The Voges-Proskauer (VP) test is a biochemical assay used in microbiology to identify and differentiate types of bacteria, particularly those belonging to the family Enterobacteriaceae. This test assesses the specific metabolic pathway a microbe uses when it ferments glucose, focusing on the production of a neutral end product. It serves to distinguish organisms that utilize the butanediol pathway from those that use the mixed-acid fermentation pathway. The VP test aids in the systematic identification of various bacteria in clinical, food, and environmental settings.
The Biochemical Mechanism of Acetoin Production
The VP test specifically looks for the ability of bacteria to convert glucose into a neutral compound called acetoin (acetylmethylcarbinol) through the butanediol pathway. This process begins when the bacterium breaks down glucose into pyruvic acid, a compound formed during sugar metabolism. Voges-Proskauer positive organisms convert this pyruvic acid into acetolactic acid, which is an unstable intermediate.
The acetolactic acid then undergoes decarboxylation, a chemical reaction that removes a carbon dioxide molecule, resulting in the formation of acetoin. Acetoin is the direct molecule the VP test is designed to detect, although it is often further reduced by the bacteria into the more stable end product, 2,3-butanediol. The production of these neutral end products contrasts sharply with the mixed-acid pathway, which yields highly acidic end products like lactate and acetate.
Because the butanediol pathway produces neutral compounds, it results in a less significant drop in the pH of the culture medium compared to the mixed-acid pathway. This difference in end products is the fundamental biochemical trait the Voges-Proskauer test capitalizes on for bacterial differentiation.
Performing the Test and Observing the Reaction
The Voges-Proskauer test is performed by inoculating a bacterial culture into a specialized medium, typically an MR-VP broth containing glucose and peptone, and incubating it for a set period. After incubation, specific chemical reagents are added to chemically detect the presence of any produced acetoin. The reagents used are known as Barritt’s reagents, consisting of alpha-naphthol (Reagent A) and potassium hydroxide (KOH) (Reagent B).
These two reagents are added sequentially to the incubated broth, with alpha-naphthol added first to serve as a color enhancer. The strong alkali, potassium hydroxide, then acts as an oxidizing agent in the presence of atmospheric oxygen, converting any acetoin present in the broth into a compound called diacetyl. Aeration, often achieved by gently shaking the tube, is necessary to supply the oxygen required for this oxidation step.
The newly formed diacetyl then reacts with guanidine compounds, which are naturally present in the peptone component of the broth medium, to produce a visible color change. This final reaction forms a complex that appears pink or red at the surface of the medium.
Interpreting Results and Application in Microbiology
The interpretation of the Voges-Proskauer test relies entirely on the color change observed at the surface of the culture medium after the addition of the reagents. A positive VP result is indicated by the development of a cherry-red or pink color within 15 to 30 minutes, confirming that the organism successfully produced and accumulated acetoin during its growth. Conversely, a negative VP result is signified by the absence of this red color, with the medium remaining yellow or turning a copper-brown color.
This color distinction is a powerful tool for classifying bacteria, particularly within the Enterobacteriaceae family, where metabolic differences are used for identification. Bacterial genera such as Klebsiella, Enterobacter, and Serratia are typically Voges-Proskauer positive, indicating they favor the butanediol pathway. In contrast, organisms like Escherichia and Shigella are generally Voges-Proskauer negative because they rely on the mixed-acid fermentation pathway.
The VP test is one component of a series of four biochemical tests collectively known as the IMViC battery, which stands for Indole, Methyl Red, Voges-Proskauer, and Citrate. This systematic approach allows clinical and environmental laboratories to narrow down the identity of an unknown microbe by comparing its metabolic profile against established standards.