Define Methyl red Test?
The Methyl Red test is a biochemical assay used to detect bacteria that produce stable acidic end-products from glucose fermentation, causing a significant drop in pH. A positive Methyl red test is indicated by a red color change after the addition of methyl red indicator, while a negative result remains yellow or orange.
Table of Contents
Purpose
The Methyl Red (MR) test is a biochemical assay designed to detect bacteria capable of producing stable acid end-products from glucose fermentation. It is one of the IMViC tests (Indole, Methyl Red, Voges-Proskauer, and Citrate), widely used to differentiate members of the Enterobacteriaceae family. The main aim of the Methyl red test is to distinguish bacteria that generate stable acids, such as Escherichia coli, from those that do not, like Enterobacter aerogenes.
Concept
The Methyl red test is based on identifying stable acidic by-products from glucose metabolism. When certain bacteria ferment glucose through the mixed-acid fermentation pathway, they release stable acids, such as lactic acid, acetic acid, and formic acid. These acids significantly lower the pH of the medium.
After incubation, the MR indicator (a pH-sensitive dye) is added to the culture. If the pH is 4.4 or lower, the indicator turns red, showing the presence of stable acids. If the pH is higher, around 6.0 or above, the medium remains yellow or orange, indicating that the bacteria did not produce enough acids to lower the pH to a highly acidic level.
Method
Media Preparation
The MR test is conducted using MR-VP broth, a nutrient-rich medium containing glucose as the carbohydrate source. It also includes peptones and buffers to regulate the pH.
Inoculation
Using a sterile loop, a small amount of the bacterial culture is transferred to the MR-VP broth.
Incubation
The inoculated broth is incubated at 35-37°C for 48 hours (or sometimes up to 72 hours depending on the bacteria). During this time, bacteria ferment the glucose present in the medium.
Methyl Red Addition
After the incubation period, 5 drops of methyl red solution (the pH indicator) are added to the broth.
Observing Results
After adding the indicator, the color of the broth is noted and interpreted within a few minutes.
Interpretation of Results
Positive Methyl red Test
Bacteria that ferment glucose through the mixed-acid pathway produce stable acids, which lower the pH of the medium to around 4.4 or below.
Adding methyl red to this culture will turn the solution red, indicating a positive MR test.
Examples of bacteria with positive MR results include Escherichia coli and Proteus vulgaris.
Negative Methyl red Test
If the bacteria do not produce stable acids in significant quantities, the pH remains above 6.0.
Adding methyl red to this culture will result in a yellow or orange color, signaling a negative MR test.
Examples of bacteria with negative MR results include Enterobacter aerogenes and Klebsiella pneumoniae.
How to Interpret the Results
Red color: A positive result, indicating the organism produces stable acids from glucose fermentation, lowering the pH significantly.
Yellow color: A negative result, showing the bacteria either do not use the mixed-acid pathway or produce neutral fermentation products, keeping the pH higher.
Orange color: An intermediate result, often interpreted as negative, suggesting that while some acid may be produced, the pH has not dropped low enough to turn the medium red.
Applications
The MR test is a key part of bacterial identification, especially within the Enterobacteriaceae family. These bacteria are gram-negative rods typically found in the intestines of humans and animals. By combining the MR test with the Voges-Proskauer (VP) test, which detects neutral by-products such as acetoin, microbiologists can classify bacteria more precisely:
Escherichia coli: MR-positive, VP-negative
Enterobacter aerogenes: MR-negative, VP-positive
The MR test is especially useful for differentiating between Escherichia coli, which produces stable acids from glucose, and Enterobacter aerogenes, which produces more neutral by-products that raise the pH.
Limitations
Extended Incubation: Some bacteria may require longer than 48 hours (up to 72 hours) to produce sufficient acid for detection.
False Negatives: Bacteria that ferment glucose but do not generate enough acid in the given time could produce false-negative results.
Sensitivity: The MR test’s accuracy can vary depending on the glucose concentration and the buffering capacity of the medium.
Conclusion
The Methyl Red (MR) test is a straightforward and reliable method for identifying bacteria that produce stable acids from glucose fermentation. It is part of the IMViC series and is crucial in distinguishing between different Enterobacteriaceae family members. A positive result is indicated by a red color change, showing strong acid production, while a negative result is seen with a yellow or orange color, indicating a lack of stable acid formation.
Frequently Asked Questions (FAQ)
What do you mean by fermentation?
Fermentation is a biological process where microorganisms, such as bacteria or yeast, break down sugars like glucose to produce energy, resulting in by-products like acids, gases, or alcohol, without using oxygen. It’s a type of anaerobic respiration used by organisms to generate energy when oxygen levels are low or absent.
Give the examples of positive Methyl Red test?
The examples of positive Methyl Red test includes Escherichia coli and Proteus vulgaris.
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