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

Last updated on June 21st, 2021

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.

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;

• MR Positive: Culture medium turns red (because pH of the medium is at or below 4.4 from the fermentation of glucose).
• MR Negative: Culture medium remains yellow (less acid is produced from the fermentation of glucose).

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 purchase from commercial providers.

The composition of MR/VP broth is as follows:

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.

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

• Organisms in the E. coli group are MR positive, and those in the Enterobacter- Klebsiella group are MR negative
• Most members of the family Enterobacteriaceae give opposite MR and VP reactions; however, certain organisms, like H. alvei and Proteus mirabilis, may give both a positive MR reaction and a positive VP reaction (often delayed)

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

• Avoid overinoculation. Bacterial growth is inhibited when the inoculum exceeds approximately 10^9 viable cells per ml
• A false-positive MR result may be obtained if the tubes are not incubated for a sufficient period of time.

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