IMViC

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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.

Contents

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.

IMViC Results IMViC Results.jpg
IMViC Results

Indole test

In this test, the organism under consideration 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 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.

Methyl red and Voges–Proskauer test

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 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 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 is used.

Citrate test

In the 1930's, S.A. Koser conducted experiments that were used to study bacterial catabolism of organic acids. [1] 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 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. [2] Bromothymol blue was added in order to reduce false positives. The citrate agar 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.

Usage

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. [3]

SpeciesIndoleMethyl RedVoges-ProskauerCitrate
Escherichia coli PositivePositiveNegativeNegative
Staphylococcus aureus NegativePositivePositiveNegative
Shigella spp. [4] NegativePositiveNegativeNegative
Salmonella spp.NegativePositiveNegativePositive
Klebsiella spp.NegativeNegativePositivePositive
Proteus vulgaris [5] PositivePositiveNegativeNegative
Proteus mirabilis NegativePositiveNegativePositive
Citrobacter freundii NegativePositiveNegativePositive
Enterobacter aerogenes NegativeNegativePositivePositive

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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.

<span class="mw-page-title-main">Thiosulfate–citrate–bile salts–sucrose agar</span>

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.

The citrate test detects the ability of an organism to use citrate as the sole source of carbon and energy.

Glucose phosphate broth is used to perform methyl red (MR) test and Voges–Proskauer test (VP).

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.

References

  1. MacWilliams, Maria P. (December 8, 2009). "Citrate test Protocol" (PDF). American Society for Microbiology. Retrieved April 16, 2023.
  2. Tankeshwar, Acharya (2013-05-17). "Citrate Utilization Test: Principle, Procedure, Results • Microbe Online". Microbe Online. Retrieved 2023-04-16.
  3. Ananthanarayan, R; Jayaram Paniker, CK (2005). Ananthanarayan and Paniker's Textbook of Microbiology. Chennai, India: Orient Longman. ISBN   8125028080.
  4. Microbiology in pictures
  5. Microbe Library "IMViC tests of Proteus vulgaris - Library". Archived from the original on 2013-01-19. Retrieved 2013-01-05.