The Triple Sugar Iron or TSI test is a microbiological test roughly named for its ability to test microorganism's ability to ferment sugars and to produce hydrogen sulfide.[1] It is often used in the selective identification of enteric bacteria including but not limited to Salmonella and Shigella.
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The TSI slant is a test tube that contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose,[2] as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate.
All of these ingredients are mixed together and allowed to solidify in the test tube at a slanted angle. The slanted shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying degrees (an aerobic environment) or not exposed to air (an anaerobic environment).
TSI agar medium was developed based on Kligler's iron agar, which had been used for the determination of lactose fermentative bacteria, by addition of sucrose to be able to detect sucrose fermentative bacteria also.
Bacteria that ferment any of the three sugars in the medium will produce byproducts.[3] These byproducts are usually acids, which will change the color of the red pH-sensitive dye (phenol red) to a yellow color. Position of the color change distinguishes the acid production associated with glucose fermentation from the acidic byproducts of lactose or sucrose fermentation. Many bacteria that can ferment sugars in the anaerobic butt of the tube are enterobacteria.
Some bacteria utilize thiosulfate anion as a terminal electron acceptor, reducing it to sulfide. If this occurs, the newly-formed hydrogen sulfide (H2S) reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a black precipitate. Examples of sulfide-producing bacteria include Salmonella, Proteus, Citrobacter and Edwardsiella species. The blackening of the medium is almost always observed in the butt (bottom) of the medium.
All lactose fermenters result in yellow slant/yellow butt (acid/acid reaction), whereas non-lactose fermenters may result in pink/yellow or yellow/yellow (if sucrose is fermented). Blackening of the butt due to H2S production may mask the acid reaction (yellow) in the butt. Salmonella enterica serovar Typhi may result in blackening of the medium at the interface of butt and slant.
Under anaerobic conditions (as occur toward the bottom of the tube) some bacteria use H+ as an electron acceptor and reduce it to hydrogen gas. This is not very soluble and may accumulate as bubbles along the inoculation track, between the agar and the glass, or in the fluid which accumulates at the bottom of the slant. Hydrogen production may lift the agar from the butt of the tube or fracture the agar. Carbon dioxide, if produced, may not show as bubbles because it is far more soluble in the medium.
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