What is a Voges Proskauer Test?

It is a procedure that helps determine the microorganism’s ability to produce acetylmethyl carbinol, which is a neutral reacting end product.

It was actually a double eponym, which is named after the two microbiologist Voges and Proskauer. In 1898, these two microbiologists observed the production of red color after adding potassium hydroxide to cultures on a particular media.

It was, later on, found out that the one responsible for the red color is not the potassium hydroxide but the production of acetylmethyl carbinol, which is a product of butylenes glycol pathway.

To make the procedure more sensitive, Barrit, in 1936 added alpha-naphthol to the medium before adding potassium hydroxide. (1, 2, 3, and 4)

Two test tubes; the other one test negative for VP test and the other one is positive

Picture 1: Two test tubes; the other one test negative for VP test and the other one is positive.

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comparison image between methyl red test and Voges Proskauer test

Picture 2: A comparison image between methyl red test and Voges Proskauer test.

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Voges–Proskauer Test Principle

The principle of Voges–Proskauer Test is to check for microorganism’s ability to produce acetylmethyl carbinol from the fermentation of glucose. If there is indeed an acetylmethyl carbinol, it will be converted to diacetyl with the help of naphthol, strong alkali, and atmospheric oxygen. (4, 5)

What are the media and reagents used?

  • MRVP broth
  • Buffered peptone
  • Glucose
  • Dipotassium phosphate (5)

Picture 3: Barritt’s reagent A used for VP test.

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There are two reagents used:

One. Barritt’s reagent A

  1. Alpha-naphthol
  2. Absolute ethanol

Procedure – You should dissolve at least six (6) grams of a-naphtholin in approximately 100 mL of ethyl alcohol (95%).

Two: Barritt’s reagent B

  1. Potassium hydroxide
  2. Deionized water (6)

 

How do you perform a VP test?

The following are the step by step guides to performing a Voges-Proskauer test:

Culture – A 24 to 48-hour tryptic soy broth culture is used.

Media – The medium to be used is MRVP medium. To create an MRVP medium, you need the following:

  • 5 grams of glucose
  • 5 grams of dipotassium hydrogen phosphate
  • 5 grams of peptone
  • Suspend the above-mentioned ingredients in a distilled water (1 liter).
  • Put at least 3 ml of media in every test tube plugged and sterilized at a temperature of 121 degree Celsius.
  • For solution B (Barritt’s reagent B), you should dissolve potassium hydroxide (16 grams) in 100 ml of water. (5, 6, and 7)

For the procedure, you will need the following materials:

  • Bunsen burner
  • Inoculating loop

Voges-Proskauer Test procedure

  1. Inoculate the microorganisms being tested using a sterile technique. Make sure to label the tube of medium.
  2. Incubate the culture at a temperature of 37 degree Celsius for 24 to 48 hours.
  3. Sterile the loop in the blue flame of the Bunsen burner and wait for the loop to turn red. Start heating from the base of the wire and move towards the tip.
  4. Get the test tube that contains the culture that has been kept for the desired number of hours.
  5. Remove the cap and put the neck of the tube into the flame.
  6. Take the loopful of microorganisms from the tryptic soy both strictly observing the aseptic method.
  7. Put the neck of the tube into the flame and replace the cap. Put the test tube back in the rack.
  8. Get two MRVP broth tubes and label each: one is labeled test and the other is labeled control.
  9. Remove the cap of the test tube labeled test and put the neck of the test tube into the flame.
  10. Inoculate the MRVP broth using the loop that contains the inoculum from the tryptic soy broth.
  11. Put the neck of the MRVP tube in the flame and put it in the rack. Make sure that you only inoculate the test tube with the label test and carefully observe the aseptic method. The test tube labeled control should be left uninoculated.
  12. Incubate the two test tubes (labeled test and control) at a 37 degree Celsius for 24 to 48 hours.
  13. Take the broths off the incubator.
  14. Remove the cap and add at least 10 drops of reagent A and 10 drops of reagent B to every broth.
  15. Shake the tube for a few minutes.
  16. If after 15 to 20 minutes a red color is formed, the result is positive. The test is negative if no red color is formed after a few minutes. (7, 8, 9, and 10)

Result interpretation

Positive Voges Proskauer Test

The Voges Proskauer test is positive if there is a pink-red color at the surface. Microorganisms that will turn positive to Voges Proskauer test are the following:

  • Listeria
  • Viridans group Streptococci with the exception of S. vestibularis
  • Enterobacter
  • Serratia marcescens
  • Klebsiella
  • Vibrio alginolyticus
  • Vibrio eltor
  • Hafnia alvei (4)

Negative Voges Proskauer Test

The test is negative if there is no pink-red color of the medium. Microorganisms that test negative of Voges Proskauer test include the following:

  • Streptococcus mitis
  • Salmonella
  • Citrobacter sp
  • Edwardsiella
  • Shigella
  • Yersinia
  • Vibrio parahaemolyticus
  • Vibrio furnissii
  • Vibrio vulnificus
  • Vibrio fluvialis (5)

Additional information

If the color changes to copper, the result is negative. On the other hand, if the color becomes rusty, the result is weak positive.

Are there any limitations?

  1. The result of Voges Proskauer test should be used along with other procedures in order to accurately differentiate the genus and species of a particular organism, especially Enterobacteriaceae.
  2. It is a must to add the reagents in proper order and in proper amount or else the result could lead to a false negative or weak positive.
  3. An hour after adding the reagent, you should check the test tube for any changes in color. A potential false positive result is possible if the color turns copper-like after adding KOH and a-naphthol.
  4. Shaking the test tube can stimulate a Voges Proskauer reaction.
  5. Some microorganisms may destroy acetoin causing the test to be invalid. (2, 4, 7, and 9)

References

  1. https://microbiologyinfo.com/voges-proskauer-vp-test-principle-reagents-procedure-and-result/
  2. https://microbeonline.com/voges-proskauer-test-principle-procedure-results/
  3. https://en.wikipedia.org/wiki/Voges%E2%80%93Proskauer_test
  4. http://www.asmscience.org/content/education/protocol/protocol.3204
  5. http://vlab.amrita.edu/?sub=3&brch=76&sim=215&cnt=2
  6. http://microbesinfo.com/2014/04/voges-proskauer-test-vp-test-principle-procedure-interpretation-and-quality-control/
  7. http://www.biologypractical.com/voges-proskauer-test-vp-test-objective-principle-procedure-and-result/
  8. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/Voges-ProskauerTestRgnts.htm
  9. https://www.sciencedirect.com/topics/immunology-and-microbiology/voges-proskauer-test
  10. https://aem.asm.org/content/15/5/1138

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