Voges Proskauer Test Principle Procedure

In this voges proskauer test principle procedure post we have briefly explained voges proskauer test principle, objectives, requirements, procedure, uses and limitations of the voges proskauer test.

The Voges Proskauer Test

Different bacteria use different metabolic processes to convert dextrose and glucose to pyruvate. Some of these processes yield acidic chemicals that swiftly degrade into neutral molecules. The butylene glycol route is used by some organisms to produce neutral end products such as acetoin and 2,3-butanediol.

Other species follow the mixed acid pathway, which results in acidic end products such lactic, acetic, and formic acid. These acidic final products have a long shelf life and will continue to be acidic. In 1898, Voges and Proskauer discovered that adding potassium hydroxide to colonies grown on certain media caused them to turn red.

Harden later demonstrated that the red color was caused by the synthesis of acetyl-methyl carbinol. Barrit improved the sensitivity of the voges proskauer test by adding alpha-naphthol to the solution before adding potassium hydroxide in 1936.


Glucose is fermented through the mixed acid fermentation pathway, which results in the production of numerous organic acids (lactic, acetic, succinic, and formic acids). A pH of less than 4.4 will arise from the stable creation of enough acid to overcome the phosphate buffer in the voges proskauer test. A red tint will occur if the pH indicator (methyl red) is applied to an aliquot of culture broth with a pH below 4.4. The pH is above 6.0 and the mixed acid fermentation pathway has not been used if the MR becomes yellow.

Instead of organic acids, the 2,3 butanediol fermentation pathway ferments glucose and produces a 2,3 butanediol end product. An aliquot of the MR/VP culture is withdrawn and a-naphthol and KOH are added to assess this route. They’re violently mixed together and placed aside for about an hour until the results are ready to be read. The presence of acetoin, a precursor of 2,3 butanediol, is detected by the Voges-Proskauer test. If acetoin is present in the culture, it will turn “brownish-red to pink”. If the culture is acetoin-free, it will turn a “brownish-green to yellow” colour.

Reagents Required

MR-VP Media

In the Voges Proskauer test, methyl red–Voges–Proskauer (MR/VP) broth is utilized. The following is the list of ingredients in MR/VP broth: Polypeptone: 7 g, Glucose: 5 g, Dipotassium phosphate: 5 g, Distilled water: 1 L Final pH; 6.9.

5% Naphthol

5 percent -naphthol in 95 percent ethyl alcohol (5 g/100 mL): The reagent should be kept in the dark at 4-8°C. The product has a shelf life of two to three weeks for the voges proskauer test.

40% KOH

Potassium hydroxide at a concentration of 40% (KOH): In a plastic container, dissolve 40 g potassium hydroxide pellets in 100 ml distilled water. During the preparation, place the bottle in a cool water bath. 


1. Using a pure culture of the test organism, inoculate a tube of MR/VP broth. Incubate at 35°C for 24 hours for the voges proskauer test.

2. Aliquot 1 mL of broth into a clean test tube at the end of this time. Add 0.6 mL of 5 percent naphthol and 0.2 mL of 40% KOH.

3. Allow 10 to 15 minutes for the tube to be undisturbed after gently shaking it to expose the medium to ambient oxygen.

Test Result

Voges Proskauer Test

Voges Proskauer Test Principle Procedure

A positive the voges proskauer test is defined as the appearance of brownish-red to pink hue on the surface 15 minutes or longer after the reagents have been added, showing the presence of diacetyl, the acetoin oxidation product. Example: Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc.

Negative the voges proskauer test cultures may create a brownish-green to yellow hue after standing for more than 1 hour; the test should not be read beyond that time. This could result in a false-positive interpretation. Example: Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus etc.

Quality Control

The voges proskauer test positive: Enterobacter aerogenes (ATCC13048)

The voges proskauer test negative: Escherichia coli (ATCC25922).


1. Some the voges proskauer test positive organisms might develop an acidic state in the medium over a long incubation period (>3 days), resulting in weak positive reactions or false-negative the voges proskauer test.

2. No more than 2 drops of KOH per 2 mL of medium should be used in the voges proskauer test. Excess KOH can generate a weakly positive reaction, which can be obscured by the production of a copper-like hue due to KOH’s interaction with -naphthol alone.

3. After adding the the voges proskauer test reagents, do not read the the voges proskauer test sample for more than 1 hour. A copper-like tint may emerge, perhaps leading to a false-positive result.

4. The voges proskauer test reagents must be added in the order stated. A VP result that is weakly positive or false-negative may result from reversing the order.

5. To differentiate genus and species within the Enterobacteriaceae, the voges proskauer test results must be utilised in conjunction with other biochemical testing.

Further Readings