Microrao

Salmonella enterica Growth; red slant, yellow butt, gas positive, black-butt (H2S produced)
Escherichia coli Growth; yellow slant, yellow butt, gas positive, no H2S produced
Pseudomonas aeruginosa Growth; red slant, red butt, no gas, no H2S produced
Shigella sonnei Growth; red slant, yellow butt, no gas, no H2S produced
Citrobacter freundiiYellow slant, yellow butt, gas production; positive reaction for H2S Blackening of medium
Enterobacter aerogenesYellow slant, yellow butt, gas production; no H2S produced
Klebsiella pneumoniaeyellow slant, yellow butt, gas positive, no H2S produced
Proteus vulgarisRed slant, yellow butt, no gas production; H2S produced
Salmonella Paratyphi ARed slant, yellow butt, gas production; no H2S produced
Salmonella TyphiRed slant, yellow butt, no gas production; H2S produced
Salmonella TyphimuriumRed slant, yellow butt, gas production; H2S produced
Shigella flexneriRed slant, yellow butt, gas negative, H2S not produced

 Uses of Triple Sugar Iron (TSI) Agar

  • It is used to determine the ability of an organism to ferment glucose, lactose, and sucrose, and their ability to produce hydrogen sulfide.
  • It is used primarily to differentiate members of the Enterobacteriaceae family from other gram-negative rods.
  • It is also used in the differentiation among Enterobacteriaceae on the basis of their sugar fermentation patterns.

Limitations of Triple Sugar Iron (TSI) Agar

  • It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
  • It is important to stab the butt of the medium. Failure to stab the butt invalidates this test. The integrity of the agar must be maintained when stabbing. Caps must be loosened during this test or erroneous results will occur.
  • TSI Agar must be read within the 18-24 hour stated incubation period. A false-positive reaction may be observed if read too early. A false-negative reaction may be observed if read later than 24 hours.
  • An organism that produces hydrogen sulfide may mask acid production in the butt of the medium. However, hydrogen sulfide production requires an acid environment, thus the butt portion should be considered acid.
  • TSI is not as sensitive in detecting hydrogen sulfide in comparison to other iron-containing mediums, such as Sulfide Indole Motility (SIM) Medium.
  • Certain species or strains may give delayed reactions or completely fail to ferment the carbohydrate in the stated manner.


  1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
  2. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He is doing his Ph.D. at the Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal. He was awarded the DAAD Research Grant to conduct part of his Ph.D. research work for two years (2019-2021) at Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. Sagar is interested in research on actinobacteria, myxobacteria, and natural products. He is the Research Head of the Department of Natural Products, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal. Sagar has more than ten years of experience in blogging, content writing, and SEO. Sagar was awarded the SfAM Communications Award 2015: Professional Communicator Category from the Society for Applied Microbiology (Now: Applied Microbiology International), Cambridge, United Kingdom (UK). Sagar is also the ASM Young Ambassador to Nepal for the American Society for Microbiology since 2023 onwards.

3 thoughts on “Triple Sugar Iron (TSI) Agar- Composition, Principle, Preparation, Results, Uses”

  1. Please concerning this ‘Blackening of the medium: Occurs in the presence of H2” the colour change rather occurs in the presence of Hydrogen sulfide (H2S) not H2

  2. Hi, I am a lab student and I have to write an riskanalysis about the TSI Agar. To make a riskanalyse, I have to choose between risk category E1-E2 or E3-E4. Do you have any idea about the risk category?
    I really appreciate your help!


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