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Methyl Red Test

Methyl red test helps us to detect the production of sufficient acid during the process of fermentation of glucose and maintenance of conditions such as pH.

The pH of an old culture is sustained below the value of 4.5 which is shown by the change in color of the indicator which is added at the end of the period on incubation.

MR-VP broth was developed by Clark and Lubs which allowed both MR and VP tests to be performed in the same medium of inoculation by aliquoting the different portions of the tube.

What is Methyl Red Test?

Methyl red test is commonly known as MR test and it is used to detect the ability of the organism to produce and maintain the stable products of acids by fermenting the glucose.

Whereas methyl red test along with VP test is always performed together; as they are physiologically related to each other and they are performed in MRVP broth.

All of the members of the family Enterobacteriaceae has the capability to convert the substance of glucose into pyruvic acid by using Embden-Meyerh-of pathway, whereas the bacteria can metabolize the pyruvic acid further.

This test is one of the commonly performed biochemical test, which is used to detect the ability of the microorganism.

Methyl Red Test Principle

Generally, some bacteria have the ability to use glucose and convert it into a stable lactic acid, acetic acid or formic acid end product.

The bacteria first metabolize through mixed acid pathway to produce stable acid.

The acid production varies from species to species depending on the specific enzymatic pathways which is seen in the bacteria.

This acid thus produced decreases the ph. value about 4.5 or in some cases even less than that, which is indicated by change in color from yellow to red when methyl red indicator is added.

During methyl red test, the bacteria which tests the growth of bacteria in a broth medium containing glucose.

When the bacteria have the ability to use the glucose by producing stable acid, the color changes from yellow to red on adding the methyl indicator in the broth culture.

Where as the mixed acid pathway gives 4 mol of acidic products which mainly involves lactic and acetic acid, 1 mol of neutral fermentation product, mostly ethanol is used here, 1 mol of carbon dioxide and 1 mol of H2 per mol of glucose is fermented.

When a large quantity of acids is producing, they cause a significant decrease in pH in the culture medium.

Methyl Red Test Reagents

MRVP Broth:

 Usually, the ph. in the MRVP broth is maintained at 6.9.

 1 litre of deionized water

 7.0 grams of Buffered peptone

 5 grams of Glucose

 5 grams of Dipotassium phosphate

Preparation of Methyl Red Solution

 Initially 0.1 gram of methyl red is dissolved in 300ml of 95% of ethyl alcohol.

 Then sufficient distilled water is added to make about 5oo ml of methyl red solution.

 Then the prepared solution is stored at a temperature of 4 to 8 ºC in a brown bottle.

 This solution which has been prepared can be stored up to one year.

Methyl Red Test Procedure

 Before inoculating the medium, it is allowed to equilibrate at a room temperature.

 Then the organisms are taken from the 18 to 24-hour pure culture, and it is lightly inoculated in the medium.

 Then the medium is incubated aerobically at a temperature of 37ºcelsius for about 24 hours.

 After incubating for 24 hours, 1 ml of aliquot broth is added into a clean test tube.

 Again, the broths are incubated for additional 24 hours.

 Two to three drops of methyl indicator are added to the aliquot.

 Then the change in color is observed immediately.

Methyl Red Test Results

 In positive reaction, the color changes to distinct red

 Some of the species like, Escherichia coli, Yersinia sps gives positive results in Methyl red test

 Whereas, in negative reaction, the color remains yellow and there will be no change in color

 Organisms such as Enterobacter aerogenes, Klebsiella pneumonia, etc. gives negative results in Methyl red test.

Methyl Red Test Uses

Basically, methyl test is paired up with Voges Proskauer test and it is used in differentiating the members of the family Enterobacteriaceae. But in recent times they are used to characterize the other groups of bacteria including those of Actinobacteria.

Methyl Red Test Limitation

 Usually, biochemical tests are recommended to perform on pure cultures for the complete identification.

 Methyl red test can be performed only if the medium is incubated for at least a minimum of 48 hours. If the tests are let to run very early them it results only in false-positive results.

 It is also important to take a note that only light inoculums are used in methyl red tests because when heavy inoculums are used the growth of bacteria will be inhibits and it results in invalid results

 Incubation can be followed for a maximum of five days, which is necessary for this test.

Methyl Red Test Citations

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