Methyl Red Test

Last reviewed by Editorial Team on February 27th, 2019.

A Methyl Red Test is a procedure that checks the ability of the microbe to perform mixed acids fermentation in the presence of glucose, which helps in differentiating enteric bacteria.

It is also used to characterize other bacteria groups such as Actinobacteria.

The Methyl Red Test is a part of the IMViC Test; a four specialized procedure used to differentiate members of the Enterobacteriaceae family.

The IMViC test includes the indole test, citrate test, Voges-Proskauer test, and the Methyl Red Test. (1, 2, 3)

methyl red test

Image 1: Two test tubes; the one in the left changes its color to red which is a positive for methyl red while the one on the left remains its color, which indicates a negative methyl red test.

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methyl red test bacteria

Picture 2: A methyl red test identifies/detects acid accumulation in the presence of glucose, which is useful in differentiating and classifying some types of bacteria.

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IMViC test

Photo 3: A methyl red test is a part of the IMViC test; a test consists of four types of specialized procedure.

(IMViC test)
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What is the principle of Methyl Red Test?

Some group of bacteria can use glucose and convert it to stable acids such as lactic acid, formic acid, and acetic acid.

It metabolizes glucose to pyruvic acid and further metabolized through mixed acid pathway until such time it reaches the stable acid state.

The type of acid produced will be determined by the species of an organism and the enzymatic pathway.

The acid produced by the organism can significantly affect the pH level. It causes a drop in the pH level (4.5 and below) as seen in the change of color of methyl red from yellow to red. (2, 3, 4, 5)

Methyl red test procedure

uninoculated MRVP broth

Image 4: The test tube on the left side contains uninoculated MRVP broth.

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methyl red solution

Picture 5: A methyl red solution is used in the standard methyl red test.

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A species being tested grown in a broth medium that contains glucose. The color of methyl red changes from yellow to red if the bacteria being tested has the ability to use glucose and produce a stable acid.

The media and reagents used for the methyl red test procedure include the following:

  • MRVP broth (glucose, buffered peptone, and dipotassium phosphate)
  • Methyl red solution (0.1 gram of methyl red dissolved in 300 ml of 95% ethyl alcohol and add 200 ml of distilled water) (4, 6, 7)

How do you perform a methyl red test?

  1. Inoculate two tubes that contain MR-VP broth containing a pure culture of the organism being tested. Before inoculation, make sure you allow the medium to equilibrate at a room temperature.
  2. Incubate at 35 to 37 degree Celsius for 24 hours or up to four days.
  3. Add five drops or at least 1 ml of methyl red indicator solution to the test tube.
  4. Re-incubate the remaining broth for at least 24 hours and add at least three drops of methyl red indicator.
  5. Observe for any changes in the color. (8, 9, 10)

Methyl red test results


The Methyl Red Test is positive if the culture medium turns red after adding methyl red.

Examples of methyl red positive organisms are Escherichia, Proteus E. coli, Salmonella, Vibrio parahaemolyticus, Streptococcus mitis, Vibrio furnissii, Citrobacter sp, Vibrio fluvialis, Shigella, Yersinia, Vibrio vulnificus, and Edwardsiella. (2, 4, 5)


If the culture medium remains yellow, it means that the result is negative. This is because less acid is produced during the fermentation of glucose. Examples of methyl red negative organisms are Enterobacter and Klebsiella. (1, 4, 6)

Are there any limitations?

Further biochemical tests need to be performed on pure culture so as to completely identify the organism.

The medium to be tested using the methyl red test should be incubated for at least 48 hours. Doing the test too early would lead to a false positive result.
A light inoculum should be used.

A too heavy inoculum will inhibit bacterial growth which could lead to invalid results.

An incubation period of up to five days is necessary. (3, 6, 10)



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