The Triple Sugar Iron (TSI) test is a microbiological test roughly named for its ability to test a microorganism's ability to ferment sugars and to produce hydrogen sulfide. [1] It is often used to differentiate enteric bacteria including Salmonella and Shigella .
The TSI slant is a test tube that contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, [2] and sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate.
All of these ingredients are mixed together, heated to sterility, 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 (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 Salmonella , Proteus , Citrobacter and Edwardsiella species. The blackening of the medium is almost always observed in the butt (bottom) of the medium.
A bacterium that is a non-lactose fermenter and ferments glucose, initially causes a yellow slant/yellow bottom (acid/acid reaction) after 8 hours but then converts to a red slant/yellow bottom after 24 hours (alkali/acid reaction). Whereas if it ferments both lactose and glucose, it results in a yellow/yellow tube and remains that way due to the large amount of acid produced in the reaction. Blackening of the bottom due to H2S production may mask the acid reaction (yellow) in the bottom of the tube. Salmonella enterica serovar Typhi may result in blackening of the medium at the interface of bottom and the slant.
Under anaerobic conditions (as occur toward the bottom of the tube) some bacteria use thiosulfate 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 (crack the agar). Carbon dioxide, if produced, may not show as bubbles because it is far more soluble in the medium.
Lactic acid fermentation is a metabolic process by which glucose or other six-carbon sugars are converted into cellular energy and the metabolite lactate, which is lactic acid in solution. It is an anaerobic fermentation reaction that occurs in some bacteria and animal cells, such as muscle cells.
An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics.
Bacteriological water analysis is a method of analysing water to estimate the numbers of bacteria present and, if needed, to find out what sort of bacteria they are. It represents one aspect of water quality. It is a microbiological analytical procedure which uses samples of water and from these samples determines the concentration of bacteria. It is then possible to draw inferences about the suitability of the water for use from these concentrations. This process is used, for example, to routinely confirm that water is safe for human consumption or that bathing and recreational waters are safe to use.
Sulfate-reducing microorganisms (SRM) or sulfate-reducing prokaryotes (SRP) are a group composed of sulfate-reducing bacteria (SRB) and sulfate-reducing archaea (SRA), both of which can perform anaerobic respiration utilizing sulfate (SO2−
4) as terminal electron acceptor, reducing it to hydrogen sulfide (H2S). Therefore, these sulfidogenic microorganisms "breathe" sulfate rather than molecular oxygen (O2), which is the terminal electron acceptor reduced to water (H2O) in aerobic respiration.
MacConkey agar is a selective and differential culture medium for bacteria. It is designed to selectively isolate Gram-negative and enteric bacteria and differentiate them based on lactose fermentation. Lactose fermenters turn red or pink on MacConkey agar, and nonfermenters do not change color. The media inhibits growth of Gram-positive organisms with crystal violet and bile salts, allowing for the selection and isolation of gram-negative bacteria. The media detects lactose fermentation by enteric bacteria with the pH indicator neutral red.
Iron(II) sulfide or ferrous sulfide is one of a family of chemical compounds and minerals with the approximate formula FeS. Iron sulfides are often iron-deficient non-stoichiometric. All are black, water-insoluble solids.
Sulfur-reducing bacteria are microorganisms able to reduce elemental sulfur (S0) to hydrogen sulfide (H2S). These microbes use inorganic sulfur compounds as electron acceptors to sustain several activities such as respiration, conserving energy and growth, in absence of oxygen. The final product of these processes, sulfide, has a considerable influence on the chemistry of the environment and, in addition, is used as electron donor for a large variety of microbial metabolisms. Several types of bacteria and many non-methanogenic archaea can reduce sulfur. Microbial sulfur reduction was already shown in early studies, which highlighted the first proof of S0 reduction in a vibrioid bacterium from mud, with sulfur as electron acceptor and H
2 as electron donor. The first pure cultured species of sulfur-reducing bacteria, Desulfuromonas acetoxidans, was discovered in 1976 and described by Pfennig Norbert and Biebel Hanno as an anaerobic sulfur-reducing and acetate-oxidizing bacterium, not able to reduce sulfate. Only few taxa are true sulfur-reducing bacteria, using sulfur reduction as the only or main catabolic reaction. Normally, they couple this reaction with the oxidation of acetate, succinate or other organic compounds. In general, sulfate-reducing bacteria are able to use both sulfate and elemental sulfur as electron acceptors. Thanks to its abundancy and thermodynamic stability, sulfate is the most studied electron acceptor for anaerobic respiration that involves sulfur compounds. Elemental sulfur, however, is very abundant and important, especially in deep-sea hydrothermal vents, hot springs and other extreme environments, making its isolation more difficult. Some bacteria – such as Proteus, Campylobacter, Pseudomonas and Salmonella – have the ability to reduce sulfur, but can also use oxygen and other terminal electron acceptors.
Sorbitol-MacConkey agar is a variant of traditional MacConkey agar used in the detection of E. coli O157:H7. Traditionally, MacConkey agar has been used to distinguish those bacteria that ferment lactose from those that do not. This is important because gut bacteria, such as Escherichia coli, can typically ferment lactose, while important gut pathogens, such as Salmonella enterica and most shigellas are unable to ferment lactose. Shigella sonnei can ferment lactose, but only after prolonged incubation, so it is referred to as a late-lactose fermenter.
Microbial metabolism is the means by which a microbe obtains the energy and nutrients it needs to live and reproduce. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe's ecological niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles.
Xylose Lysine Deoxycholate agar is a selective growth medium used in the isolation of Salmonella and Shigella species from clinical samples and from food. The agar was developed by Welton Taylor in 1965. It has a pH of approximately 7.4, leaving it with a bright pink or red appearance due to the indicator phenol red. Sugar fermentation lowers the pH and the phenol red indicator registers this by changing to yellow. Most gut bacteria, including Salmonella, can ferment the sugar xylose to produce acid; Shigella colonies cannot do this and therefore remain red. After exhausting the xylose supply Salmonella colonies will decarboxylate lysine, increasing the pH once again to alkaline and mimicking the red Shigella colonies. Salmonellae metabolise thiosulfate to produce hydrogen sulfide, which leads to the formation of colonies with black centers and allows them to be differentiated from the similarly coloured Shigella colonies.
Leuconostoc mesenteroides is a species of lactic acid bacteria associated with fermentation, under conditions of salinity and low temperatures. In some cases of vegetable and food storage, it was associated with pathogenicity. L. mesenteroides is approximately 0.5-0.7 µm in diameter and has a length of 0.7-1.2 µm, producing small grayish colonies that are typically less than 1.0 mm in diameter. It is facultatively anaerobic, Gram-positive, non-motile, non-sporogenous, and spherical. It often forms lenticular coccoid cells in pairs and chains, however, it can occasionally form short rods with rounded ends in long chains, as its shape can differ depending on what media the species is grown on. L. mesenteroides grows best at 30 °C, but can survive in temperatures ranging from 10 °C to 30 °C. Its optimum pH is 5.5, but can still show growth in pH of 4.5-7.0.
Bismuth sulfite agar is a type of agar media used to isolate Salmonella species. It uses glucose as a primary source of carbon. Bismuth and brilliant green (dye) both inhibit gram-positive growth. Bismuth sulfite agar tests the ability to use ferrous sulfate and convert it to hydrogen sulfide.
Hektoen enteric agar is a selective and differential agar primarily used to recover Salmonella and Shigella from patient specimens. HEA contains indicators of lactose fermentation and hydrogen sulfide production; as well as inhibitors to prevent the growth of Gram-positive bacteria. It is named after the Hektoen Institute in Chicago, where researchers developed the agar.
Fermentative hydrogen production is the fermentative conversion of organic substrates to H2. Hydrogen produced in this manner is often called biohydrogen. The conversion is effected by bacteria and protozoa, which employ enzymes. Fermentative hydrogen production is one of several anaerobic conversions.
Sulfur is metabolized by all organisms, from bacteria and archaea to plants and animals. Sulfur can have an oxidation state from -2 to +6 and is reduced or oxidized by a diverse range of organisms. The element is present in proteins, sulfate esters of polysaccharides, steroids, phenols, and sulfur-containing coenzymes.
Cystine tryptic agar (CTA), also known as cystine trypticase agar, is a growth medium used for the identification of microorganisms.
Thiosulfate–citrate–bile salts–sucrose agar, or TCBS agar, is a type of selective agar culture plate that is used in microbiology laboratories to isolate Vibrio species. TCBS agar is highly selective for the isolation of V. cholerae and V. parahaemolyticus as well as other Vibrio species. Apart from TCBS agar, other rapid testing dipsticks like immunochromatographic dipstick is also used in endemic areas such as Asia, Africa and Latin America. Though, TCBS agar study is required for confirmation. This becomes immensely important in cases of gastroenteritis caused by campylobacter species, whose symptoms mimic that of cholera. Since no yellow bacterial growth is observed in case of campylobacter species on TCBS agar, chances of incorrect diagnosis can be rectified. TCBS agar contains high concentrations of sodium thiosulfate and sodium citrate to inhibit the growth of Enterobacteriaceae. Inhibition of gram-positive bacteria is achieved by the incorporation of ox gall, which is a naturally occurring substance containing a mixture of bile salts and sodium cholate, a pure bile salt. Sodium thiosulfate also serves as a sulfur source and its presence, in combination with ferric citrate, allows for the easy detection of hydrogen sulfide production. Saccharose (sucrose) is included as a fermentable carbohydrate for metabolism by Vibrio species. The alkaline pH of the medium enhances the recovery of V. cholerae and inhibits the growth of others. Thymol blue and bromothymol blue are included as indicators of pH changes.
Lysine iron agar or LIA is a differential media used to distinguish bacteria that are able to decarboxylate lysine and/or produce hydrogen sulfide from those that cannot. This test is particularly useful for distinguishing different Gram-negative bacilli—especially among the Enterobacteriaceae.
Thermotoga naphthophila is a hyperthermophilic, anaerobic, non-spore-forming, rod-shaped fermentative heterotroph, with type strain RKU-10T.
Diagnostic microbiology is the study of microbial identification. Since the discovery of the germ theory of disease, scientists have been finding ways to harvest specific organisms. Using methods such as differential media or genome sequencing, physicians and scientists can observe novel functions in organisms for more effective and accurate diagnosis of organisms. Methods used in diagnostic microbiology are often used to take advantage of a particular difference in organisms and attain information about what species it can be identified as, which is often through a reference of previous studies. New studies provide information that others can reference so that scientists can attain a basic understanding of the organism they are examining.