Some bacteria have the ability to utilize glucose and convert it to a stable acid like lactic acid, acetic acid or formic acid as the end product.These bacteria initially metabolise glucose to pyruvic acid, which is further metabolized through the ‘mixed acid pathway to produce the stable acid.
The type of acid produced differs from species to species and depends on the specific enzymatic pathways present in the bacteria. The acid so produced decreases the pH to 4.5 or below, which is indicated by a change in the colour of methyl red from yellow to red.
In the methyl red test (MR test), the test bacteria is grown in a broth medium containing glucose. If the bacteria has the ability to utilise glucose with production of a stable acid, the colour of the methyl red changes from yellow to red, when added into the broth culture.
Originally the paired MR-VP tests were used to distinguish between members of the family Enterobacteriaceae, but now they are used to characterize other groups of bacteria including Actinobacteria.
Reagents And Materials Required
- Test tubes
- Conical flask
- Cotton plugs
- Inoculating loop
- Bunsen burner
- Laminar flow chamber
- Dispose jar
- MR-VP broth (methyl red-Voges Proskauer broth or glucose phosphate broth)
- Methyl red solution
- Isolated colonies or pure cultures of bacteria
- The ingredients of MR-VP broth medium (containing glucose as the main component) or its ready-made powder required for 100 ml of the broth is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling. MR-VP broth is also called glucose phosphate broth.
- Its pH is determined using a pH paper or pH meter and adjusted to 6.9 using 0.1N HCI if it is more or using 0.1N NaOH if it is less. The flask is heated, if required, to dissolve the ingredients completely.
- The broth is distributed into five test tubes (approximately 10 ml each), cotton-plugged, covered with craft paper and tied with thread or rubber band.
- The broth tubes are sterilized at 121°C (15 psi pressure) for 15 minutes in an autoclave.
- The broth tubes are allowed to cool to room temperature.
- The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, into the broth with the help of an inoculating loop sterilised over bunsen flame. The loop is sterilised after each inoculation.
- The inoculated broth tubes are incubated at 37°C for 24 to 48 hours in an incubator.
- Alcoholic solution of methyl red (3-4 drops) is dropped into each test tube.
- Red colour produced: MR positive (i.e. the bacteria has converted glucose to a stable acid, as indicated by conversion of methyl red from yellow to red colour. It indicates mixed acid fermentation).
- Red colour not produced: MR negative (i.e. the glucose in the medium has not been converted to a stable acid. This indicates butylene glycol fermentation).
Limitations of Methyl Red (MR) Test
- It is recommended that further biochemical tests be performed on pure cultures for complete identification.
- The methyl red test must not be performed unless the medium has been incubated for a minimum of 48 hours. Tests that are run too early may result in false-positive interpretation.
- It is important that a light inoculum be used. If an inoculum is too heavy, bacterial growth may be inhibited and result in invalid test results.
- Incubation periods up to 5 days may be necessary for the methyl red test.