MR-VP Test: Introduction, Principle, Procedure, Result Interpretation and Keynotes

MR-VP Test: Introduction, Principle, Procedure, Result Interpretation and Keynotes

Introduction of MR-VP Test

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

Introduction of MR Test

MR test stands for Methyl Red test. It is used for enteric gram-negative rods, as part of identification to species level. MR test positive bacteria are Escherichia coli, Klebsiella ozanae, Klebsiella rhinoscleromatis while negative are  Klebsiella pneumoniae, Enterobacter species, etc.

Principle of MR Test

The methyl red (MR) test is used 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 up to 24 h due to 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).

Composition of MR-VP test broth

  •  Buffered peptone:7.0 g
  • Glucose:5.0 g
  • Dipotassium phosphate:5.0 g
  • Deionized / Distilled water: 1000 ml
  • Final pH:6.9
  1. Generally dispense approximately 5 ml per tube.
  2. Use enough broth to cover an inverted Durham tube, if it is used.
  3. Dispense 2 ml of MR-VP broth for rapid VP testing and 0.5 ml for rapid MR testing.

Preparation of MR Solution

Methyl red solution, 0.02%

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

Requirements for MR Test

  1. MR-VP broth
  2. Test organism
  3. Inoculating wire
  4. Bunsen burner
  5. Incubator
  6. Methyl red indicator
  7. Control strains

Procedure of Methyl Red Test

  1. Inoculate 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.
  2. 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.)
  3. Add 3 to 6 drops (or 1 drop to 0.5 ml) of methyl red indicator to the aliquot.
  4.  Observe for red color immediately.

Result and Interpretation of MR 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.

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)

Keynotes on MR Test

  • A weak positive reaction is red-orange. If an orange color is present, incubate the broth further for up to 4 days and repeat the test after further incubation. In this case, it may also be helpful to set up a duplicate broth at 25°C.
  • Initially, the paired MR-VP tests were used to distinguish between members of Enterobacteriaceae, but now they are used to characterize other groups of bacteria including Actinobacteria (a phylum of mostly Gram-positive bacteria).

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

Methyl red 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 and hence a light inoculum is preferred.
  • Incubation periods up to 5 days may be necessary for the methyl red test result interpretation.

VP Test

VP Test stands for Voges Proskauer  test. It is applied for identification to the species level of the following groups of organisms-enteric gram-negative rods, Aeromonas, and Vibrio, viridans group streptococci, and staphylococci.

Principle of VP 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.

Composition Of MR-VP  Test Broth

  •  Buffered peptone:7.0 g
  • Glucose:5.0 g
  • Dipotassium phosphate:5.0 g
  • Deionized / Distilled water: 1000 ml
  • Final pH:6.9
  1. Generally dispense approximately 5 ml per tube.
  2. Use enough broth to cover an inverted Durham tube, if it is used.

Reagent preparation

Reagent 1:  5% α-Naphthol

α-Naphthol: 5 g

95% ethyl alcohol:100 ml

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

  • MR-VP broth
  • Test organism
  • Inoculating wire
  • Bunsen burner
  • Incubator
  • 5%α- naphthol
  • 40% KOH
  • Control strains
  1. Klebsiella pneumoniae ATCC 13883
  2. Escherichia coli ATCC 25922

Procedure of Voges Proskauer Test

  1. 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.
  2.  If a 5-ml broth culture is used, aliquot 2.0 ml of broth into  13 by 100-mm test tube. Hold the remainder for possible reincubation.
  3. Add 6 drops of 5% α-naphthol, and mix well to aerate.
  4. Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
  5. 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 48 hours and the test repeated.

Result and Interpretation of VP Test

  1. Voges- Proskauer  test  positive: A pink-red color at the surface is a positive reaction
  2. 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)

Keynotes on VP Test

  • A copper color should be considered negative. 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.
  • MR-VP Test is a combination of two tests.

Further Readings on MR-VP Test

  1. Lynae S. Carcia, Second Edition update, Clinical Microbiology Procedures Handbook
  2. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
  3. B.D. Skerman, A guide to the identification of the genera of bacteria, The Williams & Wilkins Co., Baltimore, MD, (1967)
  4. Cowan and Steel’s, manual for the identification of medical bacteria
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