Methyl Red (MR) test: Principle, procedure and results

Methyl Red (MR)  test determines whether the microbe performs mixed acids fermentation when supplied glucose. Types and proportion of fermentation products produced by anaerobic fermentation of glucose are one of the key taxonomic characteristics which help to differentiate various genera of enteric bacteria.

Mixed acid fermentation is one of the two broad patterns, 2-3-butanediol fermentation being another. In mixed acid fermentation, three acids (acetic, lactic and succinic) are formed in significant amounts. The mixed acid pathway gives 4 mol of acidic products (mainly lactic and acetic acid), 1 mol of neutral fermentation product (ethanol), 1 mol of CO₂, and 1 mol of H₂ per mol of glucose fermented.

These large amounts of acid results significant decrease in the pH of the medium below 4.4. This is visualized by using pH indicator, methyl red (p-dimethylaminoaeobenzene-O-carboxylic acid), which is yellow above pH 5.1 and red at pH 4.4.

The pH at which methyl red detects acid is considerably lower than the pH for other indicators used in bacteriologic culture media. Thus, to produce a color change, the test organism must produce large quantities of acid from the carbohydrate substrate being used.

MR Positive: When the culture medium turns red after the addition of methyl red, because of a pH at or below 4.4 from the fermentation of glucose.

MR Negative: When the culture medium remains yellow, which occurs when less acid is produced (pH is higher) 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. The composition of MR/VP broth is as follows:

Methyl Red pH Indicator

• Methyl red: 0.1 g in 300 mL of 95% ethyl alcohol.
• Distilled water: 200 mL

Quality Control

Positive and negative controls should be run after the preparation of each lot of medium and after making each batch of reagent. Suggested controls include the followings:

1. Positive control: Escherichia coli
2. Negative control: Klebsiella (formerly Enterobacter) aerogenes

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 up to 4 days.
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.

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