Tsi test

Biochemical Test

1. TSI TEST TRIPLE SUGAR IRON AGAR TEST

2. Background TSI stands for three sugars – glucose, sucrose and lactose. Russell – used an agar medium with two sugars to aid in the identification of intestinal gram-negative bacilli Krunweide and Kohn modified Russell’s Double Sugar agar by adding a third sugar—sucrose Difco Laboratories , Sulkin and Willet , and Hajna described a similar triple sugar ferrous sulfate medium for the identification of enteric bacilli.

3. Major Content Glucose 0.1%(used first) Sucrose 1%(used second) Lactose 1% Proteins used third Phenol red pH indicator for acid production Ferric ammonium citrate H 2 S indicator NaCl maintains osmotic pressure Agar solidifying agent

4. Purpose used to determine carbohydrate fermentation and H 2 S production in bacteria. Gas from carbohydrate metabolism can also be detected. 

5. Principle Carbohydrates pyruvate(acid) + CO 2 Peptones NH 3 (makes medium alkaline) yellow Phenol red red Sodium thiosulfate in the medium is reduced by some bacteria to hydrogen sulfide (H 2 S), a colorless gas. The hydrogen sulfide will react with ferric ions in the medium to produce iron sulfide, a black insoluble precipitate. fermentation fermentation acid alkali

6. Medium TSI Agar (Tubed slant) Formula / Liter Enzymatic Digest of Casein ............................................... 5 g Enzymatic Digest of Animal Tissue................... 5 g Yeast Enriched Peptone ................................... 10 g Dextrose ........................................................... 1 g Lactose ............................................................ 10 g Sucrose ...................................................... ..... 10 g Ferric Ammonium Citrate ................................ 0.2 g Sodium Chloride ............................................... 5 g Sodium Thiosulfate ........................................... 0.3 g Phenol Red ...................................................... 0.025 g Agar ................................................................... 13.5 g Final pH: 7.3 ± 0.2 at 25 C

7. Theory Fermentation of carbohydrates results in the production of acid which decreases the pH of the medium to change from reddish-orange to yellow. Utilization of peptones results alkalization of the medium due to the production of NH 3 . The production of hydrogen sulfide is indicated by the presence of black ppt. formed by the reaction of H 2 S with ferric ions. Slant is aerobic while butt is anaerobic. Gas production is indicated by the splitting of the agar of lifting of it to the top.

8. Interpretation Based on carbohydrate utilization and hydrogen sulfide production, a TSI slant can be interpreted in several ways :

9. Glucose fermenter tube reaction ---- alkaline over acid (K/A ) Red slant , yellow butt With H 2 S production (K/A, H 2 S + ve ) Red slant, black butt

10. Glucose, Lactose and/or Sucrose Fermenter Tube reaction --- acid over acid (A/A) Yellow slant, yellow butt With H 2 S production(A/A, H 2 S + ve ): Yellow slant, black ppt. in the butt

11. Glucose, Lactose and Sucrose Non-fermenters Tube reaction: i) alkaline over alkaline(K/K) If the bacteria can metabolize peptones both aerobically and anaerobically. both slant and butt red ii) alkaline over no change (K/NC) If peptones can only be metabolized aerobically slant red, butt no change. With H 2 S production alkaline over no change ( K/NC, H 2 S + ve ) black precipitate (H 2 S ) in the butt.

12. Expected Cultural Response Slant Butt Gas H 2 S Proteus mirabilis K A - + Pseudomonas aeruginosa K K - - E. coli A A + - Shigella flexneri K A - -