Triple Sugar Iron Test

Aim
To detect whether the given test organism can ferment sugars and produce H₂S by the triple sugar iron test.

Introduction
Triple sugar iron agar is used for differentiation of members of Enterobacteriaceae, according to their ability to ferment lactose, sucrose, dextrose, and produce H₂S. The medium contains phenyl red as a pH indicator. The indicator at different pH shows different colors in the medium. When oxidative decarboxylation of proteins take place, the H₂S produced imparts a black precipitate resulting from the reaction between H₂S and ferrous sulfate present in the medium (usually hydrogen, carbon dioxide). This is visible as bubbles in the medium or cracking of the medium.

Principle
The medium contains a small amount of dextrose as compared to lactose and sucrose, which results in the formation of very little amount of acid by dextrose fermentation. This is quickly oxidized by aerobic growth on the slant, which reverts back to the color of the slant to pink in absence of lactose and sucrose fermentation. Thus pink slant and yellow butt is dextrose fermentation.

Requirements
Test cultures test tubes, conical flasks, glass rod, inoculation loop, and triple sugar iron agar.

Procedure

1. Prepare the TSI media and distribute among all the tubes.
2. Sterilize the media in the test tubes in an autoclave for 15 minutes at 15 lb/sq inch.
3. Cool the tubes after sterilization and prepare the slants in such a way that a small butt region remains in the bottom of the tube and an upper slant region.
4. Allow this to solidify.
5. Under aseptic conditions, using an inoculation loop, take the culture of the test organisms and pierce into the butt region and streak in the slant region.
6. Incubate the tube for 24–48 hrs at 37°C and then record the observations.