Question

Discuss the biochemistry behind the MR-VP microbiology tests .

Answer 1

MRVP tests are part of IMViC tests which are used to differentiate between enterics (members of family Enterobacteriaceae). Here MR stands for Methyl Red Test and VP stands for Voges-Proskauer tests.

IMViC tests include:

• Indole test
• Methyl red test
• Voges-Proskauer test
• Citrate test

(Source: https://laboratoryinfo.com/methyl-red-test/)

MRVP tests use the same broth for bacterial growth. The broth is called MRVP 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. (Source: https://en.wikipedia.org/wiki/IMViC).

The Methyl Red (MR) test is based on the use of methyl red, a pH indicator, to detect acidity when an organism ferments glucose. Clark and Lubs, in 1915, discovered the ability of coliforms to produce and maintain acid products when cultivated in specific media. They found that cultures of Escherichia coli produced a red color upon addition of methyl red. This color development was a result of strong acid production from dextrose-fermentation. (Source: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MR-VPBroth.htm)

Voges and Proskauer, in 1898, first observed the production of a red color after the addition of potassium hydroxide to cultures grown on specific media.Harden later revealed that the development of the red color was a result of acetyl-methyl carbinol (acetoin) production. In 1936, Barrit made the test more sensitive by adding alpha-naphthol to the medium before adding potassium hydroxide. (Source: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MR-VPBroth.htm). The test is performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth which has been inoculated with bacteria. A cherry red color indicates a positive result, while a yellow-brown color indicates a negative result. (Source: https://en.wikipedia.org/wiki/Voges%E2%80%93Proskauer_test) Clark and Lubs developed MR-VP Broth which allowed both the MR and VP tests to be performed from the same inoculated medium by aliquoting portions to different tubes. (Source: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MR-VPBroth.htm)

Biochemistry of MRVP: MR-VP test is used to determine which fermentation pathway is used to utilize glucose. In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids). The stable production of enough acid to overcome the phosphate buffer will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear (first picture, tube on the left). If the MR turns yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been utilized (first picture, tube on the right). The 2,3 butanediol fermentation pathway will ferment glucose and produce a 2,3 butanediol end product instead of organic acids. In order to test this pathway, an aliquot of the MR/VP culture is removed and a-naphthol and KOH are added. They are shaken together vigorously and set aside for about one hour until the results can be read. The Voges-Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-red to pink” (tube on the left in the second picture). If the culture is negative for acetoin, it will turn “brownish-green to yellow” (tube on the left in the second picture). Note: A culture will usually only be positive for one pathway: either MR+ or VP+. Escherichia coli is MR+ and VP-. In contrast, Enterobacter aerogenes and Klebsiella pneumoniae are MR- and VP+. Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR- and VP-.(Source: https://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm#methyl_red)

(Source: https://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm#methyl_red)

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