This image contains the various reactions possible in a triple sugar iron (TSI) agar slant. The medium contains three sugars: glucose, sucrose, and lactose. The glucose is at 0.1% concentration whereas lactose and sucrose are present at 1.0% concentration in the medium. When inoculating the agar, the slants were streaked and then stabbed to the bottom of the tube. The tubes were incubated at 37 degrees Celsius for 16 hours.

Organisms present in the images are Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Shigella sonnei, and Salmonella typhi. P. aeruginosa was added as an example of what a nonfermentor would look like on a TSI slant. An uninoculated control was added to give the student a reference for what the tube looks like before incubation.

Initially, the bacteria will degrade the glucose in the medium lowering the pH. When this happens the pH indicator, phenol red, will turn yellow in the slant and the butt. About 8 to 10 hours into the incubation, the bacteria will use up the glucose. If the organisms can utilize either lactose or sucrose, they will continue to produce large quantities of acid, due to the higher lactose and sucrose concentrations, resulting in a yellow slant and butt at 16 to 24 hours of incubation. If the organisms cannot utilize sucrose or lactose, they will begin to break down the proteins in the media for energy. When this happens, amines are released and the pH rises changing the pH indicator back to red. This only happens aerobically. Therefore, glucose-fermenting organisms that cannot utilize lactose or sucrose will cause the TSI media to have a red slant and yellow butt at 16 to 24 hours of incubation. If a nonfermenting organism is placed on a TSI slant, the pH of the medium will not go down, and the medium will be red in the slant and the butt at 16 to 24 hours of incubation.

Some organisms produce H₂S gas during fermentation. This gas will reduce the ferrous sulfate in the medium causing a black precipitate to form in the butt of the tube at 16 to 24 hours of incubation. Other organisms produce gases when fermenting glucose that will cause pockets of air and cracks in the agar in the butt of the tube. In some cases enough gas is produced to cause the agar to move up from the bottom of the tube.

The organisms in the image came from various patients and stock culture collections. Knowing the TSI slant reactions helps in determining the genera of members of the family Enterobacteriaceae. This family of bacteria consists of straight rods that are Gram negative, oxidase negative, non-spore forming, and non-acid fast. They are facultative anaerobes (can grow with or without oxygen), nonhalophylic (unable to grow in high salt), and chemoautotrophs (able to grow on simple organic carbon and nitrogen compounds). They can carry out respiratory and fermentative metabolism and form both acid and gas from glucose. All genera in this family ferment glucose and will produce enough acid to cause the butt of the slant to turn yellow by 8 to 10 hours of incubation. Most strains of certain genera are able to produce acid when fermenting lactose or sucrose (Cedecea, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Kluyvera, Proteus, Providencia, Serratia, Tatumella, and Yersinia), whereas other genera are not able to ferment these sugars (Edwardsiella, Erwinia, Shigella, Salmonella, Morganella). All the genera contain gas-producing strains when fermenting sugars except Erwinia, Shigella, and Tatumella. Only Citrobacter, Edwardsiella, Proteus, and Salmonella contain H₂S-gas-producing strains.