This image depicts four test tubes each containing triple sugar iron agar (TSI) medium. Test tube 1 only contained the agar medium, hence was uninoculated. Test tube 2, contained medium that was inoculated with Shigella sp. bacteria. Test tube 3 contained medium inoculated with Providencia sp. bacteria. Test tube 4 contained medium inoculated with Pseudomonas sp. bacteria.

The TSI agar contains three sugars: lactose, sucrose, and glucose, as well as a pH-sensitive dye such as phenol red, sodium thiosulfate, ferrous sulfate, or ferrous ammonium sulfate. Due to the organism's fermentation of glucose in the absence of oxygen, an acid is produced, changing the dye from red to yellow early on in the experiment. However, after all glucose has been consumed by the Shigella bacteria, they will begin to use oxygen in their catabolic reaction, leading to the formation of alkaline end-products, thereby, changing the phenol-red dye back to a red color in the region of the slant.