Voges–Proskauer (VP) Test- Principle, Reagents, Procedure and Result

Voges and Proskauer, in 1898, first observed the production of a red color after the addition of potassium hydroxide to cultures grown on specific media. Harden later revealed that the development of the red color was a result of acetyl-methyl carbinol production. In 1936 Barrit made the test more sensitive by adding alpha-naphthol to the medium before adding potassium hydroxide.

Principle of Voges–Proskauer (VP) Test

The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethyl carbinol from glucose fermentation. If present, acetylmethyl carbinol is converted to diacetyl in the presence of ∝- naphthol, strong alkali (40% KOH), and atmospheric oxygen. The ∝-naphthol was not part of the original procedure but was found to act as a color intensifier by Barritt and must be added first. The diacetyl and quanidine-containing compounds found in the peptones of the broth then condense to form a pinkish red polymer.

2 pyruvate = acetoin + 2CO2
acetoin + NADH + H+ = 2,3-butanediol + NAD+

Media and Reagents used in Voges–Proskauer (VP) Test

MRVP broth (pH 6.9)
Ingredients per liter of deionized water:
buffered peptone= 7.0 gm
glucose= 5.0 gm
dipotassium phosphate= 5.0 gm

Voges-Proskauer Reagent A: Barritt’s reagent A
Alpha-Naphthol, 5% 50 gm
Absolute Ethanol 1000 ml
Voges-Proskauer Reagent B: Barritt’s reagent B
Potassium Hydroxide 400 gm
Deionized Water 1000 ml

Procedure of Voges–Proskauer (VP) Test

  1. Prior to inoculation, allow medium to equilibrate to room temperature.
  2. Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.
  3. Incubate aerobically at 37 degrees C. for 24 hours.
  4. Following 24 hours of incubation, aliquot 2 ml of the broth to a clean test tube.
  5. Re-incubate the remaining broth for an additional 24 hours.
  6. Add 6 drops of 5% alpha-naphthol, and mix well to aerate.
  7. Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
  8. Observe for a pink-red color at the surface within 30 min. Shake the tube vigorously during the 30-min period.

Coblentz Method for Streptococci

  1. Use 24-hour growth from blood agar plate to heavily inoculate 2 mL of MRVP broth.
  2. After 6 hours of incubation at 35°C in ambient air, add 1.2 mL (12 drops) of solution A (alpha-naphthol) and 0.4 mL (4 drops) solution B (40% KOH with creatine).
  3. Shake the tube and incubate at room temperature for 30 minutes.

Result Interpretation of Voges–Proskauer (VP) Test

Result Interpretation of Voges–Proskauer (VP) Test

Positive Reaction: A pink-red color at the surface
Examples: Viridans group streptococci (except Streptococcus vestibularis), Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc.

Negative Reaction: A lack of a pink-red color
Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus etc.

A copper color should be considered negative. A rust color is a weak positive reaction.

Quality Control of Voges–Proskauer (VP) Test

VP positive: Enterobacter aerogenes (ATCC13048)
VP negative: Escherichia coli (ATCC25922)

Limitations of Voges–Proskauer (VP) Test

  1. Results of VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae.
  2. 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 a-naphthol.
  3. Read the VP test within 1 hour of adding the reagents. The KOH and a-naphthol may react to form a copper-like color, causing a potential false-positive interpretation.
  4. Some organisms destroy acetoin, thereby rendering the VP tests invalid.
  5. Shaking the tubes enhances the VP reaction.

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