Introduction of MR Test
MR test stands for Methyl Red test. It is used for enteric gram-negative rods, as part of identiﬁcation to species level. MR test positive bacteria are Escherichia coli, Klebsiella ozanae, Klebsiella rhinoscleromatis while negative are Klebsiella pneumoniae, Enterobacter species, etc.
Principle of MR Test
The methyl red (MR) test is used to determine if an organism is able to produce stable acid end products from glucose fermentation Methyl red indicator (red color below pH 4.4; yellow color at pH 5.8) uses to determine the pH after an enteric gram-negative rod has fermented glucose to completion. All members of the Enterobacteriaceae give a positive methyl red reaction when tested up to 24 h due to conversion of glucose to pyruvic acid by the Embden-Meyerhof pathway. After further incubation (2 to 5 days) those organisms that are MR positive continue to metabolize pyruvic acid to lactic, acetic, and formic acids by the mixed acid pathway and are able to maintain the acid pH (4.4).
Composition of MR-VP broth
- Buffered peptone:7.0 g
- Glucose:5.0 g
- Dipotassium phosphate:5.0 g
- Deionized / Distilled water: 1000 ml
- Final pH:6.9
- Generally dispense approximately 5 ml per tube.
- Use enough broth to cover an inverted Durham tube, if it is used.
- Dispense 2 ml of MR-VP broth for rapid VP testing and 0.5 ml for rapid MR testing.
Preparation of MR Solution
Methyl red solution, 0.02%
- Dissolve 0.1 g of methyl red in 300 ml of 95% ethyl alcohol.
- Add sufﬁcient distilled water to make 500 ml.
- Store at 4 to 8°C in a brown bottle.
- The solution is stable for 1 year.
Requirements for MR Test
- MR-VP broth
- Test organism
- Inoculating wire
- Bunsen burner
- Methyl red indicator
- Control strains
Procedure of Methyl Red Test
- Inoculate test organism and incubate at 35C for at least 48 hours. Note: If the center of one colony is inoculated to a 0.5-ml volume of MR-VP broth, the test can be read at 18 to 24 hours.
- Remove approximately 1 ml of the 48-hours broth to a 13-by 100-mm tube. (The remainder should be reserved for testing at 3 to 5 days if necessary.)
- Add 3 to 6 drops (or 1 drop to 0.5 ml) of methyl red indicator to the aliquot.
- Observe for red color immediately.
Result and Interpretation of MR Test
The development of a stable red color on the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitutes a positive test. Because other organisms may produce smaller quantities of acid from the test substrate, an intermediate orange color between yellow and red may develop. This does not indicate a positive test. The yellow color indicates a negative test.
Result of Quality control strains
- Escherichia coli ATCC 25922—MR positive(red),VP negative(no change)
- Klebsiella pneumoniae ATCC 13883—MR negative (yellow), VP positive (red)
Keynotes on MR Test
- A weak positive reaction is red-orange. If an orange color is present, incubate the broth further for up to 4 days and repeat the test after further incubation. In this case, it may also be helpful to set up a duplicate broth at 25°C.
- Initially, the paired MR-VP tests were used to distinguish between members of Enterobacteriaceae, but now they are used to characterize other groups of bacteria including Actinobacteria (a phylum of mostly Gram-positive bacteria).
List of methyl red test positive and negative bacteria
Methyl red test positive bacteria are as follows:
- Escherichia coli
- Klebsiella ozanae
- Klebsiella rhinoscleromatis
- Klebsiella ornithiolytica
Methyl red test negative bacteria
- Klebsiella pneumoniae
- Enterobacter species
Limitations of MR Test
- For the complete identification of bacteria, further biochemical tests are also used from the pure colony of bacteria.
- The methyl red test must not be reported unless the medium has been incubated for a minimum of 48 hours. Tests that are run too early may result in false-positive result interpretation.
- If an inoculum is too heavy, bacterial growth may be inhibited that creating invalid test results and hence a light inoculum is preferred.
- Incubation periods up to 5 days may be necessary for the methyl red test result interpretation.
- Lynae S. Carcia, Second Edition update, Clinical Microbiology Procedures Handbook
- Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
- B.D. Skerman, A guide to the identification of the genera of bacteria, The Williams & Wilkins Co., Baltimore, MD, (1967)
- Cowan and Steel’s,manual for the identification of medical bacteria