TSI Test Introduction, Principle, procedure, Result , Interpretation and

TSI Test: Introduction, Principle, Procedure, Result-Interpretation and Limitation

Introduction of TSI Test

This single TSI test is useful for the following features-

To know the carbohydrate utilization ability of the organisms
Gas formation by the organisms
Hydrogen sulfide production by the bacteria

TSI stands for triple sugar iron and it is an agar medium recommended for use in the differentiation of Enterobacteriaceae by their ability to ferment glucose, lactose, and sucrose, and their ability to produce hydrogen sulfide.

Principle of TSI Test

Triple sugar iron agar contains casein and meat peptones, phenol red as the pH indicator, 0.1% glucose, 1% lactose, and 1% sucrose for fermentation. Ferric or ferrous ions and sodium thiosulphate are present to detect hydrogen sulfide production. Bacteria that are non-lactose fermenting initially produce a yellow slant due to the production of acid from the glucose. The small amount of glucose is rapidly depleted. Oxidation metabolism continues in the slant after the low concentration of glucose has been depleted, producing an alkaline pH from the aerobic breakdown of peptone; the slant turns red. There is no oxygen penetration into the butt and no oxidative metabolism; the butt remains acid and yellow. Thus, a non-lactose fermenting bacterium yields an alkaline (K) slant over an acid (A) butt ( K/A; red slant; yellow butt). Lactose/sucrose bacteria continue to produce a large amount of acid in the slant and in the butt so the reaction in both remains acid (A/A; yellow slant; yellow butt). If the slant and butt remain neutral, the organism is not capable of fermenting glucose or other sugars (K/K; red slant; red butt) Bubble, fracturing, or displacement of the medium indicates gas production by the organism due to sugar fermentation. Blackening of the medium denotes hydrogen sulfide production by the action of the bacteria with sodium thiosulfate and which is detected by the reduction of the ferric ions to produce a black precipitate.

Requirements for TSI Test

Test organism
Triple sugar iron agar
Bunsen burner and inoculating needle
BOD incubator
Test tubes rack
QC strains for quality control

Procedure of TSI Test

Warm medium to room temperature and examines for cracks and do not use if cracks appear.
Touch the center of a well-isolated colony by using a sterile inoculating needle.
Stab to within 3-5 mm from the bottom of the tube.
Place cap loosely on tube
Incubate aerobically at 35-37°C for 18-24 hours.

Observation and Interpretation of TSI Test Result

Examine the reaction in the slant and the butt also observe gas and hydrogen sulfide production.

Yellow: Acid reaction
Red: Alkaline reaction
Blackening of the medium: H₂S production
Bubbles, cracks, or displacement of the medium: Gas production

Interpretation of Carbohydrate

A/A: Glucose and lactose or sucrose fermented
K/A: Only glucose fermented or non-lactose fermenter
K/K: No carbohydrate fermented or non-glucose fermenter

Result

Tube No 1

K/K
No H₂S production
No gas formation

Tube No 2

R/A
H₂S positive
No gas formation

Tube No 3

A/A
Gas formation positive
No H₂S formation

Limitations of TSI Test

Do not read the TSI test before 18 hours, since false readings of acid in the slant may result.
H₂S production may on be inhibited on the TSI test for organisms that utilize sucrose and suppress the enzyme mechanism that results in the production of H₂S.
Sulfide indole motility (SIM) agar is more sensitive in the detection of H₂Sthan either TSI or KIA.

Keynotes

Kligler’s Iron Agar(KIA) test only differs from the Triple Sugar Iron agar (TSI) test due to lacking sucrose in its composition.
Do not attempt to interpret sugar fermentation reactions after 24 hours. Refrigerate tubes if reading will be delayed.
If desired, extend incubation only to detect H₂Sproduction. Campylobacter may take 3 days for the production of H₂S.

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