Methyl Red Test – Principle, Procedure, and Results |

IMViC Test stands for Indole, Methyl red, Voges-Proskauer, Citrate Test. This as a set of four biochemical tests used to differentiate between Escherichia coli, Klebsiella Enterobacter, and Enterobacteriaceae family organisms.

Methyl Red (MR) Test

  • Methyl Red Test was first described by Clark and Lubs in 1915. They found it useful in differentiating between the E. coli and Enterobacteraerogenes group of enteric bacteria. Previously, these two groups were divided on the basis of CO2 / H2 ratios produced when the organisms were grown in presence of glucose.
  • When grown in vacuum one group produced a ratio of 1.06 (‘low ratio’) and the other a ratio of 1.9 -3.0 (‘high ratio’). These gas ratio differences were attributed to the basic difference in metabolism between the coli-aerogenes groups, and could be an accurate means of differentiation.
  • However, determination of gas ratios is not a convenient method for identifying large numbers of cultures.
  • Therefore, Clark and Lubs Developed a simple method correlated with the differences in gas ratios. They noted that when the coli organisms (“low ratio”) were  grown in glucose broth,acid was formed until a pH between 4 and 5 and then remained stable.
  • At this point the organisms ceased activity and no pH change was seen on further metabolize, with the medium then becoming more alkaline (pH 6-7).
  • The explanation for this was that the aerogenes organisms attack the acids that cannot utilize their acid end products.
  • The higher pH obtained with the aerogenes group was also attributed to the acetoin formed in its fermentation of glucose. Acetoin is neutral, and thus the pH is higher thanin the cultures of the coli organisms where the major end products of fermentation are acids.
  • Under aerobic conditions, both E.coli and Enterobacter aerogenes have the potential to attack their respective fermentation products.
  • However E. coli produces about 130 moles of acid per 100 moles of glucose fermented, whereas E. aerogenes produces only 20 moles of acid from the same amount of glucose. Therefore, E.coli would have to neutralize by oxidation five times the amount of acid as E. aerogenes in order to raise the pH above 6.0.        
  • The concentration of glucose in the medium (0.5%) is critical as it should not be completely used up with the result that under aerobic conditions of the tests, the medium might revert to alkaline. This medium is also the recommended medium for the Voges Proskauer test which detects the presence of the butylene glycol pathways.
  • To determine the pH of the medium after incubation, methyl red indicator is used because this indicator is red at pH 4.5-5.0 & yellow at pH 6.0-7.0. A red color after addition of the indicator indicates a positive methyl red test (mixed acid fermentation), and yellow indicates a negative methyl red test (butylene glycol fermentation).  
  • This test may not be very clear at all times where colours are not always clear cut. This is because the recommended incubation time for the test (prescribed by Clark & Lubs) is 5 days at 30oC.
  • This can be overcome by using the heavy inoculums in a small volume (0.5ml) of the medium. Also, inoculated medium can now be incubated at 37oC because of the reduction in broth volume.

Principle of Methyl Red Test

The principle of the test is based on the ability of bacteria to produce organic acids during glucose fermentation, which lowers the pH of the medium.

In the methyl red test, a pH indicator called methyl red is added to the culture medium. Methyl red is yellow at a pH above 6.0, but it turns red at a pH below 4.4. After inoculating the bacteria into the medium, they ferment glucose and produce organic acids. If the bacteria produce enough acid to lower the pH of the medium below 4.4, the methyl red indicator will turn red.

Requirements of MR Test

  1. Sterile glucose phosphate broth (GPB) medium (0.5ml in each tube)
  2. Glucose phosphate broth (GPB) 
  3. Test culture suspension
  4. Methyl red indicator
  5. Dissolve 0.1 g methyl red in 300 ml 95% ethanol. Add distilled water to make up the volume to 500ml.

Procedure of MR Test

  1. Inoculate the GPB medium with culture suspension.
  2. Incubate at 37oC for 24 hours. 
  3. Add 5-6 drops of methyl red indicator. 
  4. Positive test is indicated by a bright red color of the medium.
  5. A negative test indicated by the medium remaining yellow or turning orange.

Methyl red test results

Positive MR TestNegative MR Test
E. coliEnterobacter aerogenes
Yersinia spsKlebsiella pneumoniae

Uses of MR Test

  1. Identification of bacteria: The methyl red test is used to differentiate between different types of bacteria based on their ability to produce stable acid end products during glucose fermentation. It is commonly used in the identification of enteric bacteria, such as Escherichia coli and Enterobacter aerogenes.
  2. Quality control: The methyl red test is also used as a quality control measure for microbiological media. It is a simple and reliable method to ensure that the culture media is free from contamination and that the pH of the medium is within the acceptable range for growth of microorganisms.
  3. Research: The methyl red test can be used in research to study bacterial metabolism and the pathways of glucose fermentation. It is a useful tool to investigate the effects of various treatments or mutations on bacterial metabolism.

FAQs on Methyl Red Test

  1. What is methyl red test for?

    Methyl Red Test is used to differentiate between different types of bacteria based on their ability to produce stable acid end products during glucose fermentation, such as Escherichia coli and Enterobacter aerogenes.

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