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
- Prior to inoculation, allow medium to equilibrate to room temperature.
- Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.
- Incubate aerobically at 37 degrees C. for 24 hours.
- Following 24 hours of incubation, aliquot 2 ml of the broth to a clean test tube.
- Re-incubate the remaining broth for an additional 24 hours.
- Add 6 drops of 5% alpha-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.
Coblentz Method for Streptococci
- Use 24-hour growth from blood agar plate to heavily inoculate 2 mL of MRVP broth.
- 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).
- Shake the tube and incubate at room temperature for 30 minutes.
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
- Results of VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae.
- 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.
- 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.
- Some organisms destroy acetoin, thereby rendering the VP tests invalid.
- Shaking the tubes enhances the VP reaction.