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The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into indole. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as "tryptophanase."[ citation needed ]
Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzyme.
Like many biochemical tests on bacteria, results of an indole test are indicated by a change in color following a reaction with an added reagent.
Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24–48 hours before performing the test. Following incubation, five drops of Kovac's reagent (isoamyl alcohol, para-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) are added to the culture broth.
A positive result is shown by the presence of a red or reddish-violet color in the surface alcohol layer of the broth. A negative result appears yellow. A variable result can also occur, showing an orange color as a result. This is due to the presence of skatole, also known as methyl indole or methylated indole, another possible product of tryptophan degradation.
The positive red color forms as a result of a series of reactions. The para-Dimethylaminobenzaldehyde reacts with indole present in the medium to form a red rosindole dye. The isoamyl alcohol forms a complex with rosindole dye, which causes it to precipitate. The remaining alcohol and the precipitate then rise to the surface of the medium.
A variation on this test using Ehrlich's reagent (using ethyl alcohol in place of isoamyl alcohol, developed by Paul Ehrlich) is used when performing the test on nonfermenters and anaerobes.
Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas hydrophila , Aeromonas punctata , Bacillus alvei , Edwardsiella sp., Escherichia coli , Flavobacterium sp., Haemophilus influenzae , Klebsiella oxytoca , Proteus sp. (not P. mirabilis and P. penneri), Plesiomonas shigelloides , Pasteurella multocida , Pasteurella pneumotropica, Enterococcus faecalis , Vibrio sp., and Lactobacillus reuteri .
Bacteria which give negative results for the indole test include: Actinobacillus spp., Aeromonas salmonicida , Alcaligenes sp., most Bacillus sp., Bordetella sp., Enterobacter sp., most Haemophilus sp., most Klebsiella sp., Neisseria sp., Mannheimia haemolytica, Pasteurella ureae, Proteus mirabilis , P. penneri , Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp., and Rhizobium sp.
The Indole test is one of the four tests of the IMViC series, which tests for evidence of an enteric bacterium. The other three tests include: the methyl red test [M], the Voges–Proskauer test [V] and the citrate test [C]. [1]
Enterobacteriaceae is a large family of Gram-negative bacteria. It includes over 30 genera and more than 100 species. Its classification above the level of family is still a subject of debate, but one classification places it in the order Enterobacterales of the class Gammaproteobacteria in the phylum Pseudomonadota. In 2016, the description and members of this family were emended based on comparative genomic analyses by Adeolu et al.
Tryptophan synthase or tryptophan synthetase is an enzyme that catalyses the final two steps in the biosynthesis of tryptophan. It is commonly found in Eubacteria, Archaebacteria, Protista, Fungi, and Plantae. However, it is absent from Animalia. It is typically found as an α2β2 tetramer. The α subunits catalyze the reversible formation of indole and glyceraldehyde-3-phosphate (G3P) from indole-3-glycerol phosphate (IGP). The β subunits catalyze the irreversible condensation of indole and serine to form tryptophan in a pyridoxal phosphate (PLP) dependent reaction. Each α active site is connected to a β active site by a 25 angstrom long hydrophobic channel contained within the enzyme. This facilitates the diffusion of indole formed at α active sites directly to β active sites in a process known as substrate channeling. The active sites of tryptophan synthase are allosterically coupled.
Proteus vulgaris is a rod-shaped, nitrate-reducing, indole-positive and catalase-positive, hydrogen sulfide-producing, Gram-negative bacterium that inhabits the intestinal tracts of humans and animals. It can be found in soil, water, and fecal matter. It is grouped with the Morganellaceae and is an opportunistic pathogen of humans. It is known to cause wound infections and other species of its genera are known to cause urinary tract infections.
Proteus is a genus of Gram-negative bacteria. It is a rod shaped, aerobic and motile bacteria, which is able to migrate across surfaces due its “swarming” characteristic in temperatures between 20 and 37 °C. Their size generally ranges from 0.4–0.8 μm in diameter and 1.0–3.0 μm in length. They tend to have an ammonia smell. Proteus bacilli are widely distributed in nature as saprophytes, being found in decomposing animal matter, sewage, manure soil, the mammalian intestine, and human and animal feces. They are opportunistic pathogens, commonly responsible for urinary and septic infections, often nosocomial.
A biovar is a variant prokaryotic strain that differs physiologically or biochemically from other strains in a particular species. Morphovars are those strains that differ morphologically. Serovars are those strains that have antigenic properties that differ from other strains.
Proteus mirabilis is a Gram-negative, facultatively anaerobic, rod-shaped bacterium. It shows swarming motility and urease activity. P. mirabilis causes 90% of all Proteus infections in humans. It is widely distributed in soil and water. Proteus mirabilis can migrate across the surface of solid media or devices using a type of cooperative group motility called swarming. Proteus mirabilis is most frequently associated with infections of the urinary tract, especially in complicated or catheter-associated urinary tract infections.
The oxidase test is used to determine whether an organism possesses the cytochrome c oxidase enzyme. The test is used as an aid for the differentiation of Neisseria, Moraxella, Campylobacter and Pasteurella species. It is also used to differentiate pseudomonads from related species.
Isoamyl alcohol is a colorless liquid with the formula C
5H
12O, specifically (H3C–)2CH–CH2–CH2–OH. It is one of several isomers of amyl alcohol (pentanol). It is also known as isopentyl alcohol, isopentanol, or (in the IUPAC recommended nomenclature) 3-methyl-butan-1-ol. An obsolete name for it was isobutyl carbinol.
Elizabethkingia meningoseptica is a Gram-negative, rod-shaped bacterium widely distributed in nature. It may be normally present in fish and frogs; it may be isolated from chronic infectious states, as in the sputum of cystic fibrosis patients. In 1959, American bacteriologist Elizabeth O. King was studying unclassified bacteria associated with pediatric meningitis at the Centers for Disease Control and Prevention in Atlanta, when she isolated an organism that she named Flavobacterium meningosepticum. In 1994, it was reclassified in the genus Chryseobacterium and renamed Chryseobacterium meningosepticum(chryseos = "golden" in Greek, so Chryseobacterium means a golden/yellow rod similar to Flavobacterium). In 2005, a 16S rRNA phylogenetic tree of Chryseobacteria showed that C. meningosepticum along with C. miricola were close to each other but outside the tree of the rest of the Chryseobacteria and were then placed in a new genus Elizabethkingia named after the original discoverer of F. meningosepticum.
The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic, or facultative anaerobic rod, which produces gas from lactose within 48 hours. The presence of some coliforms indicate fecal contamination.
Purple urine bag syndrome (PUBS) is a medical syndrome where purple discoloration of urine occurs in people with urinary catheters and co-existent urinary tract infection. Bacteria in the urine produce the enzyme indoxyl sulfatase. This converts indoxyl sulfate in the urine into the red and blue colored compounds indirubin and indigo. The most commonly implicated bacteria are Providencia stuartii, Providencia rettgeri, Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa.
Ehrlich's reagent or Ehrlich reagent is a reagent containing p-dimethylaminobenzaldehyde (DMAB) and thus can act as an indicator to presumptively identify indoles and urobilinogen. Several Ehrlich tests use the reagent in a medical test; some are drug tests and others contribute to diagnosis of various diseases or adverse drug reactions. It is named after Nobel Prize winner Paul Ehrlich who used it to distinguish typhoid from simple diarrhoea.
Indole is an aromatic, heterocyclic, organic compound with the formula C8H7N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environment and can be produced by a variety of bacteria. As an intercellular signal molecule, indole regulates various aspects of bacterial physiology, including spore formation, plasmid stability, resistance to drugs, biofilm formation, and virulence. The amino acid tryptophan is an indole derivative and the precursor of the neurotransmitter serotonin.
p-Dimethylaminocinnamaldehyde (DMACA) is an aromatic hydrocarbon. It is used in an acidic solution to detect indoles.
Voges–Proskauer or VP is a test used to detect acetoin in a bacterial broth culture. The test is performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth, which is a glucose-phosphate broth that has been inoculated with bacteria. A cherry red color indicates a positive result, while a yellow-brown color indicates a negative result.
Tryptophol is an aromatic alcohol that induces sleep in humans. It is found in wine as a secondary product of ethanol fermentation. It was first described by Felix Ehrlich in 1912. It is also produced by the trypanosomal parasite in sleeping sickness.
Proteus penneri is a Gram-negative, facultatively anaerobic, rod-shaped bacterium. It is an invasive pathogen and a cause of nosocomial infections of the urinary tract or open wounds. Pathogens have been isolated mainly from the urine of patients with abnormalities in the urinary tract, and from stool. P. penneri strains are naturally resistant to numerous antibiotics, including penicillin G, amoxicillin, cephalosporins, oxacillin, and most macrolides, but are naturally sensitive to aminoglycosides, carbapenems, aztreonam, quinolones, sulphamethoxazole, and co-trimoxazole. Isolates of P. penneri have been found to be multiple drug-resistant (MDR) with resistance to six to eight drugs. β-lactamase production has also been identified in some isolates.
Kovacs reagent is a biochemical reagent consisting of isoamyl alcohol, para-dimethylaminobenzaldehyde (DMAB), and concentrated hydrochloric acid. It is used for the diagnostical indole test, to determine the ability of the organism to split indole from the amino acid tryptophan. The indole produced yields a red complex with para-dimethylaminobenzaldehyde under the given conditions. This was invented by the Hungarian physician Nicholas Kovacs and was published in 1928. This reagent is used in the confirmation of E. coli and many other pathogenic microorganisms.
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.
The Morganellaceae are a family of Gram-negative bacteria that include some important human pathogens formerly classified as Enterobacteriaceae. This family is a member of the order Enterobacterales in the class Gammaproteobacteria of the phylum Pseudomonadota. Genera in this family include the type genus Morganella, along with Arsenophonus, Cosenzaea, Moellerella, Photorhabdus, Proteus, Providencia and Xenorhabdus.