MR-VP Test: Introduction, Principle, Test Requirement, Procedure

Introduction of MR-VP Test

Table of Contents

MR-VP test stands for Methyl Red –Voges Proskauer Test. MR-VP test will be discussed in two tests separately. One is for Methyl Red (MR) test and while other is for Voges Proskauer (VP)test.

Introduction of Methyl Red Test

The Methyl Red test is for enteric gram-negative rods, as part of identiﬁcation to species level. Its short name is MR test.

Principle of Methyl Red Test

The methyl red (MR) test uses to determine if an organism is able to produce stable acid end products from glucose fermentation Methyl red indicator (red color below pH 4.4; yellow color at pH 5.8) uses to determine the pH after an enteric gram-negative rod has fermented glucose to completion. All members of the Enterobacteriaceae give a positive methyl red reaction when tested for up to 24 hours due to the conversion of glucose to pyruvic acid by the Embden-Meyerhof pathway. After further incubation (2 to 5 days) those organisms that are MR positive continue to metabolize pyruvic acid to lactic, acetic, and formic acids by the mixed acid pathway and are able to maintain the acid pH (4.4).

Fig. Methyl red (MR) test reagent, positive and negative test results of MR tests

Composition of MR-VP Broth

Buffered peptone:7.0 g
Glucose:5.0 g
Dipotassium phosphate:5.0 g
Deionized / Distilled water: 1000 ml
Final pH:6.9

Generally dispense approximately 5 ml per tube.
Use enough broth to cover an inverted Durham tube, if it is used.
Dispense 2 ml of MR-VP broth for rapid VP testing and 0.5 ml for rapid MR testing.

Preparation of Methyl Red Solution

Methyl red solution, 0.02%

Dissolve 0.1 g of methyl red in 300 ml of 95% ethyl alcohol.
Add sufﬁcient distilled water to make 500 ml.
Store at 4 to 8°C in a brown bottle.
The solution is stable for 1 year.

Requirements for Methyl Red Test

MR-VP broth
Test organism
Inoculating wire
Bunsen burner
Incubator
Methyl red indicator
Control strains

Escherichia coli ATCC 25922
Klebsiella pneumoniae ATCC 13883

Procedure of Methyl Red Test

Inoculate the test organism and incubate at 35C for at least 48 hours. Note: If the center of one colony is inoculated to a 0.5-ml volume of MR-VP broth, the test can be read at 18 to 24 hours.
Remove approximately 1 ml of the 48-hours broth to a 13-by-100-mm tube. (The remainder should be reserved for testing at 3 to 5 days if necessary.)
Add 3 to 6 drops (or 1 drop to 0.5 ml) of methyl red indicator to the aliquot.
Observe for red color immediately.

Result and Interpretation of Methyl Red Test

The development of a stable red color on the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitutes a positive test. Because other organisms may produce smaller quantities of acid from the test substrate, an intermediate orange color between yellow and red may develop. This does not indicate a positive test. The yellow color indicates a negative test.

Fig. Methyl Red Test-Positive and Negative Test Results

Result of Quality Control Strains

Escherichia coli ATCC 25922—MR positive(red),VP negative(no change)
Klebsiella pneumoniae ATCC 13883—MR negative (yellow), VP positive (red)

List of Methyl Red Test Positive and Negative Bacteria

Methyl red test-positive bacteria are as follows:

Escherichia coli
Klebsiella ozanae
Klebsiella rhinoscleromatis
Klebsiella ornithiolytica
Citrobacter
Proteus
Yersinia
Edwardsiella
Salmonella

MR Test Negative Bacteria

Klebsiella pneumoniae
Enterobacter species

Limitations of MR Test

For the complete identification of bacteria, further biochemical tests are also used from the pure colony of bacteria.
The methyl red test must not be reported unless the medium has been incubated for a minimum of 48 hours. Tests that are run too early may result in false-positive result interpretation.
If an inoculum is too heavy, bacterial growth may be inhibited that creating invalid test results hence a light inoculum is preferred.
Incubation periods of up to 5 days may be necessary for the methyl red test result interpretation.

Voges-Proskauer (VP ) Test

Voges Proskauer test for identiﬁcation to the species level of the following groups of organisms

Enteric gram-negative rods, Aeromonas, and Vibrio
Viridans group streptococci
Staphylococci

Fig. Voges Proskauer Test Reagents, Negative and Positive Test Results of VP Tests

Principle of Voges Proskauer Test

Organisms utilizing the butylene glycol pathway produce acetylmethylcarbinol (acetoin) and butanediol, neutral end products that raise the pH towards neutrality (pH 6) and result in a high ﬁnal pH. Most Enterobacteriaceae demonstrate one or the other metabolic pathway but rarely both. The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethylcarbinol from glucose fermentation. If present, acetylmethylcarbinol is converted to diacetyl in the presence of α-naphthol, strong alkali (40% KOH), and atmospheric oxygen. The α-naphthol was not part of the original procedure but was found to act as a color intensiﬁer and must be added ﬁrst. The diacetyl and guanidine-containing compounds found in the peptones of the broth then condense to form a pinkish-red polymer.

Reagent Preparation

Reagent 1: 5% α-Naphthol

α-Naphthol: 5 g

95% ethyl alcohol:100 ml

Take 5-gram α-Naphthol in 100 ml of 95% ethyl alcohol and mix properly.
Store at 4 to 8°C in the dark.
Shelf life: 2 to 3 weeks.

Reagent 2: 40% Potassium Hydroxide

Potassium hydroxide( KOH) pellets: 20 gram

Distilled water: up to 100 ml

Dissolve 40 g of potassium hydroxide pellets in 100 ml of distilled water in a polyethylene bottle

Keep a bottle in a cool water bath during preparation.

Caution: KOH is hygroscopic and becomes caustic when moist. Weigh quickly in a tared beaker. Store away from acids. Avoid exposure to skin.

Requirements for Voges Proskauer Test

MR-VP broth
Test organism
Inoculating wire
Bunsen burner
Incubator
5%α- naphthol
40% KOH
Control strains

Klebsiella pneumoniae ATCC 13883
Escherichia coli ATCC 25922

Procedure of Voges Proskauer Test

Inoculate a colony of test organism in MR-VP broth and incubate at 35°C for 18 to 24 hours. Do not tighten caps. Note: Some organisms may produce acetylmethylcarbinol at room temperature and not 35°C e.g. Hafnia alvei, Yersinia, Listeria. In this case, inoculate another broth and incubate at room temperature.
If a 5-ml broth culture is used, aliquot 2.0 ml of broth into a 13 by 100-mm test tube. Hold the remainder for possible reincubation.
Add 6 drops of 5% α-naphthol, and mix well to aerate.
Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
Observe for a pink-red color at the surface within 30 min. Shake the tube vigorously during the 30-min period. Note: If the result is negative, MR-VP broth can be incubated for up to 48 hours and the test repeated.

Result and Interpretation of Voges Proskauer Test

Voges- Proskauer test positive: A pink-red color at the surface is a positive reaction
Voges- Proskauer test negative: A lack of a pink-red color is a negative reaction.

Control strains

Klebsiella pneumoniae ATCC 13883—VP positive (red)
Escherichia coli ATCC 25922—VP negative (no change)
A copper color should be considered negative. The rust color is a weak positive
Most members of the family Enterobacteriaceae give opposite MR and VP reactions; however, certain organisms, like H. alvei and Proteus mirabilis, may give both a positive MR reaction and a positive VP reaction (often delayed)
Streptococcus mitis group organisms are VP negative, whereas the other viridans group streptococci are VP positive, except Streptococcus vestibularis, which is a VP variable.
Listeria organisms are beta-hemolytic, gram-positive rods that are VP-positive at 25°C, but this test is not a key test in the identiﬁcation.