Voges-Proskauer test is one of the tests used for identification of Enterobacteriaceae. It is usually performed alongside the methyl red test since both tests are performed on cultures grown in MR-VP broth.  Both tests are based on the detection of end products from the metabolism of glucose. VP test is based on the detection of acetoin, and its detection is achieved by the addition of various reagents like creatine(for rapid VP test), 5% α-Naphthol and 40% KOH,  that lead to the formation of colored end products.

Principle of Voges Proskauer test (VP test)

Bacteria can metabolize glucose to key intermediate, pyruvic acid which can further be metabolized  to produce acetoin  (i.e., acetyl methyl carbinol or 3-hydroxybutanone) as an intermediate and can be further reduced to 2,3-butanediol.

2 pyruvate = acetoin + 2CO₂

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

It can be detected by colorimetric method. In the presence of alkali (KOH) and atmospheric oxygen, acetyl methyl carbinol is oxidized to diacetyl, a reaction which is catalyzed by alpha- naphthol. Diacetyl formed reacts with guanidine-containing compounds such as arginine contributed by peptone in the medium, to form a red colored product. The resultant red color is indicative of a positive VP test.

The second reagent, potassium hydroxide, absorbs carbon dioxide present in the medium and acts as an oxidizing agent thereby hastening the critical reaction that converts acetoin to diacetyl.

For the rapid VP test, creatine is added as an additional source of guanidine nucleus. Once added, creatine can react with diacetyl to promote color development. Also, the overall structure of creatine, which possesses an additional free NH2 group, intensifies color development.

Procedure of Voges Proskauer test (VP test)

• Inoculate a tube of MR-VP broth with the organism of interest from a overnight culture grown on Nutrient agar, MacConkey agar, Blood agar or Chocolate agar.
• Incubate for 24 hours at 35°C. (The VP test can also be performed at 48 hours)
• Remove 1.0 ml of the incubated broth to a separate tube for VP testing. (The remainder of the broth should be re-incubated for an additional 1-3 days for the methyl red test)
• Allow reagents to warm to room temperature prior to use.
• Add 0.6 mL (9 drops) of α-Naphthol Reagent to the allotted portion of MR-VP broth and gently mix.
• Add 0.2 mL (3 drops) of 40% Potassium Hydroxide.
• Shake the tube gently for 30 seconds. The broth must be exposed to oxygen for a color reaction to occur.
• Allow tube to stand for 15

Rapid Procedure for Voges Proskauer test (VP test)

• From a stock solution of MR-VP Broth, aseptically pipette 0.2 ml aliquots into sterile test tubes (10×75 mm) just prior to use.
• Take a loopful of growth from an overnight culture grown on Nutrient agar, MacConkey agar, Blood agar, or Chocolate agar and inoculate the broth.
• Incubate tube at 35°C (water bath) for 4 hours.
• Add 2 drops of Creatine Reagent and gently mix.
• Add 3 drops of α-Naphthol Reagent and gently mix.
• Add 3 drops of 40% Potassium Hydroxide and gently mix for 10 seconds.
• Allow tube to stand for 15 minutes before interpreting the color.

Interpretation of Voges Proskauer test (VP test)

• VP Positive: Pink or red color at the surface of the medium (acetoin present)
• VP Negative: Yellow or copper color at the surface of the medium (acetoin absent)

 

Quality Control

Check the medium for correct pH, color, depth, and sterility, the following organisms are used to determine the performance of the completed medium.

Organism                                                                                VP

Enterobacter aerogenes (ATCC 12453)                            + (red)

Escherichia coli (ATCC 25922)                                            – (no change)

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