TRIPLE SUGAR IRON TEST

Triple Sugar Iron Reaction

AIM:

To understand the biochemical reactions involved in the triple sugar iron agar test

PRINCIPLE:

The triple sugar iron (TSI) agar test is generally used for the identification of enteric bacteria Enterobacteriaceae). It is also used to distinguish the Enterobacteriaceae from other gram-negative intestinal bacilli by their ability to catabolize glucose, lactose, or sucrose, and to liberate sulfides from ferrous ammonium sulfate or sodium thiosulfate. (TSI agar slants contain a 1% concentration of lactose and sucrose, and a 0.1% glucose concentration. The pH indicator, phenol red, is also incorporated into the medium to detect acid production from carbohydrate fermentation. Often Kligler Iron Agar (named after I. J. Kligler in 1917), a differential medium similar to TSI, is used to obtain approximately the same information. TSI slants are inoculated by streaking the slant surface using a zig-zag streak pattern and then stabbing the agar deep with a straight inoculating needle. Incubation is for 18 to 24 hours in order to detect the presence of sugar fermentation, gas production, and H₂S production.

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:  a large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose.

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 source of nitrogen. (Remember that whenever peptone is utilized under aerobic condition ammonia is produced)

MATERIALS REQUIRED:

Triple Sugar Iron Agar tubes, Culture of enteric bacteria, Bunsen burner, inoculating needle, Incubator set at 35°C and test-tube rack

Procedure for Triple Sugar Iron Agar (TSI) Test

1. TSI slant was prepared as per the formulation given by the lab manual.

2. With a sterilized straight inoculation needle touch the top of a well-isolated colony.

3.  TSI Agar slant was inoculated by first stabbing through the center of the medium to the bottom of the tube and then streaked on the surface of the agar slant. 

4. The cap left on loosely and incubated the tube at 35°C in ambient air for 18 to 24 hours.

OBSERVATIONS:

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 -), H2s (+ or -)
4. Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or sucrose fermenter gas (+ or -), H2s (+ or -).

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 produces bubbles/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 peptone is a major constituents of TSI Agar).

4. If H₂S is produced, the black color of ferrous sulfide is seen.