Triple Sugar Iron (TSI) Agar: Principle, Results, and Interpretation

By Acharya Tankeshwar •  Updated: 06/06/22 •  4 min read

Whenever you see the name of this test i.e. ‘Triple Sugar Iron Agar‘, you have to remember that it’s a test that has three sugar (lactose, sucrose, and glucose) and also iron; and it contains agar as solidifying agent (TSI is a semi-solid media having slant and butt).

Composition of TSI Agar



Beef extract

3.0 g
Yeast extract

3.0 g

Peptone 20.0 g
Glucose 1.0 g


10.0 g

Sucrose 10.0 g
Ferrous sulfate or ferrous ammonium sulfate 0.2 g
NaCl 5.0 g
Sodium thiosulfate 0.3 g
Phenol red 0.024 g

13.0 g

Distilled water 1,000 mL

Lactose, sucrose and glucose are in the concentration of 10:10:1 (i.e. 10 part lactose (1%), 10 part sucrose (1%) and 1 part glucose (0.1%)). TSI is similar to Kligler’s iron agar (KIA), except that Kligler’s iron agar contains only two carbohydrates: glucose (0.1%) and lactose (1%).

  • 0.1% glucose: If only glucose is fermented, only enough acid is produced to turn the butt yellow. The slant will remain red.
  • 1.0 % lactose/1.0% sucrose:  If lactose or sucrose or both sugars are fermented, a large amount of acid will produce which turns both butt and slant yellow. So the appearance of yellow color in both slant and butt indicates that the isolate has the ability to ferment lactose or sucrose or both. 
  • Iron (ferrous sulfate): Indicator of H2S formation
  • Phenol red: Indicator of acidification (It is yellow in acidic conditions and red under alkaline conditions).
  • It also contains peptone which acts as a source of nitrogen (remember that whenever peptone is utilized under aerobic condition, ammonia is produced).

Why Sucrose is added to TSI Agar? 

Inoculation in TSI Agar
Inoculation in TSI agar

Addition of sucrose in TSI agar permits earlier detection of coliform bacteria that ferment sucrose more rapidly than lactose. Adding sucrose also aids the identification of certain gram-negative bacteria that could ferment sucrose but not lactose. Another basic understanding is TSI tube contains a butt-poorly oxygenated area on the bottom and a slant-angled well-oxygenated area on the top.

Preparation of TSI Agar

  • Combine the ingredients, and adjust the pH to 7.3
  • Boil to dissolve the agar
  • Dispense it into tubes
  • Sterilize by autoclaving at 121°C for 15 minutes
  • Cool in a slanted position to give a 2.5 cm butt and a 3.8 cm slant.

TSI agar is also available commercially

Procedure for TSI Agar Test

  1. With a sterilized straight inoculation needle touch the top of a well-isolated colony
  2. Inoculate TSI agar by first stabbing through the center of the medium to the bottom of the tube and then streaking on the surface of the agar slant. 
  3. Leave the cap on loosely and incubate the tube at 35°C in ambient air for 18 to 24 hours.

Principle of TSI Agar Test

  1. If lactose (or sucrose) is fermented, a large amount of acid is produced, which turns the phenol red indicator yellow both in the butt and in the slant. Some organisms generate gases, which produce bubbles/cracks in the medium.
  2. If lactose is not fermented but the small amount of glucose is, the oxygen-deficient butt will be yellow (remember that butt has comparatively more glucose than slant i.e. more media and more glucose), but on the slant, the acid produced (less acid produces in slant as media in slant is less) will be oxidized to carbon dioxide and water by the organism and the slant will be red (alkaline or neutral pH).
  3. If neither lactose/sucrose nor glucose is fermented, both the butt and the slant will be red. The slant can become a deeper red-purple (more alkaline) as a result of the production of ammonia from the oxidative deamination of amino acids (remember peptone is a major constituent of TSI agar).
  4. if H2is produced, the black color of ferrous sulfide is seen.

Expected results of TSI Agar test

TSI Agar Test results
Triple Sugar Iron Agar Test Results Image source: Clark College
  1. Alkaline slant/no change in butt (K/NC) i.e Red/Red = glucose, lactose, and sucrose non-fermenter
  2. Alkaline slant/alkaline butt (K/K) i.e Red/Red = glucose, lactose, and sucrose non-fermenter
  3. Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation only, gas (+ or -), H2 (+ or -)
  4. Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or sucrose fermenter gas (+ or -), H2 (+ or -).

Results of Triple Sugar Iron (TSI) Agar Tests

Name of the organismSlantButtGas H2S

Escherichia, Klebsiella, Enterobacter
Acid (A)Acid (A)Pos (+)Neg (-)

Shigella, Serratia
Alkaline (K)
Acid (A)
Neg (-)Neg (-)

Salmonella, Proteus
Alkaline (K)
Acid (A)

Pos (+)

Pos (+)

Alkaline (K)Alkaline (K)Neg (-)Neg (-)

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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