MR-VP Medium

Intended Use

MR-VP Medium and MR-VP Broth (Methyl Red-Voges Proskauer Medium/Broth, also known as Buffered Peptone-Glucose Broth) are used for the differentiation of bacteria by means of the methyl red and Voges-Proskauer reactions.

Summary and Explanation

Voges and Proskauer, in the latter part of the 19th century, reported the initial observations regarding the production of a red color after the addition of potassium hydroxide to specific culture media in which various organisms had grown.¹

Clark and Lubs,² in 1915, found that the addition of methyl red to cultures of Escherichia coli resulted in a red color due to the high acidity produced during the fermentation of dextrose. The smaller amount of acid produced by Klebsiella pneumoniae and Enterobacter aerogenes is converted to acetoin resulting in an alkaline reaction (negative methyl red test).

In the Voges-Proskauer test, Reagent A (5% [w/v] alphanaphthol in absolute alcohol) contains a catalyst enhancing the formation of specific metabolic products that form a red complex upon the addition of Reagent B (40% [w/v] potassium hydroxide in purified water).

MR-VP Medium/Broth was developed to enable both the MR and the VP tests to be performed in the same medium, although in different tubes or on aliquots from the same tube.

Principles of the Procedure

Methyl red-positive organisms produce high levels of acid during fermentation of dextrose, overcome the phosphate buffer system and produce a red color upon the addition of the methyl red pH indicator.

In the Voges-Proskauer test, the red color produced by the addition of potassium hydroxide to cultures of certain microbial species is due to the ability of the organisms to produce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of dextrose.³ The acetoin is oxidized in the presence of oxygen and alkali to produce a red color.³ This is a positive Voges-Proskauer reaction.

User Quality Control

Identity Specifications
MR-VP Medium
Dehydrated Appearance: Very light to light beige, free-flowing, homogeneous.
Solution:                       1.7% solution, soluble in purified water. Solution
                                    is light amber, clear.
Prepared Appearance:     Light amber, clear.
Reaction of 1.7%
Solution at 25°C:           pH 6.9 ± 0.2
Cultural Response
MR-VP Medium
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at 35 ± 2°C for 40-48 hours.

Formula

MR-VP Medium
Approximate Formula* Per Liter
Buffered Peptone......................................................... 7.0 g
Dipotassium Phosphate................................................ 5.0 g
Dextrose..................................................................... 5.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Dissolve 17 g of the powder in 1 L of purified water. Mix thoroughly.
2. If necessary, heat slightly to dissolve.
3. Dispense and autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Using a light inoculum, inoculate tubes of MR-VP media with 18- to 24-hour pure cultures. Incubate tubes aerobically at 35 ± 2°C for a minimum of 48 hours but preferably for 5 days.

Prepare the methyl red indicator by dissolving 0.1 g of methyl red in 300 mL of 95% ethyl alcohol. Add sufficient purified water to make 500 mL.

After the appropriate incubation period, aseptically remove aliquots (1 mL for the VP test) of the medium and conduct the following tests:
1. Methyl Red Test – Add 5 drops of methyl red indicator to an aliquot of the broth. Interpret the color result immediately.
2. Voges-Proskauer Test – Empty the contents (15 drops) from the reagent A dropper and 5 drops from the reagent B dropper into 1 mL of broth culture. Shake well after the addition of each reagent to aerate the sample.

Expected Results

1. Methyl Red Test
    a. Positive – red color at surface of the medium.
    b. Negative – yellow color at surface of the medium.
2. Voges-Proskauer Test

A positive reaction is indicated by the development of a distinct red color which occurs within 5 minutes.

Certain species within Enterobacteriaceae genera may react differently or give variable results. Consult appropriate texts for reactions of specific species.^3-6

Limitations of the Procedure

1. Results of the MR and VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae.
2. A precipitate may form in the potassium hydroxide reagent solution. This precipitate has not been shown to reduce the effectiveness of the reagent.
3. Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test. However, certain organisms such as Hafnia alvei and Proteus mirabilis may give a positive result for both tests.
4. Incubation time for the Methyl Red test cannot be shortened by increasing the dextrose concentration in the medium or by heavily inoculating the broth.⁷
5. Incubate MR-negative tests for more than 48 hours and test again.
6. Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with reading the results.⁷
7. VP reagents must be added in the order and the amounts specified or a weak-positive or false-negative reaction may occur. A weak-positive reaction may be masked by a copper-like color which may form due to the reaction of KOH and α-naphthol.⁷
8. Read the VP test within 1 hour of adding the reagents. The KOH and α-naphthol may react to form a copper-like color, causing a potential false-positive interpretation.⁷
9. Due to the possible presence of acetoin, diacetyl or related substances in certain raw materials,8 the use of media low in these substances (such as MR-VP media) is recommended for this test.

Free Shipping within the Continental USA