Methyl Red Test, MR - Principle, Biochemistry, Purpose, Procedure, Result, Interpretation

Introduction to Methyl Red Test (MR)

The Methyl red (MR) test is used to test the ability of an organism to produce and maintain stable adequate acid end products when it utilizes glucose through fermentation. This test also observes if the acid byproducts can overcome the buffering capacity of the system i.e. if the pH of the broth is maintained at 4.5 after 18-24 hours of bacterial culture.

Methyl Red Test (MR) test is a qualitative test for acid production which determines pH changes in bacterial cultures that vary from species to genus alike.

Principle of Methyl Red Test (MR)

The basis of the methyl red test (MR) is the use of the pH indicator methyl red which determines the hydrogen ion (H⁺) concentration present when an organism uses glucose as a source of energy via the fermentative pathway. Some bacteria produce stable acids and are able to maintain the hydrogen ions at a certain concentration before being able to tip the buffer system- decreasing the pH.

The pH indicator, methyl red, will denote changes in acidity by the color reaction within the range of 4.4 to 6. If the pH is 4.4 or less, MR remains red-colored, and if the pH is at 6 or more, the color of MR changes to yellow.

Purpose of Methyl Red Test (MR)

The Methyl Red Test (MR) is an important biochemical test to differentiate/identify the genera of the Enterobacteriaceae family.

Biochemistry of Methyl Red Test (MR)

After bacteria are inoculated into the MR/VP broth (which contains glucose-phosphate), between 18-24 hours of incubation, all microorganisms produce pyruvic acid from glucose metabolism.

However, after incubation of 2 to 5 days, Methyl Red Test (MR)-positive organisms utilize a mixed acid pathway to metabolize pyruvic acid to produce other acids such as lactic acid, acetic acid, formic acid, and succinic acid.

The formic acid is acted upon by the bacterial enzyme formic dehydrogenase to produce an equal amount of H₂ and CO₂. This results in the decreasing pH and finally surpasses the phosphate buffering system. The acidity is maintained at 4.2 or less in such cases.

Biochemistry:

2 Glucose + H₂O → 2 lactic acid + acetic acid + ethanol + formic acid + succinic acid + CO₂ + 2H₂

Formic acid → (formic dehydrogenase) → H₂ + CO₂

In Methyl Red Test (MR)-negative microorganisms, pyruvic acid undergoes a butylene glycol pathway to give end products that have neutral pH. One such end product is neutral acetyl methyl carbinol (acetoin) which increases pH and eventually decreases the medium's acidity. Thus, after 2 to 5 days of incubation, the pH of the culture media is neutral (pH 6 or greater).

Procedure of Methyl Red Test (MR)

The procedure of the Methyl Red Test (MR) is as follows:

1. Take a sterile MR/VP broth and aseptically inoculate the broth with freshly cultured test organism with the help of an inoculating loop.

2. Incubate the inoculated tube at 35-37°C for 3 to 5 days.

3. Add 3-5 drops of methyl red indicator into the test tube.

4. Observe for color change.

Result, Interpretation of Methyl Red Test (MR)

The result of the Methyl Red Test (MR) can be interpreted as follows:

Positive

A distinct red color is seen at the surface of the medium with the test bacteria.

Negative

Methyl red loses its color and yellow color is observed at the surface of the medium.

* If orange color is seen, reincubate the broth for up to 4 days and perform the test again.

Methyl Red Test (MR) positive bacteria

Some Methyl Red Test (MR) positive bacteria include

• Escherichia coli

• Proteus vulgaris

• Yersinia sp.

Methyl Red Test (MR) negative bacteria

Some Methyl Red Test (MR) negative bacteria include

• Serratia marcescens

• Enterobacter aerogenes

• Klebsiella sp.

• Pseudomonas aeruginosa