Microbiology - 013 - Triple Sugar Iron Test

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Triple sugar iron agar, or TSI, is a differential medium that tests a bacterial strain for several different properties at once. Much like the liquid medium used to test for carbohydrate fermentation, TSI contains the pH indicator phenol red to detect the production of acid from fermentation, and the medium will turn yellow in the presence of acid. However, TSI slants are solid instead of liquid, so any gas produced from fermentation will form bubbles rather than collecting in a Durham tube. These bubbles will form along the side or bottom of the tube, sometimes forming fractures in the agar. TSI also contains three different carbohydrates instead of just one – lactose, sucrose, and a small amount of glucose are included in the medium, along with ferrous sulfate to test for hydrogen sulfide production. Inoculate a TSI slant by touching a sterile needle to a pure colony of test bacteria, then stabbing the needle straight down into the agar. Before removing the needle from the tube, also streak the surface of the agar with the needle. Incubate the tube at 37 degrees Celsius overnight. After incubation, observe the color of both the top and the bottom of the agar slant. If the entire slant is yellow, the bacteria were able to ferment lactose, sucrose, or both. It’s not possible to differentiate between lactose and sucrose fermentation without additional tests. If gas was also produced, there will be bubbles within or beneath the agar. If only the bottom of the agar is yellow but the top remains red, the bacteria were only able to ferment glucose, but not lactose or sucrose. There is a much smaller amount of glucose in the media than the other two carbohydrates, so only part of the agar changes color. Bubbles in the agar would also indicate the production of gas from glucose fermentation. If the agar remains red after incubation, the bacteria were not able to ferment any of the carbohydrates in the medium. Another result to check is the appearance or absence of a black precipitate in the agar. If there is a black color, the bacteria produced hydrogen sulfide, which reacted with the ferrous sulfate in the medium to form ferrous sulfide, which is black. This result is separate from the carbohydrate fermentations, and the black precipitate can make it difficult to check the color of the top and bottom of the agar. A shorter incubation time may be required to prevent the precipitate from spreading so quickly. Alternatively, it is sometimes necessary to incubate a TSI tube for up to 48 hours to see the first hints of hydrogen sulfide production.

Triple sugar iron agar, or TSI, is a differential medium that tests a bacterial strain for several different properties at once. It tests for acid and gas production from the fermentation of glucose and sucrose and/or lactose and for the production of hydrogen sulfide.

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