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Methyl Red for MR-VP Tests

Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC tests as the medium in which both the Methyl Red and Voges-Prosakuer tests can be performed. It is a simple broth that contains peptone, buffers, and dextrose or glucose.

Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways. Some of these pathways produce unstable acidic products which quickly convert to neutral compounds. Some organisms use the butylene glycol pathway, which produces neutral end products, including acetoin and 2,3-butanediol. Other organisms use the mixed acid pathway, which produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are stable and will remain acidic.

The Methyl Red test involves adding the pH indicator methyl red to an inoculated tube of MR-VP broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red.

Test Procedure:
1. Lightly inoculate the tube from a single colony, preferably an 18-24 hour culture.
2. Slightly loosen the cap and incubate the tubes at 35-37°C for 48 hours.
3. After incubation, use a sterile pipette to remove two - 1mL aliquots and place into two small tubes.
• One tube is for the methyl red test and the other for the Voges-Proskauer test.
• You do not want to contaminate your original broth tube in case you have to do further incubation.
4. Add 5 drops of methyl red to one tube. Read the result immediately. Do NOT mix the tube.
5. For the Voges-Proskauer test add 15 drops of Voges-Proskauer A reagent. Mix well to aerate the sample. Oxygen is needed to complete the reaction.
6. Add 5 drops of Voges-Proskauer B to the tube and mix well to aerate the sample.
7. Read the results within 5-15 minutes.
Methyl Red - A red color at the surface is considered a positive result. A negative test is indicated by a yellow color at the surface.
Voges-Proskauer - A positive test is indicated by a pink-red color developing within 5 minutes.
Limitations of Procedure:
• Other tests are needed to definitively identify the Enterobacteriaceae.
• The VP test should be done at 48 hours. Longer incubation times could result in false positives.
• The VP reagents must be added in the order listed and with mixing to avoid weak-positive or falsenegative results.
• The broth must be incubated for a minimum of 48 hours for the MR test. Negative MR tests should be incubated for an additional 48 hours.

NOTE: Methyl red differs from Phenol red (which is used in the fermentation test and the MSA plates) in that it is yellow at pH 6.2 and above and red at pH 4.4 and below. Phenol red turns yellow below a pH of 6.8. If you get these two pH indicators confused, you will have a difficult time interpreting test results.

The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a copper color is present, the result is negative.

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