Objectives of the Methyl Red (MR) Test
- Based on the generation of acid, to distinguish between two primary categories of facultative anaerobic enteric bacteria.
- Using a mixed-acid fermentation of glucose, it assesses the bacterial ability to produce stable acid end products.
Principle of Methyl Red (MR) Test
The MR test, also called the methyl red test, is used to determine an organism’s capacity to create and retain stable acid end products from the fermentation of glucose. Due to their physiological similarities and the use of MRVP broth, MR and VP tests are carried out concurrently. By using the Embden-Meyerhof pathway, all Enterobacteriaceae can convert glucose to pyruvic acid. However, there are two more methods by which bacteria may metabolise pyruvic acid.
By using the mixed acid route to break down pyruvic acid, organisms may maintain an acidic environment and create additional acidic by products like lactic acid and acetic acid. Methyl red is used to identify mixed acid fermentation, which causes the broth’s pH to decrease. After incubation, the MR indicator—which is red at pH 4.4 and yellow at pH 6.2—is added. The broth medium will stay red even after adding methyl red, a pH indicator, if the organism generates a lot of organic acids from the fermentation of glucose, such as formic acid, acetic acid, lactic acid, and succinic acid.
The initial fermentation products are further metabolised by MR-negative organisms through the process of decarboxylation to create neutral acetyl methylcarbinol (acetoin), which lowers the medium’s acidity and increases the pH towards neutrality (pH 6.0 or above). The broth medium will turn yellow for organisms that do not create the acid end products, giving a negative test result.
Buffered peptone 7.0 gm/L, Dextrose 5.0 gm/L, Dipotassium phosphate 5.0 gm/L, Final pH (at 25 °C) 6.9±0.2
Procedure of Methyl Red (MR) Test
- MRVP broth should be inoculated with a pure culture of the organism.
- For a minimum of 48 hours, incubate in room air at 35°–37 °C.
- Add 5 or 6 drops of the methyl red solution per 5 mL of broth.
- Keep an eye out for the broth medium’s colour to change.
Results Interpretation of the Methyl Red (MR) Test
Positive: Bright red colour
Weakly positive: red-orange colour.
Negative: Yellow colour
Limitations of Methyl Red (MR) Test
- Since some organisms might not have produced sufficient products during the fermentation of glucose within 48 hours, the MR test shouldn’t be read before that time.
- Additionally, MR-negative organisms could not have had enough time to convert those substances, which causes them to test positive for MR.
- If the methyl red test results are still inconclusive (orange) after 48 hours, incubate the broth for an additional three days and re-test the broth culture.
- To distinguish between distinct Enterobacteriaceae species, MR-VP testing should be used in combination with additional confirmatory assays.
Testing for Methyl Red (MR): Quality Control
MR Positive: test for Escherichia coli (ATCC25922)
MR negative: Enterobacter aerogenes (ATCC13048).
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- Aneja K.R (2003), Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.
- Barry AL, Bernsohn KL, Adams AB, Thrupp LD. Improved 18-hopur methyl red test. Appl Micro 1970; 20:866-70.
- Dalynn Biologicals (2002), Methly red reagent, catalogue no. RM65. http://www.dalynn.com/dyn/ck_assets/files/tech/RM65.pdf