To detect whether the given test organism can ferment sugars and produce H2
by the triple sugar iron test.
Triple sugar iron agar is used for differentiation of members of Enterobacteriaceae,
according to their ability to ferment lactose, sucrose, dextrose, and produce H2
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 H2
S produced imparts a black precipitate resulting from
the reaction between H2
S and ferrous sulfate present in the medium (usually
hydrogen, carbon dioxide). This is visible as bubbles in the medium or cracking
of the medium.
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.
Test cultures test tubes, conical flasks, glass rod, inoculation loop, and triple
sugar iron agar.
- Prepare the TSI media and distribute among all the tubes.
- Sterilize the media in the test tubes in an autoclave for 15 minutes at
15 lb/sq inch.
- 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
- Allow this to solidify.
- 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
- Incubate the tube for 24–48 hrs at 37°C and then record the observations.