Voges Proskauer (VP) Test Overview
Objective of Voges Proskauer (VP) Test
Based on the generation of neutral products, to distinguish between two primary kinds of facultative anaerobic enteric bacteria.
Principle of Voges Proskauer (VP) Test
The Voges Proskauer test, frequently referred to as the VP test, evaluates an organism’s ability to produce 2, 3-butanediol or acetoin, which are neutral by-products of the fermentation of glucose. By using the Embden-Meyerhof pathway in all Enterobacteriaceae, glucose could be converted to pyruvic acid. However, there are two more methods by which bacteria may metabolise pyruvic acid.
By using the mixed acid route to break down pyruvic acid, organisms may maintain an acidic environment and create additional acidic by-products like lactic acid and acetic acid. If the organism generates numerous organic acids from the fermentation of glucose, like formic acid, acetic acid, lactic acid, and succinic acid, the pH indicator methyl red, which is added to the broth medium, will not change the colour of the medium.
But, MR-negative organisms continue to break down the early fermentation products by decarboxylation to produce neutral acetyl methylcarbinol (acetoin). That reduces the acidity of the medium and raises the pH towards neutrality (pH 6.0 or above). The MR and VP tests are run simultaneously because of the physiological commonalities between them and the use of MRVP broth.
The intermediate acetoin, also known as acetyl methyl carbinol or 3-hydroxybutanone, is produced by bacteria when they ferment carbohydrates through the butanediol route. Acetoin can then be further reduced to 2,3-butanediol.
2 pyruvate = acetoin + 2CO 2
acetoin + NADH + H + = 2,3-butanediol + NAD +
The intermediate acetoin is transformed into diacetyl in the presence of KOH, a process that is accelerated by alpha naphthol. A pinkish-red substance is produced when diacetyl combines with the guanidine group linked to molecules provided by peptone in the medium. Barritt’s adaptation of the VP test uses alpha naphthol as a colour intensifier.
Media Used in Voges Proskauer (VP) Test
Final pH (at 25 °C): 6.90.2, Buffered Peptone: 7.0 gm/L, Dextrose: 5.0 gm/L, Dipotassium Phosphate: 5.0 gm/L.
Procedure of VP Test
- MRVP broth should be inoculated with a pure culture of the organism.
- For a minimum of 48 hours, incubate in room air at 35°–37°C.
- Alpha napthol (VP Reagent I, 6 drops) and 40% KOH (VP Reagent II, 2 drops) should be added.
- Keep an eye out for the broth medium’s colour to change.
Result Interpretation of VP Test
Positive: Crimson to ruby pink (red) color
Negative: No change in coloration
Limitations of VP Test
- Gram-negative bacteria can be identified using the Voges-Proskauer test. For full identification, further biochemical testing using pure cultures is advised.
- Certain Enterobacteriaceae VP-positive strains may cause false-negative results when incubated for an extended period of time (more than 72 hours at 35 °C) because of the breakdown of acetylmethyl carbiol.
- It is crucial to introduce the reagents in the correct order. Weak positive responses or false-negative reactions might occur if the order is reversed.
- To distinguish between distinct Enterobacteriaceae species, MR-VP testing should be used in combination with additional confirmatory assays.
Quality control of VP Test
VP positive: Enterobacter aerogenes (ATCC13048)
VP negative: Escherichia coli (ATCC25922)
- Tille P.M (2014)Bailey and Scott’s diagnostic microbiology, Thirteen edition, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
- Aneja K.R (2003), Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.
- McDevitt S. 2009. Methyl red and voges-proskauer test protocols. http://www.asmscience.org/content/education/protocol/protocol.3204