Voges Proskauer Test: Introduction, Principle, Test Requirements, Test Procedure, Result-Interpretation, Positive and Negative Bacteria, and Limitations

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

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.

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

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  Voges Proskauer 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 a 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 to 48 hours and the test repeated.

Result and Interpretation of Voges Proskauer 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)
• 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.

Further Readings on Voges Proskauer 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