Triple Sugar Iron (TSI) Agar

Test

• Prepare the TSI culture medium according to the instructions in the tube.
• Inoculate the desired sample on the surface of the slant anf botton of the tube.
• Partially close the tube lid and incubate at 35±2°C for 18-24 hours under aerobic conditions.

Results and Interpretation

• After the incubation period, compare the reactions of test samples with standard assays.
• The sugar fermentation reaction in this medium is indicated by a color change in the medium. If the bottom of the culture medium turns yellow (acidic reaction) but the slanted part of the medium remains red (alkaline reaction), it means that the bacteria are only capable of fermenting glucose.
• If the entire culture medium in the tube turns yellow, it means that the bacteria are capable of fermenting glucose, lactose, and/or sucrose.
• If the entire culture medium remains red, it indicates the presence of non-fermenting bacteria.
• The production of hydrogen sulfide in this medium is indicated by the blackening of the bottom of the culture medium in the tube and the production of gas by the rising of the medium or by cracking.

Note

• Production of hydrogen sulfide on Kligler Iron Agar medium may be better detectable than TSI medium. Research has shown that the use of sucrose by bacteria in TSI medium can suppress enzymatic reactions for hydrogen sulfide production. Also, not all H₂S positive Salmonella species are capable of producing hydrogen sulfide in TSI medium.
• Adding sucrose to TSI culture medium is performed to limit some non-lactose fermenting bacteria such as Proteus and Citrobacter species.
• Biochemical tests and serological typing should also be performed for more accurate and definitive identification of microorganisms.
• When culturing bacteria on this medium, the loop should not be used because if it is cultured deeply, the agar may crack, causing false positive gas production results.
• Fresh cultures should be used for culturing bacteria. Using mixed cultures (multiple colonies) results in unreliable results.
• The tube caps should be half-open. In this way, air exchange is better facilitated and alkaline reactions are more likely to occur on the slanted part.