Methyl Red and Voges-Proskauer Tests

Biochemical Reactions/Pathway

Discussion: In addition to discussing your lab results; include in your discussion, the biochemical reactions/pathway we detected in this experiment. Also, include the names of the chemicals added to this experiment & why they were added.

Methyl red and Voges-Proskauer (MR-VP) broth is a combination medium used for both methyl red (MR) and Voges-Proskauer (VP) tests. It is a simple solution containing only peptone, glucose, and a phosphate buffer. The peptone and glucose provide protein (with its nitrogen) and fermentable carbohydrate, respectively, and the potasshun phosphate resists pH changes in the medium.

The MR test is designed to detect organisms capable of performing a mixed acid fermentation, which overcomes the phosphate buffer in the medium and lowers the pH. The acids produced by these organismstend to be stable, whereas acids produced by other organisms tend to be unstable and subsequently are converted to more neutral products.

Mixed acid fermentation is verified by the addition of methyl red indicator dye following incubation. Methyl red is red at pH 4.4 and yellow at pH 6.2. Betweed these two pH values, it is various shades of orange. R.ed color is the only true indication of a positive result. Orange is negative or inconclusive. Yellow is negative. The Voges-Proskauer test was designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butanediol. Adding VP reagents to the medium oxidizes the acetoin to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color. A positive VP result, therefore, is red. No color change (or development of copper color) after the addition of reagents is negative. The copper color is a result of interactions between the reagents and should not be confused with the true red color of a positive result.

 Use of positive and negative controls for comparison is usually recommended. Glycolysis To avoid conflicting results of the two tests, two 1 mL volumes are transferred from the MR-VP broth to separate test tubes after incubation. Methyl red indicator reagent is added to one tube, and VP reagents are added to the other. Color changes then are observed and documented. Refer to the procedural diagram in Figure 5.14.

Time 5.9 pH Changes in MR-VP Broth The MR test identifies organisms that perform a mixed acid fermentation and produce stable acid end products. MR ( +) organisms lower the broth's pH

permanently. The VP test is used to identify organisms that perform a 2,3-butanediol fermentation. VP (-) organisms initially may produce acid and temporarily lower the pH, but because the 2,3butanediol fermentation end products are neutral, the pH at completion of the test is near neutral. Lactate (CH3CHOH·COO")

Application The methyl red and Voges-Proskauer tests are components of the IMViC battery of tests (Indole, Methyl red, Voges-Proskauer, and Citrate) used to distinguish between members of the family Enterobacteriaceae and differentiate them from other Gram-negative rods. For more information about these fermentations, refer to Figure. In This Exercise Today you will perform methyl red and Voges-Proskauer tests on Escherichia coli and Enterobacter aerogenes. After a 48 hour incubation period, you will observe the 5.10 Methyl Red Test Results The tube on the left is MR positive. The tube on the right is MR negative. Acetaldehyde 1 f . d 'd fermentation and a 2,3butanediol resu ts o a mJXe ac1 . f t . The exercise involves transfer of a portion ermentali ving organisms and the measure o rot contammg to complete the observable reactions. a ltlon o reagen s Pl . transfers and in your measurements. ease use care m your For help with the procedure, refer to Figure 5· For help interpreting your results, refer to Figures and Tables 5-3 and 5-4.

Materials Per Student o Gloves u Chemical eye protection Per Student Group o Three MR-VP broths Methyl red reagent o VP reagents A and B Six nonsterile test tubes Three nonsterile 1 mL pipettes Fresh cultures of:

1. Escherichia coli

2. Enterobacter aerogenes