Voges Proskauer (VP) Test

Last updated on June 21st, 2021

Results of Voges-Proskauer (VP) Test
Results of Voges-Proskauer (VP) Test

The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethylcarbinol from glucose fermentation. Voges-Proskauer is a double eponym, named after two microbiologists working at the beginning of the 20th century. They first observed the red color reaction produced by appropriate culture media after treatment with potassium hydroxide.

It was later discovered that the active product in the medium formed by bacterial metabolism is acetyl methyl carbinol, a product of the butylenes glycol pathway.

Principle

Pyruvic acid, the pivotal compound in the fermentative degradation of glucose, is further metabolized through various metabolic pathways, depending on the enzyme systems possessed by different bacteria. One such pathway results in the production of acetoin (acetyl methyl carbinol), a neutral-reacting end product.

If present, acetylmethylcarbinol is converted to diacetyl in the presence of α-naphthol, strong alkali (40% KOH), and atmospheric oxygen. The diacetyl and guanidine-containing compounds found in the peptones of the broth then condense to form a pinkish-red polymer.

Voges Proskauer (VP) Test Principle

Organisms such as members of the Klebsiella-Enterobacter-Hafnia-Serratia group produce acetoin as the chief end product of glucose metabolism and form smaller quantities of mixed acids.

Media and Reagents

  1. Media:
    Methyl red-Voges-Proskauer (MR/VP) broth (formulated by Clark and Lubs) is used in Voges–Proskauer test. The composition of MR/VP broth is as follows:  
    Ingredient MR/VP broth (g/L)
    Polypeptone 7 g
    Glucose 5 g
    Dipotassium phosphate 5 g
    Distilled water 1 L
    Final pH 6.9
  2. Reagents:
    1. 5% α-naphthol (5 g/100 mL) in 95% ethyl alcohol.
      The reagent should be stored at 4-8°C in the dark. The shelf life is 2-3 weeks.
    2. 40% potassium hydroxide (KOH)
      Dissolve 40 g of potassium hydroxide pellets in 100 ml of distilled water in a polyethylene bottle. Keep the bottle in a cool water bath during preparation.

Quality Control

Examine broth for signs of contamination, dehydration, and deterioration prior to use. Perform QC on each new lot of media and reagent prior to use with one organism known to demonstrate a positive reaction and one organism known to give a negative reaction.
Organisms

  • Klebsiella pneumoniae ATCC 13883—VP positive (red)
  • Escherichia coli ATCC 25922—VP negative (no change)

Procedure of Voges Proskauer Test

  1. Inoculate a tube of MR/VP broth with a pure culture of the test organism.
  2. Incubate for 24 hours at 35°C
  3. At the end of this time, aliquot 1 mL of broth to a clean test tube.
  4. Add 0.6mL of 5% α-naphthol*, followed by 0.2 mL of 40% KOH.
    (Note: It is essential that the reagents be added in this order.)
  5. Shake the tube** gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 minutes.

*The α-naphthol was not part of the original procedure but was found to act as a color intensifier by Barritt and must be added first.

** Shaking the tubes increases the VP reaction.

Results and Interpretation

A positive VP test is represented by the development of a pink-red color at the surface within 15 minutes or more after the addition of the reagents indicating the presence of diacetyl, the oxidation product of acetoin. The test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a copper-like color, potentially resulting in a false-positive interpretation.

If the result is negative, the glucose or MRVP broth can be incubated for up to 48 h and the test repeated.

Reporting results

  • Most members of the family Enterobacteriaceae give opposite MR and VP reactions; however, certain organisms, like H. alvei and Proteus mirabilis, may give both a positive MR reaction and a positive VP reaction (often delayed).
  • Streptococcus mitis group organisms are VP negative, whereas the other viridans group streptococci are VP positive, except Streptococcus vestibularis, which is VP variable

Voges-Proskauer (VP) Positive Organisms of Enterobacteriaceae family are:

  1. Klebsiella species
  2. Enterobacter species
  3. Hafnia species
  4. Serratia species

Limitations

  1. With prolonged incubation (>3 days), some VP-positive organisms can produce an acid condition in the medium, yielding weak positive reactions or false-negative VP reactions.
  2. Do not add more than 2 drops of KOH per 2 ml of medium. Excess amounts of KOH can give a weakly positive reaction, which may be masked by the formation of a copper-like color because of the reaction of KOH with α-naphthol alone.
  3. Do not read the test more than 1 h after adding the VP reagents. A copper-like color may develop, resulting in a potential false-positive interpretation.
  4. Reagents must be added in the specified order. A reversal of order may result in a weakly positive or false-negative VP result.

References and further readings

  1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
  2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
About Acharya Tankeshwar 476 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

8 Comments

  1. I am taking a microbiology review course and the class notes seem to be contradictory to what I have found on line. My question is in regards to the gram negative bacteria Aeromonas hydrophila. Are these bacteria lactose fermenters on MacConkey agar? I am studying for my micro MT exam and want to make sure I have my facts straight

    • Dear Cathy
      Thank you so much for sharing comment/question. As per my knowledge most of the species of Aeromonas are non-lactose fermenting; however, some lactose fermenting Aeromonas spp. have been isolated.

  2. Hello sir,
    I am writing a Practical book for my students and find your blog on Biochemical tests very helpful. Please can you provide how best to reference you, especially the blogs from microbe online.com.
    Secondly, what is RYU stain as I will like to try some of the test for the students. I am a lecturer in a University in Nigeria.

  3. “…result in the production of acetion (acetyl methyl carbinol), a neutral-reacting…”
    Sorry to be that guy, just almost put “acetion” in a report.

    Also appreciate this site, really helped.

  4. GRAM POSITIVE COCCI,CATALASE NEGATIVE, COAGULASE NEGATIVE AND ITS NOT GROUPING WITH LANCEFIELD.WHAT CAN BE THE PROBLEM HERE

  5. Hello Sir,
    i am RIcky i would like to know what can cause a false negative result on the API 20 E VP test.
    Cause after adding the two reagents and waiting for 10mins as required, i had a pale pink colour which is normally supposed to be Negative but my colleaques are saying it was suppose to be positive. So i would like to know the possible mistakes i encounter during the praticals.

  6. Why alpha naphthol is used 1st and followed by potasium hydroxide in 2nd…..why we add in this order ,why not in opposite order..n what is the reason behind that…plz give an brief explanation…..

  7. A PUS SAMPLE OF 53 YEAR MALE HAS CULTURED ON BLOOD AGAR AND MACKONKEY AGAR
    FOLLOWNG ARE THE RESULTS OF VARIOUS TESTS:
    1) PINK, SMALL, ROUND, MUCOID COLONY ON MACKONKEY AGAR
    2) Y-HEMOLYSIS ON BLOOD AGAR
    3) GRAM NEGATIVE BACILLI
    4) CATALASE POSITIVE
    5) INDOLE POSITIVE
    6) METHYL RED POSITIVE
    7) VP NEGATIVE
    8) CITRATE POSITIVE
    9) UREASE NEGATIVE
    10) TSI- A/A
    11) MANNITOL POSITIVE
    NOW PLEASE ANSWER ME WHAT ORGANISM IS THIS?

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