Triple Sugar Iron Agar (TSIA) test - Introduction, Media, Principle, Biochemistry, Procedure, Result, Interpretation

Introduction to Triple Sugar Iron Agar test (TSIA)

The biochemical test triple sugar iron agar (TSIA) test is used to identify and/or differentiate gram-negative bacteria- especially the members of the Enterobacteriaceae family. This test is used to determine the ability of the organism to produce gas from sugar fermentation, the production of hydrogen sulfide gas, and the ability to ferment lactose and sucrose.

Media for Triple Sugar Iron Agar test (TSIA)

Although the Triple Sugar Iron Agar test (TSIA) is widely used, Kligler's iron agar (KIA) can be used. The TSIA contains three types of sugars- glucose (dextrose), lactose, and sucrose. Glucose is present in low concentrations while lactose and sucrose are present in high concentrations (10 times more).

Biochemistry of Triple Sugar Iron Agar test (TSIA)

Microorganisms, usually of the Enterobacteriaceae family, first metabolize glucose in both the slant (aerobically as oxygen is present to serve as the final electron acceptor) and in the butt where an anaerobic condition is prevalent. After all the glucose has been converted to pyruvate, it is further metabolized through the aerobic Krebs cycle in the slant and through an anaerobic cycle in the butt.

As a result, acidic end products are released which changes the pH indicator present in the medium - phenol red - to change to yellow. Thus both slant and but of the Triple Sugar Iron Agar test (TSIA) inoculated with glucose fermenters appear yellow. In cases of non-glucose fermenters, both the butt will remain red or even turn redder due to the release of alkaline end products.

After glucose is used up, the organisms may begin to metabolize ample lactose and sucrose present in the Triple Sugar Iron Agar test (TSIA). As the concentration of lactose and sucrose is high, the test organism will continue to break them down, making continuous acid end products. This reaction is coined as (A/A) and the organism is a lactose as well as sucrose fermenter.

If there is a production of gas, it will cause the media to break. In high gas producers, the entire media may have been raised in the tube.

In cases of the test organism not being able to use lactose and sucrose, it must utilize the protein present in the media. The protein metabolism, which primarily occurs on the slant where oxygen is plentiful, produces by-products such as NH₃ which is alkaline in nature. As a result, the phenol red indicator reverts back from yellow to red in color. Thus a non-lactose fermenter shows a red slant while the but remains yellow due to anaerobic glucose utilization. This reaction is termed alkaline/acid (Alk/Acid).

Moreover, glucose non-fermenters may metabolize peptone to produce alkaline end products and such reactions are termed alkaline/alkaline (alk/alk).

The Triple Sugar Iron Agar test (TSIA) can also detect hydrogen sulfide gas production. Some organisms are able to reduce amino acids with sulphur such as cysteine cystine, methionine while some can metabolize inorganic sulphur compounds including thiosulphate, sulphate, and sulphites. As a byproduct hydrogen sulfide gas (H₂S) having a characteristic rotten egg smell is released.

The H₂S gas if released can be detected by adding heavy metal salt with iron (FE²⁺) or lead (Pb²⁺) to the medium where sulphur substrates such as cysteine and sodium thiosulphate have been added. Even if the H₂S gas is colorless, it reacts with metal salt (FeSO₄) to produce an insoluble black FeS precipitate.

Biochemistry:

Cysteine → (cysteine desulfurase) → α amino-acrylic acid + H₂S

H₂S + FeSO₄ (Ferrous Sulphate) → FeS (Ferrous Sulphide) + H₂SO₄

Na₂S₂O₃ → (thiosulphate reductase) → Na₂SO₃ + 2 H₂S

Fe⁺⁺⁺ + H₂S → FeS (Ferrous Sulphide)

Procedure of Triple Sugar Iron Agar test (TSIA)

The procedure of Triple Sugar Iron Agar test (TSIA) includes:

1. Take a sterile Triple Sugar Iron Agar test (TSIA) and inoculate the test organism by stabbing the but followed by streaking the slant in a zic-zac path.

2. Incubate the inoculated tube at 35-37°C for 18-24 hours.

3. Observe for changes in color in the slant and butt.

Result, Interpretation of Triple Sugar Iron Agar test (TSIA)

A total of six types of reactions/results and interpretation are seen in the Triple Sugar Iron Agar test (TSIA) test:

1. A/A, G+ = glucose fermentation with acid and gas production; lactose and/or sucrose also fermented. Eg: E. coli, Klebsiella

   A/A, G+, H₂S+= glucose fermentation with acid, gas, and H₂S production; lactose and/or sucrose also fermented. Eg: Citrobacter

2. Alk/A, G+ = glucose fermentation with acid, gas production; lactose and/or sucrose not fermented. Eg: Salmonella paratyphi A

   Alk/A, G+, H₂S+ = glucose fermentation with acid, H₂S, and gas production; lactose and/or sucrose not fermented. Eg: Salmonella paratyphi B, Salmonella paratyphi C, Proteus

3. Alk/A = glucose fermented with no gas production; lactose and/or sucrose not fermented. Eg: Shigella

4. Alk/NC = glucose, lactose, sucrose non fermenter. Eg: Pseudomonas

5. NC/NC = glucose, lactose, sucrose non fermenter. Eg: Alkaligenes, Acenetobacter

6. A/NC = glucose metabolized oxidatively

*NC = no change