Procedure of the Methyl Red Test: A Guide to Detecting Acidic Fermentation

The Methyl Red test, also known as the MR test, is an essential tool in the field of microbiology and biochemistry. It serves as a diagnostic tool for identifying bacteria that are capable of performing mixed acid fermentation of glucose. This test is particularly valuable in distinguishing between different types of microorganisms based on their metabolic characteristics. In this article, we will delve into the procedure of the Methyl Red test, its significance, and its applications in microbiological analysis.

Understanding the Principle:

The Methyl Red test is based on the ability of certain bacteria to produce and maintain stable levels of acidic metabolic products during glucose fermentation. Bacteria that can ferment glucose through mixed acid pathways produce a significant amount of stable acids, primarily lactic, acetic, succinic, and formic acids. The Methyl Red indicator, a pH-sensitive dye, changes its color from yellow to red when the pH of the medium drops below a certain threshold, which is around pH 4.4. This color change indicates the presence of a significant amount of acidic end-products, confirming mixed acid fermentation.

Materials Required:

1. MR-VP broth (Methyl Red-Voges-Proskauer broth)

2. Inoculating loop

3. Incubator

4. Methyl Red indicator solution


1. Inoculation: Start by inoculating the MR-VP broth with a pure culture of the microorganism under investigation. The broth contains glucose as the fermentable carbohydrate source.

2. Incubation: Allow the culture to incubate at the appropriate temperature, usually around 37°C for 24 to 48 hours, depending on the specific organism’s growth rate.

3. Preparation of Methyl Red Indicator: While the culture is incubating, prepare the Methyl Red indicator solution. This solution is composed of Methyl Red dye dissolved in a suitable solvent. The exact composition may vary depending on the laboratory protocol.

4. Testing for Acid Production: After the incubation period, take a sample of the culture and add a few drops of the Methyl Red indicator solution to it. Mix the solution gently but thoroughly to ensure proper mixing of the indicator with the sample.

5. Observation: Observe the color change in the solution. If the pH of the medium has dropped below the threshold (around pH 4.4), the solution will turn red. If there is no significant pH drop, the solution will remain yellow.


The interpretation of the Methyl Red test is quite straightforward:

– Positive Result: A positive result is indicated by a stable red color in the solution after the addition of the Methyl Red indicator. This suggests that the organism has produced a substantial amount of stable acidic products, confirming its ability to perform mixed acid fermentation.

– Negative Result: A negative result is indicated by a yellow color in the solution after the addition of the Methyl Red indicator. This suggests that the organism has not produced sufficient quantities of stable acidic products through mixed acid fermentation.


The Methyl Red test has several applications in microbiology and related fields:

1. Microbial Identification: The test aids in the identification and classification of microorganisms based on their metabolic pathways, which can be crucial for medical and environmental assessments.

2. Quality Control: In food and beverage industries, the test can be used to detect the presence of certain bacteria that may indicate contamination or spoilage.

3. Environmental Monitoring: The Methyl Red test can be employed to assess the metabolic activities of microorganisms in various ecosystems, contributing to environmental research.

4. Clinical Diagnostics: Some bacteria associated with urinary tract infections and other clinical conditions can be identified using the Methyl Red test, aiding in diagnosis and treatment.

In conclusion, the Methyl Red test is a valuable tool in microbiology laboratories for identifying bacteria capable of mixed acid fermentation. Its simple yet effective procedure makes it a fundamental technique in microbial characterization, enabling researchers and professionals to make informed decisions in various applications, from healthcare to industry and beyond.

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