A pH indicator incuded in the medium can detect acid production from fermentation of these carbohydrates. A yellow color change indicates acid in the medium while no color change indicates an alkaline surrounding. Inoculation of the tube is a two step procedure. First, a loop of bacteria is spread across the surface of the agar. Carbohydrate uilization can be determined through analysis of the extent of acid production.
Acid production limited to only the butt of the tube is indicative of glucose utilization.
This is because the concentration of glucose is lower than that of other sugars, thus the acid production is not very extensive. Acid production in the slant and butt indicates sucrose or lactose fermentation because of the relatively high concentrations of these sugars, thus leading to extensive acid production. TSI agar can also detect reduction of sodium thiosulfate to hydrogen sulfide. Hydrogen sulfide production will turn parts of the agar black. Production of other gases is marked by cracks in the agar as well as an air gap at the bottom of the test tube.