Voges Proskauer Test

Voges Proskauer Test Definition

Voges Proskauer (VP) Test confirms the butylene glycol type of the fermentation by the organism. In general, two pathways of glucose fermentation exist among the Enterobacteriaceae.

Mixed acid fermentation resulting in large amount of formic, acetic, lactic and succinic acids and ethanol

Butylene glycol fermentation resulting in little or nor of the acids, but a large amount of a polar butylene glycol and neutral ethanol. Acetyl methyl carbinol or acetoin, is an intermediate in the production of butylene glycol. 

The VP test detects the presence of acetoin in the culture medium, thus confirming the butylene glycol pathway.    

Voges and Proskauer in 1898 grew the organisms in a medium containing sugar and then added potash to the culture. After 24 hours or longer at room temperature, a red colour developed in the medium, indicating what they called a positive reaction.

Hearden in 1906 showed that the red colour developed was due to the production of acetylmethylcarbinol. In the presence of potash and air, this compound is oxidized diacetyl, which reacts with some constituent of the medium to form a red colour (Diacetyl alone in the presence of potash does not yield red colour). This medium constituent was identified to be free NH2 group on a guanidine moiety, such as that available from arginine present in the peptone.

In 1931 O’Meara found that the colour was intensified by the addition of creatinine, which provides a greater amount of the guanidine groups. With this modification, the red colour developed in 15 minutes.

In  1936,  Barritt  made  the  test  more  sensitive  by  adding  α-napthol.  With  this  the sensitivity of the test was increased 50-fold (without napthol, at least 10 ppm of diacetyl was detectable). The exact role of α-napthol is not identified.

Some precautions necessary for the VP test are:

  1. Meat infusion broth should not be used to grow the culture because the presence of acetoin, diacetyl and related substances in the muscle extract would give a false positive test. 
  2. α-napthol should be added before the KOH.

Voges Proskauer VP Test Principle

The principle of the VP test is based on the ability of these bacteria to convert glucose into acetoin via a series of enzymatic reactions.

In the VP test, the bacteria are inoculated into a glucose-containing medium and allowed to ferment the glucose. After incubation, the culture is treated with a solution containing alpha-naphthol and potassium hydroxide (KOH), which react with the acetoin to produce a reddish-brown color. The intensity of the color is proportional to the amount of acetoin produced, and thus indicates the presence of the fermenting bacteria.

Rapid VP tests, in general, consist of inoculation of heavy growth of organism from a solid medium into a small amount (0.3-0.5ml) of GPB medium. After incubation of 4 hours at 37oC sufficient acetoin can be produced to be detectable. If the organism has already been grown on a medium containing glucose, acetoin produced in that medium may be carried over and can result in a red color immediately. Another rapid test utilizes reagent impregnated strips.

2 pyruvate = acetoin + 2CO2
acetoin + NADH + H+ = 2,3-butanediol + NAD+

Requirements VP Test

  1. Sterile glucose phosphate broth (GPB) medium (0.5-1.0ml in each tube). 
  2. Test culture suspension (Enterobacter / Klebsiella). 
  3. 5%- α-napthol in absolute ethanol. 
  4. 40% KOH solution (containing 0.5% creatinine, if desired). 

Procedure of VP Test

  1. Inoculate GPB medium with the culture suspension. 
  2. Incubate at 37oC for 24 hours. 
  3. Add 0.6 ml of 5% α-napthol and mix well. 
  4. Add 0.2 ml of 40% KOH solution, shake well. 
  5. Positive VP test is indicated by a red color of the medium, within 5 minutes. A negative VP test is indicated by the medium remaining brown.

VP Test Results

VP Test PositiveVP Test Negative
Viridans group streptococciStreptococcus mitis
ListeriaCitrobacter Spp.
EnterobacterShigella Spp.
Serratia marcescensEdwardsiella
Hafnia alveiSalmonella
Vibrio eltorVibrio fluvialis
Vibrio alginolyticusVibrio vulnificus

Uses of VP Test

  1. The VP test is often used to differentiate between members of the Enterobacteriaceae family, which includes important human pathogens such as Escherichia coli, Salmonella, and Shigella. This test helps in the identification of these organisms in clinical settings.
  2. The VP test is used in the production of fermented beverages, such as beer and wine, to determine the presence of acetoin-producing bacteria. It is also used in the food industry to detect certain spoilage organisms.
  3. The VP test can be used to identify acetoin-producing bacteria in environmental samples, such as soil and water. This can be useful in monitoring the quality of water and soil for potential bacterial contamination.
  4. The VP test is commonly used in microbiological research to investigate the metabolic pathways and physiology of bacteria. It is often used in conjunction with other tests to identify bacterial strains and study their characteristics.