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16-468: The term "IMViC" is an acronym for each of these tests. "I" is for indole test ; "M" is for methyl red test ; "V" is for Voges-Proskauer test, and "C" is for citrate test . The lower case "i" is merely for "in" as the Citrate test requires coliform samples to be placed "in Citrate". These tests are useful in distinguishing members of Enterobacteriaceae . In this test, the organism under consideration

32-438: A negative test, meaning the butanediol fermentation is used. The VP test uses alpha-naphthol and potassium hydroxide to test for the presence of acetylmethylcarbinol (acetoin), an intermediate of the 2,3-butanediol fermentation pathway. After adding both reagents, the tube is shaken vigorously then allowed to sit for 5–10 minutes. A pinkish-red color indicates a positive test, meaning the 2,3-butanediol fermentation pathway

48-447: 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

64-500: Is L -tryptophan indole-lyase (deaminating; pyruvate-forming) . Other names in common use include L -tryptophanase , and L -tryptophan indole-lyase (deaminating) . This enzyme participates in tryptophan metabolism and nitrogen metabolism . It has 2 cofactors : pyridoxal phosphate , and potassium . As of late 2007, 3 structures have been solved for this class of enzymes, with PDB accession codes 1AX4, 2C44, and 2OQX. This EC 4.1 enzyme -related article

80-417: 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 ." Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes

96-416: Is actually used to determine ability of an organism to separate indole from amino acid tryptophan and it is added after incubation. A positive result is indicated by a pink/red layer forming on top of the liquid. These tests both use the same broth for bacterial growth. The broth is called MR-VP broth. After growth, the broth is separated into two different tubes, one for the methyl red (MR) test and one for

112-527: Is green before inoculation, and turns blue, because of BTB as a positive test indicator, meaning citrate is utilized. The test is also prepared on a slant to maximize bacterial growth for an even better indication of the use of citrate. These IMViC tests are useful for differentiating the family Enterobacteriaceae , especially when used alongside the Urease test . The IMViC results of some important species are shown below. Indole test The indole test

128-482: Is grown in peptone water broth. It contains tryptophan , which under the action of enzyme tryptophanase is converted to an Indole molecule, pyruvate and ammonium. The indole is then extracted from the broth by means of xylene. The broth is sterilized for 15 minutes at around 121 °C. To test the broth for indole production, Kovac's reagent . Kovac's reagent consist of amyl alcohol and para-dimethylaminobenzaldehyde and concentrated hydrochloric acid . Kovac's reagent

144-592: 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]. Tryptophanase The enzyme tryptophanase ( EC 4.1.99.1 ) catalyzes the chemical reaction This enzyme belongs to the family of lyases , specifically in the "catch-all" class of carbon-carbon lyases. The systematic name of this enzyme class

160-406: Is used. In the 1930's, S.A. Koser conducted experiments that were used to study bacterial catabolism of organic acids. Koser found that citrate metabolism could be an indicator for bacteria found in natural environments. Additionally, citrate could be used to distinguish bacterial coilforms found in soil, and aquatic environments, such as Enterobacteiacea , and coilforms with fecal contamination. It

176-463: The Voges-Proskauer (VP) test . The methyl red test detects production of acids formed during metabolism using mixed acid fermentation pathway using pyruvate as a substrate. The pH indicator Methyl Red is added to one tube and a red color appears at pH's lower than 4.2, indicating a positive test (mixed acid fermentation is used). The solution remaining yellow (pH = 6.2 or above) indicates

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192-509: The deamination reaction, during which the amine (-NH 2 ) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid , ammonium (NH 4 ) 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

208-437: 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

224-754: 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 , Vibrio sp., and Lactobacillus reuteri . Bacteria which give negative results for

240-399: 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

256-445: Was found that coilforms without fecal contamination grew, while the coilforms with fecal contamination did not grow. This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate as its sole carbon and energy source. The agar contains citrate and ammonium ions (nitrogen source) and bromothymol blue (BTB) as a pH indicator. Bromothymol blue was added in order to reduce false positives. The citrate agar

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