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Triple Sugar Iron Agar Test

Triple Sugar Iron Agar test is a combo test and useful for identification of mainly gram-negative bacteria, on the basis of following tests-

• fermenter or oxidizer or non-utilizer of carbohydrates like glucose, lactose, or sucrose
• production of hydrogen sulfide (H2S)
• formation of gas

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

Preparation of Triple Sugar Iron Agar

1. Dissolve 65 grams of the medium in 1000 ml of distilled water.
2. Sterilize by autoclaving at 115ºC pressure for 15 minutes.
3. Allow the medium to set in slope form with a butt about 2.5 inches in length.

Principle of Triple Sugar Iron (TSI) Agar Test

The 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.

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 glucose utilization, while acid production in 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.

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. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. 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.
4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
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. 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, Melborne, Sans Franscisco 1996.
7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.