Voges Proskauer (VP) Test: Principle, Procedure, Results

By Acharya Tankeshwar •  Updated: 05/04/22 •  4 min read
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 butylene glycol pathway.

Principle of VP Test

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.

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 into 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

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

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

Limitations of VP Test

  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

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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11 responses to “Culture media for Salmonella typhi and paratyphi”

  1. paul says:

    one of the best student learning and review webs by far…..SPECIFIC to the point and NO JUNK thrown around to fill in space !!!

  2. joshua mogaka says:

    I like this site for precise and specific facts about microbes.

  3. Soumi Datta says:

    If we incubate any bacterial pure culture like E.coli,B.cereus,P.aeruginosa more than 16 to 18 hours. What is the impact of it on the survival of the culture.

  4. phily says:

    Sor… I love so much to do culture to the stool sample fo s. Typhi and other diseases, urine fo the chronic U. T. I and H. V. S…, so can help me to get clear whole procedure,result and how to treat the infection of a such diseases.
    Diagram will be best for the each test

  5. Noel says:

    Can you please let me know for standard environmental swab samples in poultry production should enrichment broth be used or dry agar according to ISO 6579:2002 amended 2007.
    It would seem the method involving enrichment broth prior to the amended standard would likely find salmonella and is not appropriate and is why the amended 2007 version as I understand it does not use enrichment.
    I would be grateful if you could clarify what is the correct test method. Thank you.

  6. moses ludoru says:

    I like the sit and do culture of stool for confirmation of salmonella typi and paratypi
    so can u classify for weather the use of rapid salmonella typi test are correct in stool diagnosis
    can u make the interpretation of biochemical test for the identification of salmonella typi easy

  7. sneha says:

    I like ur site.. .havng some confusion??

  8. Hafsa Said says:

    can you please write about molecular techniques for identifying bacteria, fungi or maybe specifically for diseases such as salmanellosis

  9. Muhmmad Khizar Khan says:

    Tankeshwar sir, I’ve a question. I have acquired blood samples from the patients suffering from typhoid fever and done VIDAL test to confirm the presence of S. typhi, I want to culture the microbe on MacConkey Agar from blood. I am a bit confused as how to do it because its my first time doing so. Your help is highly appreciated.

    • tankeshwar says:

      Muhmmad, you have to first inoculate the blood sample in Broth (Brain Heart Infusion Broth or Bactec Blood Culture System). Find more about Blood Culture here. Sub-culture is done in solid culture medium (Blood Agar, Chocolate Agar and MacConkey Agar) only if growth is noted in the broth medium.

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