3.2.1: Triple Sugar Iron Agar Test

Triple sugar iron agar is an agar utilized to assess the ability of a bacterium to ferment sugars and produce hydrogen sulfide gas (H2S). Bacteria differ in their abilities to metabolize sugars. Some metabolize a greater variety of sugars than others. In metabolizing the sugars some species will produce gases. One such gas that may be produced during sugar metabolism is hydrogen sulfide gas.
Triple sugar iron agar contains three different sugars glucose (0.1%), sucrose (1%), and lactose (1%). In addition to the sugars the media contains the pH indicator phenol red, peptones, sodium thiosulfate, and ferrous ammonium sulfate.

After inoculation and incubation of the media, some bacteria may ferment glucose, sucrose, and/ or the lactose present in the media. In doing so they produce acidic products which lower the pH of the medium, causing the phenol red to turn yellow. Some bacteria however, can only metabolize the glucose. Glucose is present in a much lower concentration than the sucrose and lactose, therefore the bacteria would exhaust the sugars rather quickly as they ferment them. This would turn the media yellow initially. After exhausting the glucose, the bacteria can then metabolize the peptones present in the media producing ammonium as a result. Ammonium would increase the pH of the medium making it more alkaline, resulting in the media changing from yellow to red. This would occur in regions of the tube that are exposed to oxygen. The areas of the media that are anaerobic (lacking oxygen) would remain acidic, and thus continue to appear yellow.

In addition to assessing sugar metabolism the media can also reveal whether the bacterium produces hydrogen sulfide gas during the fermentation of the sugars. If the bacteria is capable of hydrogen sulfide gas production it will convert the sodium thiosulfate to hydrogen sulfide gas. Hydrogen sulfide gas is a colorless gas, so in order to detect its presence, the ingredient ferrous ammonium sulfate is included. The hydrogen sulfide gas interacts with the ferrous ammonium sulfate to produce a black precipitate. Other gases produced by fermentation, such as carbon dioxide or oxygen gas, will be detected due to bubbles or cracks in the media.

This test is an important test in helping differentiate members of the family Enterobacteriaceae.

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