Voges-Proskauer Test - Principle, Biochemistry, Purpose, Procedure, Result, Interpretation

Introduction to Voges-Proskauer Test

Voges-Proskauer (VP) test is a biochemical test used in the differentiation/identifying bacteria by detecting the presence or absence of acetoin (acetylmethyl carbinol). Acetoin is a neutral end product formed when glucose is metabolized to form pyruvic acid which in turn undergoes mixed acid fermentation.

Purpose of Voges-Proskauer Test

The Voges-Proskauer Test is primarily used to differentiate E. coli (VP negative) from the Klebsiella-Enterobacter groups (VP positive). However, many genera and species of the Enterobacteriaceae family are VP-positive.

Principle, Biochemistry of Voges-Proskauer Test

The VP-negative microorganisms do not produce 2,3 butanediol after mixed acid fermentation but produce acidic products such as formic acid, acetic acid, succinic acid, ethanol, hydrogen, and carbon dioxide.

On the other hand, the bacteria which are VP-positive undergo mixed acid fermentation to produce 2,3 butanediol, which is the major end product of pyruvate utilization.

Although the VP test detects acetoin and not 2,3 butanediol, it is still an important product as 2,3 butanediol and acetoin formation is a reversible education of the oxidation system.

The neutral end products acetoin and 2,3 butanediol are oxidized to diacetyl in the presence of oxygen and alkali.

Biochemistry

2,3 butanediol → (oxidation by 2,3 butanediol dehydrogenase) → Acetoin

Acetoin → (reduction by diacetyl acetoin reductase) → 2,3 butanediol

Barrett's reagent A, which contains alpha-naphthol, and Barrett's reagent B, which contains potassium hydroxide, is the reagent used for the VP test and detects acetoin.

Barrett's reagent A (5% alpha-naphthol) is an alcoholic solution that acts as a color intensifier by combining with the product of diacetyl and arginine present in peptone early in the reaction.

Barrett's reagent B is 40% KOH which aids in the absorption of CO₂ which acts to rapidly oxidize acetoin to diacetyl. Diacetyl is the essential reactant for color development in the VP test reaction.

Procedure of Voges-Proskauer Test

The procedures of the Voges-Proskauer Test are as follows:

1. Take a sterile MR/VP broth and aseptically inoculate the broth with freshly cultured test organism with the help of an inoculating loop.

2. Incubate the inoculated tube at 35-37°C for 18-24 hours.

3. Add 0.6 ml of Barrett's reagent A, and 0.2 ml of Barrett's reagent B to the incubated tube.

4. Shake gently to mix and expose the medium to atmospheric oxygen.

5. Observe for a color change for 10-15 minutes.

Result, Interpretation of Voges-Proskauer Test

The result of the Voges-Proskauer Test can be interpreted as follows:

Positive

Pinkish-red color is seen on the surface of the medium.

Negative

Yellow color is seen on the surface of the medium. If copper color is seen, due to actions of reagents when mixed, and is still negative.

Voges-Proskauer Test (VP) negative bacteria

A list of Voges-Proskauer Test (VP) negative bacteria includes:

• Enterobacter

• Klebsiella

• Serratia marcescens

• Hafnia alvei

• Vibrio cholerae biotype El Tor

• Vibrio alginolyticus

Voges-Proskauer Test (VP) positive bacteria

A list of Voges-Proskauer Test (VP) negative bacteria includes:

• Citrobacter sp.

• Shigella

• Yersinia

• Edwardsiella

• Salmonella

• Vibrio furnissii

• Vibrio fluvialis

• Vibrio vulnificus

• Vibrio parahaemolyticus