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General laboratory information for lab and clinic staff; not medical advice and not a laboratory procedure.
Short answer
The methyl red (MR) test shows whether a bacterial culture grown in glucose broth produced enough acid by mixed-acid fermentation to hold the medium at or below pH 4.4, where methyl red turns red (MR positive). A yellow result is MR negative. It is one of four tests in the IMViC battery and is normally read alongside the Voges-Proskauer (VP) test, which grows in the same MR-VP broth.
Definition and scope
The American Society for Microbiology (ASM) protocol by Sylvia McDevitt describes MR and VP as two tests that belong to the IMViC group: indole, methyl red, Voges-Proskauer and citrate (the “i” is added for pronunciation). The battery was developed to separate members of the Enterobacteriaceae and is now also used to characterize other groups, including Actinobacteria.
The test is historical as well as practical. Clark and Lubs introduced methyl red as a pH indicator in 1915 to differentiate colon-type from aerogenes-type bacteria, and the alpha-naphthol intensifier that most labs use for the VP reaction goes back to Barritt in 1936.
How the test works
Mixed-acid fermentation and the pH indicator
Organisms that ferment glucose by the mixed-acid pathway produce enough acid to lower the pH of the broth sharply. Methyl red is a pH indicator that is red at or below pH 4.4 and yellow at about pH 6.0, so a red culture is MR positive and a yellow one is MR negative. ASM lists Escherichia coli as a suggested MR-positive control and Enterobacter aerogenes as a suggested MR-negative control.
Why it is paired with the Voges-Proskauer test
Organisms that instead use the butanediol pathway make acetoin (acetyl methyl carbinol, 3-hydroxybutanone) and keep the broth less acidic. In the VP reaction, potassium hydroxide oxidizes acetoin to diacetyl, a step catalyzed by alpha-naphthol, and diacetyl reacts with a guanidine group from the peptone to give a pinkish-red color. ASM suggests E. coli as MR+/VP- and E. aerogenes as MR-/VP+. The pairing is a useful contrast, but it is not a strict opposition: Vaughn and colleagues (1939) studied an “intermediate” group (Enterobacter, Klebsiella, Hafnia, Proteus, Serratia) in which an organism can be MR positive and VP positive at once.
Medium and reagents (as supply items)
The test uses a buffered peptone-glucose broth, sold as MR-VP broth, plus indicator and Barritt reagents. The ASM protocol gives the following compositions.
| Item | Composition per ASM | Note from ASM |
|---|---|---|
| MR-VP broth | Buffered peptone 7.0 g, dipotassium phosphate 5.0 g, dextrose 5.0 g per litre; final pH 6.9 ± 0.2 at 25°C (0.5% glucose) | Commercially available as MR-VP broth; the medium is a modification of the original Clark and Lubs formulation with reduced glucose and peptone |
| Methyl red solution | 0.1 g methyl red in 300 ml of 95% ethanol plus 200 ml water, i.e. 0.05% (w/v) in 60% ethanol | Stored at 4°C |
| Barritt A (VP-1) | 5% alpha-naphthol in absolute ethanol | Color intensifier; reagents must be prepared fresh |
| Barritt B (VP-2) | 40% potassium hydroxide (ASM notes 40% NaOH may be used) | Reagents must be prepared fresh |
ASM notes that two tubes of MR-VP broth can be used instead of splitting one culture between the two tests, and that small screw-cap tubes reduce leakage. It refers readers to its safety guidelines for teaching laboratories and to safety data sheets for the broth, reagents, alpha-naphthol, methyl red and ethanol.
Reading results: a validity checklist
A clear red or yellow is straightforward; most disputed results trace back to a handful of conditions that ASM names explicitly.
- Incubation length and temperature. ASM specifies 35°C ± 2°C for 48 hours. An orange result, or a false negative, may occur if incubation was too short; ASM advises repeating after a further 24 to 48 hours.
- Inoculum. ASM specifies a light inoculum from a fresh 18 to 24 hour pure culture; a heavy inoculum can give aberrant results.
- Glucose level. More glucose in the medium than specified can cause a false-positive MR.
- VP timing. A VP incubation shorter or longer than 48 hours may give false negatives, and the VP result should be read within one hour because a delayed reading can develop a copper-like color.
- Temperature-variable organisms. Hafnia alvei and Enterobacter can be VP variable at 37°C but positive at 25 to 30°C.
- Reagent age. ASM states that the reagents must be prepared fresh.
Where it matters for labs and supply decisions
For stocking and quality systems, the practical point is that MR-VP broth is a differential culture medium. Under 21 CFR 866.2320, a differential culture medium is a device made mostly of liquid biological material used to grow and identify different types of pathogenic microorganisms, where a specific biochemical component is added and organisms are identified by a visible change such as a color change that shows certain metabolic reactions. The regulation states that results help diagnose disease and provide epidemiological information. It is classified as Class I (general controls) and is exempt from premarket notification under subpart E of part 807, subject to the limitations in 866.9.
Because the reagents are prepared fresh and the broth is a sterile culture medium, a lab running the test also depends on sterilization capacity and sound aseptic practice. See CASRAI’s guides on what an autoclave is, lab incubators and identifying and preventing culture contamination, and the dictionary entries for biosafety cabinet and Biosafety Level 2.
Common misunderstandings
“MR and VP are opposites, so one result predicts the other”
Often they contrast, as with E. coli and E. aerogenes, but the intermediate group studied by Vaughn et al. can be positive for both.
“An orange tube means a weak positive”
ASM treats an inconclusive orange color as a possible sign of insufficient incubation, with repeat testing after more time, not as a result to report.
“More glucose gives a stronger test”
Excess glucose can cause a false-positive MR, which is why the medium is specified at 0.5% glucose.
FAQ
What does a red methyl red result mean?
Red indicates the culture acidified the broth to pH 4.4 or below by mixed-acid fermentation, so the organism is MR positive. Yellow is MR negative (ASM).
What is the Voges-Proskauer test?
It detects acetoin, produced through the butanediol pathway. With Barritt reagents it gives a pinkish-red color when positive, and it should be read within one hour (ASM).
What is MR-VP broth?
A buffered peptone-glucose broth (peptone, dipotassium phosphate and 0.5% glucose, final pH 6.9 ± 0.2) used for both the MR and VP tests (ASM).
Sources
- McDevitt S. “Methyl Red and Voges-Proskauer Test Protocols.” American Society for Microbiology, created 8 December 2009. https://asm.org/protocols/methyl-red-and-voges-proskauer-test-protocols. Date checked: 2026-10-30.
- 21 CFR 866.2320, Differential culture medium. https://www.ecfr.gov/current/title-21/section-866.2320. Date checked: 2026-10-30.
