thumb
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 sulfideHydrogen sulfide is the chemical compound with the formula H
2S. This colorless, toxic and flammable gas is partially responsible for the foul odor of rotten eggs and flatulence....
. It is often used in the selective identification of enteric bacteria including but not limited to
SalmonellaSalmonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which project in all directions...
and
ShigellaShigella is a genus of Gram-negative, non-spore forming rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agent of human shigellosis, Shigella cause disease in primates, but not in other mammals. It is only naturally found in humans and apes. During infection,...
.
The
TSI slant is a test tube that contains
agarAgar or agar agar is a gelatinous substance derived from seaweed. Historically and in a modern context, it is chiefly used as an ingredient in desserts throughout Japan, but in the past century has found extensive use as a solid substrate to contain culture medium for microbiological work...
, a pH-sensitive dye (
phenol redPhenol red is a pH indicator that is frequently used in cell biology laboratories.-Chemical structure and properties:...
), 1%
lactoseLactose is a sugar that is found most notably in milk. Lactose makes up around 2–8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey...
, 1%
sucroseSucrose, commonly called table sugar, is a moosaccharide of glucose and fructose with the molecular formula C12H22O11. This white, odorless, crystalline powder has a pleasing, sweet taste. It is best known for its role in human nutrition...
, 0.1%
glucoseGlucose , a monosaccharide also known as - grape sugar, blood sugar, or corn sugar, is a very important carbohydrate in biology. The living cell uses it as a source of energy and metabolic intermediate...
, as well as
sodium thiosulfateSodium thiosulfate , also spelled sodium thiosulphate, is a colorless crystalline compound that is more familiar as the pentahydrate, Na2S2O3•5H2O, an efflorescent, monoclinic crystalline substance also called sodium hyposulfite or “hypo.”The 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.
thumb
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 sulfideHydrogen sulfide is the chemical compound with the formula H
2S. This colorless, toxic and flammable gas is partially responsible for the foul odor of rotten eggs and flatulence....
. It is often used in the selective identification of enteric bacteria including but not limited to
SalmonellaSalmonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which project in all directions...
and
ShigellaShigella is a genus of Gram-negative, non-spore forming rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agent of human shigellosis, Shigella cause disease in primates, but not in other mammals. It is only naturally found in humans and apes. During infection,...
.
Composition
The
TSI slant is a test tube that contains
agarAgar or agar agar is a gelatinous substance derived from seaweed. Historically and in a modern context, it is chiefly used as an ingredient in desserts throughout Japan, but in the past century has found extensive use as a solid substrate to contain culture medium for microbiological work...
, a pH-sensitive dye (
phenol redPhenol red is a pH indicator that is frequently used in cell biology laboratories.-Chemical structure and properties:...
), 1%
lactoseLactose is a sugar that is found most notably in milk. Lactose makes up around 2–8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey...
, 1%
sucroseSucrose, commonly called table sugar, is a moosaccharide of glucose and fructose with the molecular formula C12H22O11. This white, odorless, crystalline powder has a pleasing, sweet taste. It is best known for its role in human nutrition...
, 0.1%
glucoseGlucose , a monosaccharide also known as - grape sugar, blood sugar, or corn sugar, is a very important carbohydrate in biology. The living cell uses it as a source of energy and metabolic intermediate...
, as well as
sodium thiosulfateSodium thiosulfate , also spelled sodium thiosulphate, is a colorless crystalline compound that is more familiar as the pentahydrate, Na2S2O3•5H2O, an efflorescent, monoclinic crystalline substance also called sodium hyposulfite or “hypo.”The 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 also sucrose fermentative bacteria.
Interpretation of results
Bacteria that ferment any of the three sugars in the medium will produce byproducts. 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 sulfideHydrogen sulfide is the chemical compound with the formula H
2S. This colorless, toxic and flammable gas is partially responsible for the foul odor of rotten eggs and flatulence....
(H
2S) reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a black precipitate. Examples of sulfide-producing bacteria include
SalmonellaSalmonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which project in all directions...
,
ProteusIn Greek mythology, Proteus is an early sea-god, one of several deities whom Homer calls the "Old Man of the Sea", whose name suggests the "first", as protogonos is the "primordial" or the "firstborn". He became the son of Poseidon in the Olympian theogony In Greek mythology, Proteus (Πρωτεύς) is...
,
CitrobacterCitrobacter is a genus of Gram-negative coliform bacteria in the Enterobacteriaceae family.The species C. amalonaticus, C. koseri, and C. freundii use solely citrate as a carbon source. Citrobacter species are differentiated by their ability to convert tryptophan to indole, ferment lactose, and...
and
EdwardsiellaEdwardsiella is a Gram negative, fermentative bacteria of the family Enterobacteriaceae. It was first discovered in snakes in 1962....
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 H
2S production may mask the acid reaction (yellow) in the butt.
Salmonella entericaSalmonella enterica is a rod shaped, flagellated, aerobic, Gram-negative bacterium, and a member of the genus Salmonella.-Serovars:...
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.