A basic protocol of TSIA test

1. Ms. Sadhana Gupta
Lecturer Bhilai Mahila Mahavidyalaya

2. AIM: Identification of Enteric Pathogen by TSIA medium
REQUIREMENTS –
Microbial culture, test tube, inoculating loop, Bunsen burner , TSIA
medium , conical flask, pipette.
PRINCIPLE-
The triple sugar- iron agar test employing Triple Sugar Iron Agar is
designed to differentiate among organisms based on the differences in
carbohydrate fermentation patterns and hydrogen sulfide production.
Carbohydrate fermentation is indicated by the production of gas and a
change in the colour of the pH indicator from red to yellow. If gas is
produced during fermentation, bubbles will form along the stab line,
fracturing or displacing the medium

3. TSI Agar composition

4. PROCEDURE-
1)Prepare TSIA medium and pour it in test tubes , allow it to solidify.
2)With a straight inoculation needle, touch the top of a well-isolated
colony.
3)Inoculate TSI by first stabbing through the center of the medium to the
bottom of the tube and then streaking the surface of the agar slant.
4)Leave the cap on loosely and incubate the tube at 35°-37°C in ambient
air for 18 to 24 hours.
5)Examine the reaction of medium

5. OBSERVATION –
Following observation may be seen-
An alkaline/ acid (red slant/yellow butt) reaction: It is indicative of
dextrose fermentation only.
An acid/acid (yellow slant/yellow butt) reaction: It indicates the
fermentation of dextrose, lactose and/or sucrose.
An alkaline/alkaline (red slant, red butt) reaction: Absence of
carbohydrate fermentation results.
Blackening of the medium: Occurs in the presence of H2 S
Gas production: Bubbles or cracks in the agar indicate the production of
gas ( formation of CO2and H2)

6. Inoculated tubes of TSIA medium

7. Result and interpretation - Tubes were observed for acid and gas
production and it can be interpreted by following table
S.N RESULT (SLANT/BUTT) INTERPRETATION
1
Red/yellow
Glucose fermentation only,
peptone catabolized.
2
Yellow/yellow
Glucose and lactose and/or
sucrose fermentation
3
Red/red
No fermentation, peptone
catabolized under aerobic
and/or anaerobic conditions
4 Yellow/yellow with bubbles Glucose and lactose and/or
sucrose fermentation, gas
produced
5
Red/yellow with bubbles
Glucose fermentation only, gas
produced.

8. Cont……..
S.N RESULT (SLANT/BUTT) INTERPRETATION
6 Red/Yellow with bubbles and
black precipitate
Glucose fermentation only, Gas
produced, H2S produced
7 Yellow/Yellow with bubbles and
black precipitate Glucose and lactose and/or sucrose
fermentation, Gas produced, H2S
produced.
8 Red/Yellow with black precipitate Glucose fermentation only, H2S
produced
9 Yellow/Yellow with black
precipitate Glucose and lactose and/or sucrose
fermentation, H2S produced

9. PRECAUTIONS –
1. Sterilize the glassware properly.
2. Inoculate the tubes in butt region carefully.
3. Loosely cover the test tubes for proper reaction.

10. thank you