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

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

Did you Know? Clark and Lubs formulated the media for MR and VP tests.

Principle of Methyl Red (MR) Test

In mixed acid fermentation, bacteria form significant acids (acetic, lactic, and succinic), decreasing the medium’s pH below 4.4. It is visualized by using a pH indicator, methyl red (2-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid), a 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 bacteriological culture media. Thus, the test organism must produce large quantities of acid from the supplied carbohydrate source to produce a color change. Change in the MR-VP broth’s color is observed after adding a pH indicator, and the result is categorized as;

• MR Positive: The culture medium turns red (because the 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

The media used in the test is methyl red-Voges-Proskauer (MR/VP) broth. Media and reagents can be prepared in-house or purchased from commercial providers.

The composition of MR/VP broth is as follows:

Ingredients	MR/VP broth (g/L)
Polypeptone	7 g
Glucose	5 g
Dipotassium phosphate	5 g
Distilled water	1 L
Final pH	6.9

Methyl Red (0.02%) pH Indicator

1. Firstly, dissolve 0.1 g of MR in 300 ml of 95% ethyl alcohol.
2. After that, add sufficient distilled water to make 500 ml.
3. Then, store at 4 to 8°C in a brown bottle.
4. The solution is usable for one year.

Quality Control for MR Test

After preparing or procuring each lot of medium, examine the broth for signs of contamination, dehydration, and deterioration. Perform performance testing of media and reagent before use with one organism known to demonstrate a positive reaction and one 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 and VP tests. Only the addition of reagents differs, and both tests are carried out consecutively.

1. Firstly, bring the MR-VP broth to room temperature.
2. Then, inoculate two tubes containing MR-VP broth with a pure culture of the microorganisms under investigation.
3. After that, incubate the broth medium at 35 °C for at least 48 hours.
4. Then, add five drops of the methyl red indicator solution to the first tube. The second tube is for the Voges-Proskauer test. Barritt’s reagent is used for the test.
5. Finally, observe for change in color in any of the tubes. 

Expected results of MR Test

The development of a stable red color on the surface of the medium with MR reagent 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. It does not indicate a positive test.

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

Reporting results

• Organisms in the E. coli group are MR positive, whereas 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 Hafnia 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 over-inoculation because it increases the number of viable cells. Bacterial growth is inhibited when the inoculum exceeds 10⁹ viable cells per ml.
• Also, incubating the tubes for insufficient time may result in a false-positive MR test.

References and further readings

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