Voges Proskauer (VP) Test

Last updated on June 21st, 2021

The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethylcarbinol from glucose fermentation. Voges-Proskauer is a double eponym, named after two microbiologists working at the beginning of the 20^th century. They first observed the red color reaction produced by appropriate culture media after treatment with potassium hydroxide.

It was later discovered that the active product in the medium formed by bacterial metabolism is acetyl methyl carbinol, a product of the butylenes glycol pathway.

Principle

Pyruvic acid, the pivotal compound in the fermentative degradation of glucose, is further metabolized through various metabolic pathways, depending on the enzyme systems possessed by different bacteria. One such pathway results in the production of acetoin (acetyl methyl carbinol), a neutral-reacting end product.

If present, acetylmethylcarbinol is converted to diacetyl in the presence of α-naphthol, strong alkali (40% KOH), and atmospheric oxygen. The diacetyl and guanidine-containing compounds found in the peptones of the broth then condense to form a pinkish-red polymer.

Organisms such as members of the Klebsiella-Enterobacter-Hafnia-Serratia group produce acetoin as the chief end product of glucose metabolism and form smaller quantities of mixed acids.

Media and Reagents

1. Media:
   Methyl red-Voges-Proskauer (MR/VP) broth (formulated by Clark and Lubs) is used in Voges–Proskauer test. The composition of MR/VP broth is as follows:  

   Ingredient	MR/VP broth (g/L)
   Polypeptone	7 g
   Glucose	5 g
   Dipotassium phosphate	5 g
   Distilled water	1 L
   Final pH	6.9

2. Reagents:
   1. 5% α-naphthol (5 g/100 mL) in 95% ethyl alcohol.
      The reagent should be stored at 4-8°C in the dark. The shelf life is 2-3 weeks.
   2. 40% potassium hydroxide (KOH)
      Dissolve 40 g of potassium hydroxide pellets in 100 ml of distilled water in a polyethylene bottle. Keep the bottle in a cool water bath during preparation.

Quality Control

Examine broth for signs of contamination, dehydration, and deterioration prior to use. Perform QC on each new lot of media and reagent prior to use with one organism known to demonstrate a positive reaction and one organism known to give a negative reaction.
Organisms

• Klebsiella pneumoniae ATCC 13883—VP positive (red)
• Escherichia coli ATCC 25922—VP negative (no change)

Procedure of Voges Proskauer Test

1. Inoculate a tube of MR/VP broth with a pure culture of the test organism.
2. Incubate for 24 hours at 35°C
3. At the end of this time, aliquot 1 mL of broth to a clean test tube.
4. Add 0.6mL of 5% α-naphthol*, followed by 0.2 mL of 40% KOH.
   (Note: It is essential that the reagents be added in this order.)
5. Shake the tube** gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 minutes.

*The α-naphthol was not part of the original procedure but was found to act as a color intensifier by Barritt and must be added first.

** Shaking the tubes increases the VP reaction.

Results and Interpretation

A positive VP test is represented by the development of a pink-red color at the surface within 15 minutes or more after the addition of the reagents indicating the presence of diacetyl, the oxidation product of acetoin. The test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a copper-like color, potentially resulting in a false-positive interpretation.

If the result is negative, the glucose or MRVP broth can be incubated for up to 48 h and the test repeated.

Reporting results

• 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 VP variable

Voges-Proskauer (VP) Positive Organisms of Enterobacteriaceae family are:

1. Klebsiella species
2. Enterobacter species
3. Hafnia species
4. Serratia species

Limitations

1. With prolonged incubation (>3 days), some VP-positive organisms can produce an acid condition in the medium, yielding weak positive reactions or false-negative VP reactions.
2. Do not add more than 2 drops of KOH per 2 ml of medium. Excess amounts of KOH can give a weakly positive reaction, which may be masked by the formation of a copper-like color because of the reaction of KOH with α-naphthol alone.
3. Do not read the test more than 1 h after adding the VP reagents. A copper-like color may develop, resulting in a potential false-positive interpretation.
4. Reagents must be added in the specified order. A reversal of order may result in a weakly positive or false-negative VP result.

References and further readings

1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition