Microbiologists use the Methyl red test to identify bacteria that can ferment glucose to stable-acid by-products. Methyl red (MR) is a pH indicator that turns red below 4.4, orange between 4.4 and 6.2, and yellow above 6.2.
Methyl red (2-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid) is a dark red crystalline azo dye.
The MR-VP medium provides the glucose substrate, while methyl red is the indicator.
They use MR-VP tests to distinguish between members of the Actinobacteria group and the family Enterobacteriaceae.
MR test forms part of the iMViC tests that consist of (Indole, MR, Voges Proskauer, and Citrate)
Methyl red test principle
Bacteria use different metabolic pathways to break down glucose or sugars into acid. Some bacteria produce unstable acids, which quickly change to neutral compounds. Some use the butylene glycol pathway, whose end products are neutral compounds like 2,3-butanediol or acetoin.
Other bacteria use the mixed acid pathway, which produces stable acids like acetic, succinic, lactic, and formic acid. These products remain acidic.
MR detects acid at a considerably low pH (<4.4) compared to other indicators used in culture media. The test bacteria must produce stable acid to produce a color change.
If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the media buffers and create an acidic environment. Methyl red will stay red if acidic end products are present.
Without stable acids, the MR indicator turns yellow in pH over 6.2 and orange between 4.4 and 6.2.
| Ingredient | Quantity |
|---|---|
| Distilled water | 1 L |
| Dipotassium phosphate | 5 g |
| Glucose | 5 g |
| Polypeptone | 7 g |
| Final pH | 6.9 |
Test guidelines
MR should not be confused with Phenol red. Phenol red turns yellow below a pH of 6.8, while MR turns red below a pH of 4.4. Make sure to distinguish the two.
Always conduct performance testing of both media and reagents with known bacteria to demonstrate positive and negative reactions. Also, check for signs of media contamination, such as deterioration and dehydration.
Avoid over-inoculation, and you inhibit bacterial growth when the inoculum exceeds 109 viable cells/ ml.
Allow ample time for incubation to prevent false positive results.
Microbiologists use MR-VP broth for both Methyl Red and Voges-Proskauer tests. The tests only differ on the reagent used; in most cases, they carry out the tests consecutively.
Most Enterobacteriaceae give opposite VP and MR reactions. Others like Hafnia alvei and Proteus mirabilis give similar results.
The test should be carried out in a sterile environment with personal protective equipment to prevent contamination.
Test requirements include MR/VP medium, MR reagent, test tubes, inoculating wire, and your specimen culture.
Methyl red test Procedure
- Bring the MR-VP broth medium in a test tube to room temperature.
- Inoculate a pure culture of the bacteria in question into the medium.
- Incubate the preparation at 37℃ for 24 hours.
- Add three drops of MR reagent into the test tube, and observe the color change.
- If you observe a red-orange color, repeat the procedure but incubate for four days at 37oC to prevent reporting false positive results.
- Report MR positive if you observe a red color and MR negative for the yellow or orange color.
Methyl-red positive bacteria
Methyl red-positive bacteria include Proteus, Shigella, Salmonella, Citrobacter, Proteus, Yersinia species, and Escherichia coli.
Methyl-red negative bacteria
Methyl red-positive bacteria include Hafnia and Enterobacter species, Serratia marcescens, Klebsiella pneumoniae, and Klebsiella aerogenes.
