Methyl Red (MR) Test- Principle, Procedure and Results Interpretation

 

AIM

This test is used to determine the ability of an organism to produce and maintain stable acid end products from glucose fermentation. This test is helpful in differentiating members of Enterobacteriace.

Principle

Methyl red is a pH indicator, with a range between 6.0 (yellow) and (red). 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. The methyl red test is a quantitative test for acid production, requiring positive organisms to produce strong acids (lactic, acetic, formic) from glucose through the mixed acid fermentation pathway. Because many species of the Enterobacteriaceae may produce sufficient quantities of strong acids that can be detected by methyl red indicator during the initial phases of incubation, only organisms that can maintain this low pH after prolonged incubation (48–72 hours), overcoming the pH-buffering system of the medium, can be called methyl red–positive

Media and Reagents

The medium most commonly used is methyl red–Voges-Proskauer (MR/VP) broth, as formulated by Clark and Lubs. This medium also serves for the performance of the Voges–Proskauer test.

MR/VP Broth Composition
Polypeptone	7 g
Glucose	5 g
Dipotassium phosphate	5 g
Distilled water	1 L
Final pH	6.9

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 preparation of each lot of medium and after making each batch of reagent. Suggested controls include the following:

Positive control: Escherichia coli

Negative control: Klebsiella pneumoniae

Test Procedure

Inoculate the MR/VP broth with a pure culture of the test organism. Incubate the broth at 37°C for 48–72 hours (no fewer than 48 hours).

After incubation, add 5 drops of the methyl red reagent directly to the broth and observe the result.

Results Interpretation

Positive:  Bright Red color        

Negative:  Yellow/Orange color

The development of a stable red color in 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.

Limitation of Methyl Red test

Avoid testing an extremely turbid broth inoculum; bacterial growth is inhibited if the inoculum is excessively large.  With each logarithmic decrease in inoculum size, there is an increase in time required for the MR-positive organisms to accumulate enough acidic products to overcome the buffer system.

The MR-VP tests should not be relied upon as the only means of identification for differentiation of genera within the family Enterobacteriaceae. Citrate and indole tests must be performed in conjugation with the MR-VP tests.  It is possible for some organisms to destroy acetoin, making the MR-VP invalid for identification.

VP-positive organisms are not necessarily MR-negative.  Certain organisms such as Enterobacter hafniae and Proteus mirabilis may give both a positive MR and VP reaction, although the VP may be delayed.

Reference

Koneman’s Color Atlas and Text book of Diagnistic Microbiology.  Bailey and Scott’s Diagnostic Microbiology.

Parker RH, Hoeprich PD. Disk method for rapid identification of Haemophilus sp. Am J Clin Pathol 1963; 37:319–327.

Barry AL, Bernsohn KL, Adams AP, et al. Improved 18-hour methyl red test. Appl Microbiol 1970; 20:866–870.

Blazevic DJ, Ederer GM. Principles of Biochemical Tests in Diagnostic Microbiology. New York, NY: Wiley, 1975:75–77.