Voges Proskauer test: Use, Principle, Procedure and Result Interpretation

Voges Proskauer Test: Use, Principle, Procedure and 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

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

Generally dispense approximately 5 ml per tube.
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

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 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 48 h 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. 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.

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