Methyl Red (MR) Test: Principle, Procedure, Results

By Acharya Tankeshwar •  Updated: 05/03/22 •  4 min read

Methyl Red (MR) test determines whether an organism performs mixed acid fermentation and produces stable acid end products. MR indicator is used to determine the pH after an enteric Gram-negative rod has fermented glucose to completion.

Principle

In mixed acid fermentation, three acids (acetic, lactic, and succinic) are formed in significant amounts decreasing the pH of the medium below 4.4. This is visualized by using a pH indicator, methyl red (p-dimethylaminoazeobenzene-O-carboxylic acid) pH indicator which is red at pH ≤ 4.4, and yellow color at pH 5.8.

Fig: Methyl Red (MR) Test Reaction
Fig: Methyl Red (MR) Test Reaction

The pH at which methyl red detects acid is considerably lower than the pH indicators used in bacteriologic culture media. Thus, to produce a color change, the test organism must produce large quantities of acid from the supplied carbohydrate source. Change in the color of the MR-VP broth is observed after the addition of pH indicator and the result is categorized as;

Media and Reagents

Methyl red-Voges-Proskauer (MR/VP) broth (formulated by Clark and Lubs) is used in this test. Media and reagents can be prepared in-house or purchased from commercial providers.

The composition of MR/VP broth is as follows:

IngredientsMR/VP broth (g/L)
Polypeptone7 g
Glucose5 g
Dipotassium phosphate5 g
Distilled water1 L
Final pH6.9

Methyl Red (0.02%) pH Indicator

  1. Dissolve 0.1 g of MR in 300 ml of ethyl alcohol, 95%.
  2. Add sufficient distilled water to make 500 ml.
  3. Store at 4 to 8°C in a brown bottle.
  4. Solution is stable for 1 year.

Quality Control

After the preparation or procurement of each lot of medium, examine the broth for signs of contamination, dehydration, and deterioration. Perform performance testing of media and reagent prior to use with one organism known to demonstrate a positive reaction and one organism known to give a negative reaction.

Organisms

  1. Escherichia coli ATCC 25922—MR positive (red)
  2. Klebsiella pneumoniae ATCC 13883—MR negative (yellow)

Procedure for Methyl Red (MR) Test

MR-VP broth is used for both MR Test and VP test. Only the addition of reagent differs, and both tests are carried out consecutively.

  1. Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms under investigation.
  2. Incubate at 35 °C for at least 48 hours.
  3. Add about 5 drops of the methyl red indicator solution to the first tube (for Voges-Proskauer test, Barrit’s reagent is added to another tube).
  4. A positive reaction is indicated if the color of the medium changes to red within a few minutes.
Methy Red Test Left: Negative Right: Positive
Methyl Red Test
Left: Negative
Right: Positive

Expected results: 

The development of a stable red color on the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitutes a positive test.  Because other organisms may produce smaller quantities of acid from the test substrate, an intermediate orange color between yellow and red may develop. This does not indicate a positive test.

  1. Escherichia coli: MR test positive- the appearance of red color after the addition of methyl red reagent.
  2. Klebsiella (formerly Enterobacter) aerogenes: MR test negative- the lack of color change after the addition of methyl red.

Reporting results

Methyl Red (MR) positive organisms

  1. Escherichia coli
  2. Shigella species
  3. Salmonella species
  4. Citrobacter species
  5. Proteus species
  6. Yersinia species

Methyl Red (MR) negative organisms

  1. Enterobacter species
  2. Hafnia species
  3. Serratia marcescens
  4. Klebsiella pneumoniae

Limitations

References and further readings

  1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
  2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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23 responses to “IMViC Tests: Principle, Procedure, Results”

  1. Ashish Kumar says:

    Wonderful orientation…great answer indeed !!!

  2. Abhishek says:

    ITS REALLY GOOD AND CONCISE. SUPERLIKE

  3. Tasha says:

    Something to note about the tests mentioned:
    SIM test, its is 3 tests in one:
    Indole
    Motility
    Hydrogen sulfide production

    http://www.microbelibrary.org/library/2-associated-figure-resource/3488-sulfur-indole-motility-test-results-from-various-microbes

    MR/VP testing is done with one media for 2 test. Grow the organism in the broth then remove 1 ml to a clean test tube to do the vp test

    http://www.microbeworld.org/component/jlibrary/?view=article&id=12634

    Citrate, organism uses nitrate as sole source of carbon:

    http://www.microbelibrary.org/library/2-associated-figure-resource/2534-citrate-test-on-escherichia-coli-and-enterobacter-aerogenes

  4. Shenly Legaspina says:

    Thank you so much sir 🙂

  5. What is the importance of Imvic?

  6. c.satisha says:

    very helpfull of thank u so much sir your obidient c.satisha

  7. Nice information what ever your providing through online, nice to understand process and mechanism of IMVIC test

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