Voges Proskauer (VP) Test: Principle, Procedure and Results 4.53/5 (19)

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.

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 pathways result in the production of acetion (acetyl methyl carbinol), a neutral-reacting end product.

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. In the presence of atmospheric oxygen and 40% potassium hydroxide, acetoin is converted to diacetyl, and alpha-naphthol serves as a catalyst to bring out a red complex.

 

Media and Reagents

1. Media: The medium is MR/VP broth
2. Reagents:
   1. Alpha-naphthol, 5% color intensifier
      1. Alpha Naphthol-5g
      2. Absolute ethyl alcohol- 100 mL
   2. Potassium Hydrooxide, 40%, oxidizing agent
      1. Potassium hydroxide 40g
      2. Distilled water to: 100 mL

Quality Control

Positive and negative controls should be run after preparation of each lot of medium and after making each batch of reagent. Suggested controls include the following:

• Positive Control: Enterobacter aerogenes
• Negative Control: Escherichia coli

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^oC
3. At the end of this time, aliquot 1 mL of broth to clean test tube.
4. Add 0.6mL of 5% alpha 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.

Results and Interpretation

A positive test is represented by the development of a red color 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.

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