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Objective of VP Test
To differentiate two major types of facultative anaerobic enteric bacteria based on production of neutral products.
Principle of VP Test
Voges Proskauer test, commonly known as VP test is used to determine the ability of some organisms to produce neutral end products (e.g., 2, 3-butanediol or acetoin) from glucose fermentation. All members of the Enterobacteriaceae can convert glucose to pyruvic acid by the Embden-Meyerhof pathway, but bacteria can further metabolize pyruvic acid by two different pathways. Organisms metabolizing pyruvic acid by the mixed acid pathway will produce more acid end products, such as lactic acid and acetic acid, and maintain an acidic environment. If the organism produces large amount of organic acids that includes formic acid, acetic acid, lactic acid, and succinic acid from glucose fermentation, the broth medium will remain red after the addition of methyl red, a pH indicator. However, MR-negative organisms further metabolize the initial fermentation products by decarboxylation to produce neutral acetyl methylcarbinol (acetoin), which results in decreased acidity in the medium and raises the pH towards neutrality (pH 6.0 or above). MR test along with VP test is performed simultaneously because they are physiologically related and are performed on MRVP broth.
Bacteria fermenting sugars via the butanediol pathway produce acetoin (i.e., acetyl methyl carbinol or 3-hydroxybutanone) as an intermediate which can be further reduced to 2,3-butanediol.
2 pyruvate = acetoin + 2CO 2
acetoin + NADH + H + = 2,3-butanediol + NAD +
In the presence of KOH the intermediate acetoin is oxidized to diacetyl, a reaction which is catalyzed by alpha naphthol. Diacetyl reacts with the guanidine group associated with molecules contributed by peptone in the medium, to form a pinkish-red-colored product. The alpha naphthol in the Barritt’s modification of the VP test serves as a color intensifier.
Media Used in VP Test
Buffered peptone 7.0 gm/L, Dextrose 5.0 gm/L, Dipotassium phosphate 5.0 gm/L, Final pH ( at 25°C) 6.9±0.2
Procedure of VP Test
- Inoculate MRVP broth with a pure culture of the organism.
- Incubate at 35°-37°C for a minimum of 48 hours in ambient air.
- Add 6 drops of VP reagent I (alpha napthol) and 2 drops of VP reagent II(40% KOH).
- Observe for the color change in the broth medium.
Result Interpretation of VP Test
Positive: Crimson to ruby pink (red) color
Negative: No change in colouration
Limitations of VP Test
- The Voges-Proskauer test may be used in the identification of gram-negative bacteria. Additional biochemical testing using pure culture is recommended for complete identification.
- Prolonged incubation (greater than 72 hours at 35ºC) of certain VP-positive strains of Enterobacteriaceaemay result in false-negative reactions due to the breakdown of acetylmethyl carbiol.
- The order of the addition of reagents is extremely important. Reversal of the order may result in weak positive reactions or false-negative reactions.
- MR-VP testing should be used in conjunction with other confirmatory tests to differentiate organisms among the Enterobacteriaceae.
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