Triple Sugar Iron (TSI) Agar Test: Introduction, Composition, Principle, Test Requirements, Procedure, Result- Interpretation, Keynotes, and TSI Test Footages

Triple Sugar Iron Agar Test

The triple Sugar Iron (TSI) Agar test is a combo test and is useful for the identification of mainly gram-negative bacteria, on the basis of the following tests-

  • fermenter or oxidizer or non-utilizer of carbohydrates like glucose, lactose, or sucrose
  • production of hydrogen sulfide (H2S)
  • formation of gas
Triple Sugar Iron (TSI) Agar Test: Introduction, Composition, Principle, Test Requirements, Procedure, Result- Interpretation, Keynotes, and TSI Test Footages
Various bacterial biochemical reactions in Triple Sugar Iron (TSI) Agar Test

Composition of Triple Sugar Iron Agar

Triple Sugar Iron Agar (TSI) (Oxoid, England)

Ingredients      gm/liter
Lab-lemco powder   3.0
Yeast Extract 3.0             
Peptone         20.0
Lactose                10.0
Sucrose            10.0
Glucose             1.0
Ferric Citrate 0.3
Sodium Chloride   5.0
Sodium Thiosulphate 0.3
Phenol Red   0.024
Agar 12.0
Final pH (at 25ºC)     7.4±0.2
Table: Triple Sugar Iron Agar (TSI)

Preparation of Triple Sugar Iron Agar

  • Dissolve 65.0 grams of the medium in 1000 ml of distilled water.
  • Sterilize by autoclaving at 115ºC pressure for 15 minutes.
  • Allow the medium to set in slope form with a butt about 2.5 inches in length as shown above image.

Principle of Triple Sugar Iron (TSI) Agar Test

The Triple Sugar Iron (TSI) Agar contains casein and meat peptones, phenol red as the pH indicator, 0.1% glucose, 1% lactose, and 1% sucrose for fermentation. Ferric or ferrous ions and sodium Thiosulphate are present to detect hydrogen Sulphide production. Organisms that are non-lactose fermenting initially produce a yellow slant due to the production of acid from glucose. The small amount of glucose is rapidly depleted. Oxidative metabolism continues in the slant after the low concentration of glucose has been depleted, producing an alkaline pH from the aerobic breakdown of peptone; the slant turns red. There is no oxygen penetration into the butt and no oxidative metabolism; the butt remains acid and yellow.

Thus, a non-lactose fermenting organism yields an alkaline (K) slant over an acid (A) butt (K/A); red slant; yellow butt). Lactose fermenting and/or sucrose-fermenting bacteria continue to produce a large amount of acid in the slant and butt so the reaction in both remains acid (A/A; yellow slant; yellow butt). If the slant and butt remain neutral, the organism is not capable of fermenting glucose or other sugars (K/K; red slant; red butt). Gas production from sugar fermentation is indicated by bubbles, the fracturing of the medium, or displacement of the medium. Hydrogen Sulfide is produced by the action of the bacteria with sodium Thiosulphate. This is detected by the reduction of ferric ions to produce a black precipitate.

Test Requirements

  • TSI agar
  • Test organisms (bacteria)
  • BOD Incubator
  • Inoculating loop and bunsen burner
  • Control strains

Procedure of Triple Sugar Iron Agar Test

  1. Stab and streak the test organism on the surface of TSI.
  2. Incubate at 37ºC for 24 hours.
  3. Acid production limited only to the butt region of the tube is indicative of glucose utilization, while acid production in the slant and butt indicates sucrose or lactose fermentation.

Result and interpretation of Triple Sugar Iron Agar Test

  1. Yellow (Acid)/ Yellow (Acid), Gas, H2S: Lactose/Sucrose fermenter, H2S producer.
  2. Red (Alkaline)/ Yellow (Acid), No gas, No H2S: Only Glucose, not Lactose/Sucrose fermenter, anaerogenic, No H2S production.
  3. Red (Alkaline)/ No change: Glucose, Lactose, and Sucrose Non-fermenter.
  4. Yellow (Acid)/ No change: Glucose oxidizer
  5. No change/No change: Non-fermenter.

Limitations of the TSI Test

  1. Do not read the TSI  test before 18 hours, since false readings of acid in the slant may result.
  2. H2S production may on be inhibited on the TSI test for organisms that utilize sucrose and suppress the enzyme mechanism that results in the production of H2S.
  3. Sulfide indole motility (SIM) agar is more sensitive in the detection of H2S than either TSI or KIA.


  1. Kligler’s  Iron Agar (KIA) test only differs from the Triple Sugar Iron agar (TSI) test due to lacking sucrose in its composition.
  2. Do not attempt to interpret sugar fermentation reactions after 24 hours. Refrigerate tubes if the reading will be delayed.
  3. If desired, extend incubation only to detect H2Sproduction. Campylobacter may take 3 days the production of H2S.

TSI Test Footages

Escherichia coli in TSI agar

Escherichia coli in TSI agar
Fig. Escherichia coli in TSI agar

Klebsiella pneumoniae TSI test

Klebsiella pneumoniae TSI test
Fig. Klebsiella pneumoniae TSI test

Acinetobacter TSI reaction

Acinetobacter TSI reaction
Fig. Acinetobacter TSI reaction

Proteus in TSI agar

Proteus in TSI agar
Fig. Proteus in TSI agar

Salmonella Typhi Biochemical Reaction

Salmonella Typhi Biochemical Reaction
Fig. Salmonella Typhi Biochemical Reaction

Pseudomonas aeruginosa in TSI medium

Pseudomonas aeruginosa in TSI medium
Fig. Pseudomonas aeruginosa in TSI medium

Further Readings

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  2. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  4. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  6.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  7. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melbourne, Sans Francisco 1996.

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