Methyl Red Test for Bacteria: Procedure & Principle

Instructor: Laura Foist

Laura has a Masters of Science in Food Science and Human Nutrition and has taught college Science.

E-coli is a dangerous bacteria, and there are several tests that can be performed to identify if E-coli is present. Methyl red is one of these tests. In this lesson, we will learn how to perform this test.


If you have ever had the stomach flu, then you probably know that it is not a pleasant experience. But if you were infected with E-coli, it would be a particularly unpleasant experience, since it causes extreme diarrhea, vomiting, cramping, and bloody stools. E-coli can lead to death, especially in young children, the elderly, pregnant women, and those with weakened immune systems.

Food manufacturers often test food to ensure it does not contain E-coli. One of the tests that can be performed is the methyl red test. The methyl red test tests for bacteria that perform mixed acid fermentation of glucose. Since E-coli is in the family of bacteria that performs mixed acid fermentation of glucose, it can be identified by this test.

Methyl Red Test Principles

When some types of bacteria, such as E-coli, come in contact with glucose, the bacteria use the glucose as an energy source. The process of breaking down glucose for energy will ferment the glucose and form acetic acid, lactic acid, and succinic acid.

The large amounts of acid produced greatly decreases the pH of a mixture to below 4.4. This change in pH can be seen with an indicator. Methyl red turns red at a pH below 4.4, but is yellow at a higher pH.

At a pH below 4.4, methyl red is a red color. At a pH above 4.4, methyl red is a yellow color
Methyl red indicator colors

Methyl Red Test Procedures

In order to perform the test, first a test tube with MR-VP broth is prepared. MR-VP broth is methyl red-Voges-Proskauer broth; it contains glucose, peptone, and a buffer. The buffer is included to ensure that a sample can produce enough acid to not only drop the pH, but also to overcome the buffer. The peptone is included simply because this is a multi-functional broth that can also be used for Voges-Proskauer tests at the same time.

This test tube of broth is heat treated to ensure it starts out clean. Then, it is inoculated with the sample in question. It is then incubated for 2-4 days at 35 degrees Celsius. It is incubated at 35 degrees Celsius because this is the ideal temperature for E-coli to grow, and if there is any E-coli in the sample, we want to ensure that we can actually see it growing.

MR-VP broth is inoculated with the sample in a test tube.
Test tubes

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