Methyl red test differentiates whether the organism can undergo mixed acid fermentation or not, by the supply of glucose. Organisms that can ferment glucose will give positive MR test by producing a large amount of acid, and some organisms give negative MR test. It also helps to identify and classify between the different genera of enteric bacteria, based on the glucose fermentation. In simple words, the methyl red test checks the amount of acid formed by an organism after fermenting glucose.
Due to the production of acid, the pH of the media falls down from 6.9 to 4.5. The acid formation can be visualized by the addition of a “Methyl red” pH indicator. If the test organism in the media causes the fermentation of glucose, then methyl red solution will impart a red colour to it.
Content: Methyl Red Test
Methyl red test sometimes abbreviated as MR-test. It is one of the procedures of an IMViC analysis, where ‘M’ is an acronym for the method named “Methyl red test”. It can define as a quantitative test that is indicated by the appearance of red colour in the media depending upon the amount of acid production.
Things to Keep in Mind
Before jump onto the theory of methyl red test, we must know about the basic things. On studying the MR test, we will go through the following terms that we should keep in mind:
Methyl red is an acidic pH indicator, which contains p-dimethylaminoaeobenzene-O-carboxylic acid that quantifies the H+ ion concentration in the media. If the media includes a sufficient amount of acids, then the methyl red gives off red colour. And if there is no acid formation, the media will remain yellow even by the addition of methyl red indicator.
MRVP broth is a standard media to perform methyl red and Voges Proskauer test. This media is supplemented with glucose, which is exploited by the test organisms to carry out anaerobic fermentation or mixed acid fermentation into a variety of end products.
Gram-negative bacteria of the Enterobacteriaceae family generally undergo mixed acid fermentation. Mixed acid fermentation pathway is a process of converting glucose (6-C compound) into products like four stable acids (lactic, acetic, formic, succinic acids), one molecule each of ethanol, carbon dioxide and hydrogen.
Principle of Methyl Red Test
In the methyl red test, MRVP broth is used that contains a sufficient amount of glucose. The fermentative enteric bacteria will catalyse the conversion of glucose into pyruvic acid by a number of enzymes. Then, pyruvic acid undergoes mixed acid pathway that generally forms four stable acidic products, and one molecule of ethanol, CO2, H2 by the fermentation of a single glucose molecule.
There are four stable acidic products are produced, namely lactic acid, formic acid, succinic acid, acetic acid etc. as an end products of a mixed acid pathway that differs from species to species. The production of stable acids falls down the pH to 4.5 or below. The decrease in pH will finally result in a colour change of MRVP broth from yellow to red indicated by adding methyl red indicator.
The methyl red uses a standard media (MRVP broth) and an indicator reagent (methyl red).
Composition of MRVP broth:
Polypeptone: 7 g
Glucose: 5 g
Dipotassium phosphate: 5 g
Distilled water: 1 L
Final pH: 6.9
The protocol for performing the methyl red test includes the following steps:
- First, take all the ingredients required to prepare MRVP broth and methyl red indicator.
- Then, prepare MRVP broth by adding distilled water to the weighed ingredients and autoclave for 15 minutes at 121 degrees Celsius temperature.
- Under aseptic conditions, pour 3 ml of MRVP broth into the dust-free tubes.
- Inoculate 24-hour old culture of the test organism via red hot inoculating loop into the MRVP broth.
- Incubate the test tubes for 2-5 days at a temperature between 35-37 degrees Celsius.
- Add a few drops of methyl red solution to observe any colour change in the broth.
Positive result: Confirms by the appearance of red colour on the top of MRVP broth.
Examples: Escherichia coli, Proteus vulgaris, Yersinia sp etc.
Negative result: Here, the colour of the media do not undergo any change.
Examples: Enterobacter aerogenes, Klebsiella pneumoniae, Serratia marcescens etc.