Whenever you see the name of this test i.e. Triple Sugar Iron Agar, you have to remember that it’s a test which 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 Triple Sugar Iron Agar (TSI)
Lactose, Sucrose and Glucose in the concentration of 10:10:1 (i.e. 10 part Lactose (1%), 10 part Sucrose (1%) and 1 part Glucose (0.1%)).
- 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 sugar 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 condition 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 in TSI?
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. Other basic understanding is TSI Tube contains butt (poorly oxygenated area on the bottom) slant (angled well-oxygenated area on the top).
Procedure for Triple Sugar Iron Agar (TSI) 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.
Interpretation of Triple Sugar Iron 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 butt and in the slant. Some organisms generate gases, which producesbubbles/cracks on 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 comparatively have more glucose compared to slant i.e. more media more glucose), but on the slant the acid (less acid as media in slant is very 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 production of ammonia from the oxidative deamination of amino acids (remember peoptone is a major constituent of TSI Agar).
4) if H2S is produced, the black color of ferrous sulfide is seen.
- red/red: glucose, lactose and sucrose nonfermenter
- red/yellow: glucose fermentation only
- yellow/yellow: glucose, lactose and/or sucrose fermentor
- black: in the butt indicates production of H2S.
- bubles or crack in the tube: indicate the production of CO2 or H2
In 1917 Sulkin and Willett described a medium containing the carbohydrates, glucose, lactose and sucrose, and iron salts.
The medium showed fermentation of these carbohydrates, as well as hydrogen sulfide production.
Hajna modified the medium in 1945 to contain phenol red as the pH indicator, and is the formulation still in use today.
MMIZ, ErasmusMC, Rotterdam