Clark and Lubs (1) found that the addition of methyl red to cultures of Escherichia coli resulted in a red colour due to high acidity produced during dextrose fermentation. Voges-Proskauer (2) reported red colouration after addition of potassium hydroxide to specific culture media with organisms grown in it. The investigators developed MR-VP Broth which enables both tests to be performed in same medium in different tubes. The red colour produced by the addition of potassium hydroxide to cultures is due to the ability of organisms to produce a neutral product acetoin (acetyl methyl carbinol) from dextrose (3). The acetoin is oxidized in the presence of oxygen and alkali to produce diacetyl which reacts with creatine to produce a red color. This formulation is also recommended by BIS (5) and ISO committee (8) for the detection of coli-aerogenes group. A slightly modified formulation (M070S) is recommended by BIS (4,6,7) for the detection of E. coli, Vibrio parahaemolyticus and Bacillus cereus responsible for food poisoning. To test V.parahaemolyticus for VP, addition of 2-3% Sodium chloride to the medium is required.
The Methyl Red (MR) test is performed after maximum of 5 days of incubation at 30°C (9) and Voges-Proskauer test (VP) cultures are incubated at 30°C for 24-48 hours (10). Various other tests have been suggested by Werkman (11), OMeara (12) Levine, Epstein and Voughn (13) and Voughn, Mitchell and Levine (9). Werkmans Test (8): 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 color developing in a few minutes is positive reaction. OMeara Test (8): Add of 25 mg of solid creatine to 5ml 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 (13) modified OMeara technique by dissolving the creatine in a concentrated solution of potassium hydroxide. Voughn, Mitchell and Levine (9) recommended the method of Barritt (14) as, addition of 1 ml of 40% potassium hydroxide and 3 ml of 5% a - naphthol in absolute ethanol to 5 ml culture. Positive test is indicated by eosine pink color within 2-5 minutes.
Storage and Shelf-life:
Store below 30°C in tightly closed containers and the prepared medium at 2 - 8°C. Use before expiry date on the label.
References:
1. Clark and Lubs, 1915, J. Inf. Dis., 17 : 160.
2. Voges and Proskauer, 1898, Zeit, Hyg., 28 : 20.
3. MacFaddin, J.F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.
4. Bureau of Indian Standards, IS : 5887 (Part I) 1976, reaffirmed 1986.
5. Bureau of Indian Standards, IS : 5887 (Part - III) 1976.
6. Bureau of Indian Standards, IS : 5887 (Part IV) 1976.
7. Bureau of Indian Standards, IS : 5887 (Part - V) 1976, reaffirmed 1986.
8. International Organization for Standardization (ISO), 1993, Draft ISO/DIS 6597.
9. Vaughn, Mitchell and Levine, 1939, J. Am. Water Works Association, 31:993.
10. Kallas, Chinn and Coulter, 1931, J. Bact., 22 : 125.
11. Werkman, 1930, J. Bact., 20 : 121.
12. OMeara, 1931, J. Path. Bacteriol., 34 : 401.
13. Levine, Epstein and Voughn, 1934, Am. J. of Publ. Health, 24 : 505.
14. Barritt, 1936, J. Path. Bacteriol., 42 : 441.