Methyl Red & Vogues-Proskauer Tests

Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC test panel. It is the medium in which both the Methyl Red and Voges-Proskauer tests can be performed. 

MR-VP broth is a simple broth that contains peptone and dextrose or glucose as nutrients. It also contains buffers.

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 acids. 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.

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.

Instead of making you aliquot inoculated broth into sterile test tubes, we have already divided the broth. Inoculate two MR-VP broths for each organism you wish to test. For the lab exercise, you would inoculate two MR-VP broths with Escherichia coli and two MR-VP broths with Enterobacter cloacae. After incubation, you would perform an MR test on one tube of Escherichia coli and one tube of Enterobacter cloacae, and you would perform a VP test on the other tube of Escherichia coli and the other tube of Enterobacter cloacae.

The MR and VP tests are part of the IMViC panel and are particularly useful in the identification of the Enterobacteriaceae. You will perform the IMViC panel very early in the identification of your Gram negative unknown.