Voges Proskauer Test

introduction image

Voges Proskauer test depends upon the formation of a neutral end product “Acetoin” in the MRVP media, which directly relies on the breakdown of glucose into an intermediate product “Pyruvic acid”. The pyruvic acid undergoes butylene glycol pathway and produces an end product named acetyl methyl carbinol or acetoin. It is named after the contribution of the two bacteriologists named Voges and Proskauer who first observed the formation of red-pink colour in the media by the addition of potassium hydroxide.

Later, Harden studied the reason for the colour change and concluded that the colour changed due to acetoin formation. In 1936, a scientist named Baritt used two reagents (KOH and α-naphthol) to perform the VP test. Both α-naphthol and KOH are now being very popular and named as Baritt’s-I and Baritt’s-II reagent. Further, two more scientists (Barry and Feeney) came and introduced a rapid method of VP test by using creatine reagent.

Content: Voges Proskauer Test

  1. Definition
  2. Principle
  3. Media and Reagents
  4. Procedure
  5. Result Interpretation
  6. Limitations

Definition

It is also abbreviated as VP test, and a part of the IMViC test indicated by an acronym ‘V’. Voges Proskauer test ascertains the organism’s potential to form a neutral end product “Acetyl methyl carbinol” by the supply of glucose. It makes the use of standard media (MRVP broth) and two reagents (KOH and α-naphthol). A VP positive organism will exploit glucose and degrade it into a metabolic product pyruvic acid by following the EMP pathway. Then the pyruvic acid will degrade by the test organism through the butylene glycol pathway into AMC or acetoin.

Principle of Voges Proskauer Test

Voges Proskauer test includes a series of reaction, where primary substrate glucose metabolizes into intermediate product pyruvate. Pyruvate enters the butylene glycol pathway to form neutral by-product acetoin. Then oxidation of acetoin occurs by the treatment with ‘KOH’ (an oxidizing agent).

principle of VP test

Potassium hydroxide will react with acetoin to form a compound named Diacetyl. On further treatment, diacetyl condenses with the α-naphthol and the guanidine containing compounds to give ruby pink colour to the media. Creatine contains free –NH2 group that condenses with diacetyl to intensify the colour of a product formed.

Media and Reagents

Voges Proskauer test uses MRVP broth as a standard media and two reagents, namely VP-I and VP-II:

Composition of MRVP broth:
Polypeptone: 7g
Glucose: 5g
Dipotassium phosphate: 5g
Distilled water: 1L
Final pH: 6.9

Composition of VP-I reagent: It also refers as “Barrit’s reagent-I” that includes α-naphthol as a colour intensifier. To prepare 5% of α-naphthol, we require:
α-naphthol: 5 grams
Absolute ethanol: 100 ml

Composition of VP-II reagent: It also refers as “Barrit’s reagent-II” that includes potassium hydroxide as an oxidizing reagent. To prepare 40% of KOH solution, we require:
KOH: 40grams
Distilled water: 100ml

Nowadays, creatine reagent also uses as a colour intensifier and to prepare 0.5% of creatine, we require:
Creatine: 0.5 grams
Distilled water: 100 ml

Procedure

The procedure of the VP test includes the following steps:

  1. Prepare MRVP broth and the reagents required to perform the experiment.
  2. Autoclave the MRVP broth, pour this liquid media into sterilized test tubes.
  3. Take little inoculum and inoculate into the broth via a sterilized inoculating loop.
  4. Then incubate the test tubes for a minimum of 2 days.
  5. After incubation, add 0.6 ml of VP-I reagent and then 0.2 ml VP-II reagent.
  6. Allow the test tubes to stand for at least 10 minutes and then observe the test tubes for any colour change.

Result Interpretation

Positive: A positive VP test confirms the formation of a ruby pink colour on the top of the broth.
Examples: Serratia marcescens, Vibrio eltor, Listeria sp, Enterobacter sp etc.

result interpretation of VP test

Negative: No colour change in the broth indicates a negative VP test.
Examples: Streptococcus mitis, Salmonella sp, Citrobacter sp, Yersinia sp, Shigella sp etc.

Limitations

Voges Proskauer test includes the following limitations:

  • The VP-I and VP-II reagent should be added sequentially and in a proper ratio.
  • For the observation of result, the test tubes must be shaken.
  • Some test organism degrades “Acetoin” and interprets false results.
  • Sometimes α-naphthol and KOH reagent react to give off a copper-like colour and gives a negative result.

Therefore, Voges Proskauer test differentiates the two major types of anaerobic enteric bacteria based on the production of a neutral product, and it is performed alongside with a methyl test.

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