Methyl Red Test Principle Procedure and Results

In this methyl red test principle procedure and results post we have briefly explained methyl red test principle, objectives, requirements, procedure, uses and limitations of methyl red test procedure.

Methyl Red Test Principle Procedure and Results

Methyl red test procedure is a biochemical test used to determine whether a bacterial species has the potential to create stable acid end products (Mixed-acid fermentation) from glucose. Organisms that can ferment glucose will produce a considerable amount of acid, resulting in a positive methyl red test, whilst others will result in a negative methyl red test.

Methyl red test procedure also aids in the identification and classification of distinct enteric bacteria genera based on glucose fermentation. Methyl red test procedure determines how much acid an organism produces following glucose fermentation. The pH of the media drops from 6.9 to 4.5 as a result of the acid generation.

Definition

Methyl red test procedure is also known as the MR-test. It’s a quantitative test that, depending on the amount of acid produced, is proven by the presence of red colour in the medium.

Principle

Methyl red test procedure is performed with MRVP broth, which offers an appropriate amount of glucose. Through their complicated enzyme system, the fermentative intestinal bacteria convert glucose to pyruvic acid. 

The pyruvic acid next enters the mixed acid pathway, where it ferments a single glucose molecule to produce four stable acidic metabolites and one molecule of ethanol, CO2, and H2. Lactic acid, formic acid, succinic acid, and acetic acid are the four stable acidic compounds, and the end products of a mixed acid pathway differ from species to species. 

The pH drops to 4.5 or below when stable acids are produced. The drop in pH will eventually cause the colour of MRVP broth to change from yellow to red, which we can see by using a methyl red indicator.

Media and Reagents

Composition

Polypeptone: 7 g

Glucose: 5 g

Dipotassium phosphate: 5 g

Distilled water: 1 L

Final pH: 6.9

Indicator

Methyl red: 50 mg

0.1M NaOH: 1.86 ml

95% ethyl alcohol: 50 ml

Distilled water: Take distilled water to make the volume up to 100 ml.

Methyl Red Test Procedure

1. Then, add distilled water to the weighed materials and autoclave for 15 minutes at 121 degrees Celsius to make Methyl red test procedure broth.

2. Fill the dust-free tubes with 3 mL of MRVP broth under aseptic conditions. Inoculate the Methyl red test procedure broth with a 24-hour-old culture of the test organism using a red hot inoculating loop.

3. Incubate the test tubes at a temperature of 35-37 degrees Celsius for 2-5 days. To see if the soup changes colour, add a few drops of methyl red solution.

Result

A positive methyl red test is when the surface of the medium develops a stable red colour, indicating adequate acid production to drop the pH to 4.4.

Other organisms producing smaller amounts of acid from the test substrate may create an intermediate orange colour between yellow and red.

This does not imply that the test was positive methyl red test. A negative test is shown by the colour yellow.

Methyl Red test

Methyl Red Test Principle Procedure and Results

Quality Control

Escherichia coli ATCC 25922: MR positive (red), VP negative (no change)

Klebsiella pneumoniae ATCC 13883: MR negative (yellow), VP positive (red).

Applications

1. Do not read the MR test before 48 hours, as some organisms will not have created enough compounds from glucose fermentation.

2. MR-negative organisms may not have had enough time to transform those products, resulting in an MR positive appearance.

3. If the methyl red test procedure results are inconclusive (orange) after 48 hours, leave the broth incubating for another three days and retest the broth culture.

4. To differentiate Enterobacteriaceae species, methyl red test procedure should be utilised in conjunction with additional confirmatory assays.

Limitations

1. Do not read the methyl red test procedure before 48 hours, as some organisms will not have created enough compounds from glucose fermentation.

2. MR-negative organisms may not have had enough time to transform those products, resulting in an MR positive appearance.

3. If the methyl red test results are inconclusive (orange) after 48 hours, leave the broth incubating for another three days and retest the broth culture.

4. To differentiate Enterobacteriaceae species, MR-VP testing should be utilised in conjunction with additional confirmatory assays.

Further Readings

Reference