Voges Proskauer Test: Use, Principle, Procedure And Result Interpretation

Introduction

Voges–Proskauer commonly VP is a test used to detect acetoin in a bacterial broth culture. The test is performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth which has been inoculated with bacteria. A cherry red color indicates a positive result, while a yellow-brown color indicates a negative result.The test depends on the digestion of glucose to acetylmethylcarbinol. In the presence of oxygen and strong base, the acetylmethylcarbinol is oxidized to diacetyl, which then reacts with guanidine compounds commonly found in the peptone medium of the broth. Alpha-naphthol acts as a color enhancer, but the color change to red can occur without it.

Voges Proskauer  test is used for identification to the species level of the following groups of organisms

• Enteric gram-negative rods, Aeromonas, and Vibrio
• Viridans group streptococci
• Staphylococci

Principle  of Voges Proskauer Test

Organisms utilizing the butylene glycol pathway produce acetylmethylcarbinol (acetoin) and butanediol,neutral end products that raise the pH towards neutrality (pH 6) and result in a high final pH. 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.

The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethylcarbinol from glucose fermentation. If present, acetylmethylcarbinol is converted to diacetylinthepresence of α-naphthol, strong alkali (40% KOH), and atmospheric oxygen. The α-naphthol was not part of the original procedure but was found to act as a color intensifier  and must be added first. The diacetyl and quanidine-containing compounds found in the peptones of the broth then condense to form a pinkish red polymer.

Media And Reagents Used in VP Test

Reagents

1. Alpha-naphthol, 5% color intensifier
   1. Alpha α-5g
   2. Absolute ethyl alcohol- 100 mL
2. Potassium Hydrooxide, 40%, oxidizing agent
   1. Potassium hydroxide 40g
   2. Distilled water: 100 mL

Media

Material Required

• Bunsen burner.
• Inoculating loop.

Quality Control

Positive and negative controls should be run after preparation of each lot of medium and after making each batch of reagent. Suggested controls include the following:

• Positive Control: Klebsiella (formerly Enterobacter) aerogenes
• Negative Control: Escherichia coli

Procedure

1. Using sterile technique, inoculate each experimental organism to the appropriately labeled tube of medium by means of loop inoculation. Incubate the cultures for 24-48 hours at 37°C.
2. Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot. Heat from the base of the wire first and slowly move towards the loop (tip). Heat the wire until it is red-hot.
3. From the rack, take the test tube containing the Tryptic Soy Broth(TSB) cultures that has been kept for 24 – 48 hours.
4. Remove the cap from the TSB tube and flame the neck of the tube.
5. Using aseptic technique take a loop full of the organism from the TSB (tryptic soy broth).
6. Again flame the neck of the tube and replace the cap and place the tube in the test tube rack.
7. Take two sterile MR-VP broth tubes, one named Test and the other Control.
8. Remove the cap of  the MR-VP broth tube named ‘Test’ and flame the neck of the tube.
9. Inoculate the MR-VP broth with the inoculation loop containing the inoculum from the TSB.
10. Again flame the neck of the MR-VP tube and place it in the test tube rack. Inoculate only the broth in the tube named ‘Test’ using aseptic technique. Leave the broth in the tube named ‘Control’ uninoculated.
11. Incubate both the tubes (Test and  Control) for 24 to 48 hours at 37°C.
12. Remove the broths from the incubator.
13. Remove the cap and add 10 drops of Barritt’s A reagent and 10 drops of Barritt’s B reagent to each broth.
14. Shake gently for several minutes.

Results Interpretation

Positive Results

• Red color formation within 15 to 20 minutes is a positive result. 

Examples: Viridans group streptococci (except Streptococcus vestibularis), Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc.

Negative Results

• No red color formation after 15 to 20 minutes is a negative result.

Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus etc.

Limitations Of Voges Proskauer Test

1. Results of the MR and VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae.
2. A precipitate may form in the potassium hydroxide reagent solution. This precipitate has not been shown to reduce the effectiveness of the reagent.
3. Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test. However, certain organisms such as Hafnia alvei and Proteus mirabilis may give a positive result for both tests.
4. Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with the readings of the results.
5. VP reagents must be added in the order and the amounts specified or a weak-positive or false-negative reaction may occur. A weak-positive reaction may be masked by a copper-like color which may form due to the reaction of KOH and a-naphthol.
6. Read the VP test within 1 hour of adding the reagents. The KOH and a-naphthol may react to form a copper-like color, causing a potential false-positive interpretation.
7. Due to the possible presence of acetoin, diacetyl or related substances in certain raw materials, the use of media low in these substances (such as MR-VP media) is recommended for this test.

Further References

1. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.
2. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
4. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.
5.  Tille, P., et al. Bailey and Scott’s Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.
6. Lynae S. Carcia, Second Edition update, Clinical Microbiology Procedures Handbook.
7. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.