Triple sugar iron agar test is designed in such a way, to differentiate the different groups of the Enterobacteriaceae family, that are all gram-negative and are capable of fermenting glucose which initiates the production of acid and to differentiate them from the other species of gram-negative bacilli present in the intestine.

This differentiation is purely based on the fermentation of the monosaccharides like glucose, fructose and lactose which results in production of one of the acids, hydrogen sulfide.

This Triple Sugar Iron medium usually consists of about 10 parts of lactose, 10 parts of sucrose and 1 part of glucose and peptone. Here, phenol red and ferrous sulphate acts as an indicator which detects the presence of carbohydrates and it expresses it by the action of color change.

Usually here in the presence of carbohydrate in the medium, the colors change is from organ red to yellow, at the time of presence of acids. In case of oxidative decarboxylation of the peptone, the alkaline products in the medium built and results in increase in the ph. This change is indicated by change in color of the medium to orange red to deep red.

Whereas the sodium thiosulphate and the ferrous sulphate that are being present in the medium, detects the production of hydrogen sulphide which is indicted by formation of black color butt in the tube.

Initially glucose is utilized by a fermentative organism present in the medium, thus changes the entire medium into yellow when it becomes acidic within 8 to 12 hours. But this remains acidic even after 18 to 24 hours due to the presence of the organic acids and results in fermentation of glucose under the anaerobic conditions in the butt of the tube.

However, the slant reverts to the alkaline by change in color to red as of oxidation of the fermentation products under aerobic conditions on the slant. This change results in the formation of the carbon dioxide and water and oxidation of peptones in the medium to the alkaline amines. On addition to monosaccharides such as glucose, lactose and sucrose gets fermented.

Due to this fermentation products large amount of fermentation products are formed along the slant, thus neutralizing the alkaline amines and renders the slant acid, which is yellow in color because of 18-to-24-hour reaction. In case, if the slant and butt convert in alkaline form which shows that glucose is not yet fermented.

The organisms showing this reaction are usually called as non-fermenters and they derive their nutrition from the peptones that are present in the medium. And the presence or synthesis of carbon dioxide and hydrogen gas in the reaction is indicated by formation of bubbles or by observing the crack in the medium of the agar or in some cases it may also lead to separation of agar from the sides or from the bottom of the tube.

Whereas the production of hydrogen gas in the medium requires an acidic environment which leads to the blackening of the agar butt on reaction with ferric ammonium citrate in the tube.