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Methyl Red and Voges-Proskauer test are among the two various tests used in the biochemical identification of bacterial species. These tests were originally studied by Voges, Proskauer (1) and subsequently by Clark and Lubs (2) to differentiate between members of the coli- aerogens group. Both the tests are based on the detection of specific breakdown products of carbohydrate metabolism.
All members of Enterobacteriaceae are, by definition, glucose fermenters. In MR-VP Broth, after 18-24 hours of incubation, fermentation produces acidic metabolic byproducts. Therefore initially all enterics will give a positive MR reaction if tested (3, 4, 5). However, after further incubation, required by the test procedure (2-5 days), MR - positive organisms continue to produce acids, resulting in a low pH (acidic) that overcomes the phosphate buffering system and maintains an acidic environment in the medium (pH 4.2 or less). MR-negative organisms further metabolize the initial fermentation products by decarboxylation to produce neutral acetyl methylcarbinol (acetoin), which results in decreased acidity in the medium and raises the pH towards neutrality (pH 6.0 or above) (6). In the presence of atmospheric oxygen and alkali, the neutral end products, acetoin and 2, 3-butanediol, are oxidized to diacetyl, which react with creatine to produce a red color.
The Methyl Red (MR) test is performed after 5 days of incubation at 30°C (8). The Voges-Proskauer test (VP) cultures are incubated at 30°C for 24-48 hours (9). Various test procedures have been suggested for performing the VP test by Werkman (10), OMeara (11) Levine, et al (12) and Voughn et al (8).
Werkmans Test (10): Add 2 drops of a 2% solution of ferric chloride to 50 ml culture and 5 ml of 10% sodium hydroxide. Shake the tube to mix well. Stable copper colour developing in a few minutes is positive reaction.
OMeara Test (11): Add 25 mg of solid creatine to 5 ml culture and then add 5 ml concentrated (40%) sodium hydroxide. Red color development in a few minutes after shaking the tube well is a positive reaction.
Levine, Epstein and Voughn (12) modified OMeara technique by dissolving the creatine in a concentrated solution of potassium hydroxide (R031, OMeara Reagent). Voughn, Mitchell and Levine (8) recommended the method of Barritt (13) as, addition of 1 ml of Barritt Reagent B (R030 - 40% potassium hydroxide) and 3 ml of Barritt Reagent A (R029 - 5% a-naphthol in absolute ethanol) to 5 ml culture. Positive test is indicated by eosin pink colour within 2-5 minutes.
The MR and VP tests should not be relied upon as the only means of differentiating E.coli from the Klebsiella - Enterobacter groups. Also occasionally a known acetoin-positive organism fails to give a positive VP reaction. To overcome this possibility, gently heat the culture containing the VP reagents (7).
Storage and Shelf-life:
Store below 30°C in a tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label.
1.Voges O. and Proskauer B., 1898, Z. Hyg. Infektionskr., 28:20.
2.Clark W. M. and Lubs H. K., 1915, J. Infect. Dis., 17:160.
3.Barry A. L., Bernsohn K. L., Adams A. B., Thrup L. D., Appl. Microbiol., 1970, 20 (6), 866-870.
4.Branson D., Methods in Clinical Bacteriology, Springerfield, IL: Charles C Thomas, 1972, 32-33.
5.Cowan S. T., Cowan and Stuls Manual for the Identification of Medical Bacteria, 2nd Ed., Cambridge, Cambridge University Press, 1974, 37,48.
6.MacFaddin J. F., 2000, Biochemical tests for Identification of Medical Bacteria, 3rd Ed., Lippincott, Williams and Wilkins, Baltimore.
7.MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.
8.Vaughn R. H., Mitchell N. B. and Levine M., 1939, J. Am. Water Works Association, 31:993.
9.Ruchhoft C. C., Kallas J. G., Chinn B. and Coulter E. W., 1931, J.Bacteriol., 22 : 125.>br />
10.Werkman C. H., 1930, J. Bact., 20: 121.
11.OMeara R. A. Q., 1931, J. Path. Bacteriol., 34 : 401.
12.Levine M., Epstein S. S. and Voughn R. H., 1934, Am. J. Publ. Health, 24: 505.
13.Ewing W. H., 1986, Edwards and Ewings Identification of Enterobacteriaceae, 4th Ed., Elsevier Science Publishing Co., Inc., New York.
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