Fish Microbiology and Quality Assurance

9. Triple sugar iron agar (TSI) test

The TSI test is designed to differentiate among different groups/genera of ennterobacteriaceae which ferment glucose with the production of acid, and also distinguish enterobacteriaceae from other Gram negative intestinal bacilli. This differentiation is based on the differences in carbohydrate fermentation pattern and hydrogen sulphide production by various groups of intestinal bacteria.
The TSI agar slant contains three sugars (1% each of gucrose and lactose, and 0.1% glucose), and phenol red indicator which indicates the acid production due to carbohydrate fermentation by change in colour from orange red to yellow. Upon incubation both the butt and slant are observed for colour change (fermentative activities), gas production and hydrogen sulphide production

1. Alkaline slant (red) and acid butt (yellow) with or without gas production:

This reaction indicates fermentation of only glucose. The organism has preferentially utilized glucose present in small concentration and produce small amount of acid on slant surface, owering the pH initially (indicated by indicator turning yellow). But the utilization of peptone increases pH making the slant turn to red colour. In the butt, the initial acid reaction is maintained because of reduced oxygen and slow growth, and this makes tit appear yellow.

2. Acid slant (yellow) and acid butt (yellow) with or without gas production:

This reaction is indicative of utilization of lactose and/or sucrose, after initial exhaustion of glucose, which serve as substrates for continued fermentation and maintenance of acid reaction both in slant and butt.

3. Alkaline slant (red) and alkaline butt (red) or no change (orange red) butt.:

This reaction is indicative of inability of test organisms to ferment carbohydrate But utilization of peptones under aerobic and/or anaerobic condition results in ammonia production resulting in alkaline condition both in slant and butt. The degradation of peptone only by aerobic condition results in alkaline pH in slant.
Procedure:
The TSI slants are stab-streak inoculated with test bacterial culture using sterile inoculation loop, incubated for 18-24 hours at 37⁰C and observed for colour change both in slant and butt.

Triple sugar iron agar

Beef extract : 3.0 g
Yeast extract : 3.0 g
Peoptone : 15.0 g
Proteose peptone : 5.0 g
Lactose : 10.0 g
Saccharose : 10.0 g
Dextrose : 1.0 g
Ferrous sulphate : 0.2 g
Sodium chloride : 5.0 g
Sodium thiosulphate : 0.3 g
Phenol red (15 aqueous solution) : 2.5 ml
Agar : 15.0 g
Distilled watr : 1 l
pH : 7.4

Dissolve the ingredients by boiling and transfer 5 ml volume to test tubes, autoclave at 121⁰C for 15 minutes, and allow setting in a slope to get a butt and slant.