History of Voges Proskauer (VP) Test
In 1898, Voges as well as Proskauer (16)characterized the process of fermentation of sugars by a variety of bacteria. They demonstrated that the gas that was produced during the process was a mixture of H2 and CO2 that when they added KOH to the cultures been grown in glucose peptone medium to allow for a prolonged period of incubation in presence of oxygen certain organisms developed a red fluorescent color. While the exact nature of the color was not known in the early days, this technique was suggested to differentiate between the bacterial strains that produced it and ones that didn’t. in 1906 Arthur Harden analyzed the fermentation products of Enterobacter aerogenes. He discovered that when it was surrounded by glucose, the organism made two compounds, acetoin as well as 2,3-butanedio.
Testing both compounds in with KOH both did not produce the red-pinkish color as described in Voges as well as Proskauer. The addition of peptone in the reaction produced the observed coloration when combined with Acetoin. Harden believed that acetoin was converted to diacetyl that then reacted with substances in the peptone mix and produced a colorless product. It was later discovered it was the existence of a guanidine guanidine group (on an element in peptone) is necessary for the color-changing reaction. In 1915, Clark and Lubs presented a new method to differentiate colon-aerogenes bacteria. Prior to that, the bacteria in this group were distinguished according to the gas ratio (CO2/H2) generated in the course of fermentation. In the presence of buffered peptone and glucose broth, bacteria can be distinguished in “low-ratio organisms” and “high-ratio organisms,”depending the amount of H2 and CO2 that were produced in almost identical amounts or the quantity of CO2 was greater.
The third ” ratio group” refers to those that don’t produce H2. In the same way that Clark and Lubs noted that the techniques used in determination of gas ratios were not modified to be used in routine tests and they therefore tried other methods for separating the various groups. Three years prior, Michaelis and Marcora discovered thatEscherichia bacteria produced hydrogen ions while growing inside lactose broth. With this knowledge, Clark and Lubs tested low-ratio and high-ratio species to determine their capacity to produce hydrogen ions while growing on dextrose. They found that in glucose-peptone medium , the low-ratio species generated 100-times more hydrogen ions as high-ratio strains.
Clark and Lubs modified the culture medium (0.5 percent peptone 0.5 per cent dextrose 0.5 percent K2HPO4) to permit the differentiation of low-ratio organisms from high-ratio organisms. This medium’s volume of hydrogen ions produced by low-ratio organisms resulted in an acidity that hindered their growth, whereas high ratio organisms produced significantly less hydrogen ions. Following growth in this medium the final concentration of hydrogen ions permitted the distinction between high-ratio and low-ratio ones. For a test of the hydrogen ion concentration using pH indicators. The pH indicators used were paranitrophenol as well as methyl red The test was conducted in 1916. Levine observed that bacteria that were negative in the test for methyl red proved positive for the Voges-Proskauer test.
This discovery established the general observation of a correlation between the creation of hydrogen ions as well as Acetoin. Organisms that produce glucose by fermenting it and producing large amounts of hydrogen ions, do not produce acetoin in the form of a fermentation intermediate. In 1916 this Voges-Proskauer (VP) testing method was still not fully optimized. Following the addition of KOH into the test medium the positive reaction was evident by a faint pink hue and took around 24 hours for the reaction to take place. The procedure was improved with adding ferric chloride prior making use of NaOH (instead that of KOH). The new assay was more sensitive and led to a solid copper tone, instead of the original Eosin pink.
O’Meara has further enhanced the Voges-Proskauer test by adding Creatine and NaOH to the grown-up cultivars. Within a few minutes after adding the ingredients and stirring the red color was evident and indicated the presence Acetoin and a positive Voges Proskauer test. The speed reduction and intensity of the reaction led to the widespread acceptance of the O’Meara modification as the norm test for VP test at the time. However, the modification of O’Meara didn’t produce the same results. In 1936, Barritt published his modifications to the test of the Voges-Proskauer, where he demonstrated that the adding anaphthol to the test intensified the positive reaction, making the test more sensitive. Today, Barritt’s modification to the Voges-Proskauer test has become the procedure that is used to determine Acetoin’s presence, an intermediate in metabolic sugar fermentation process via the process of butanediol.
During the development of the methyl red and Voges-Proskauer tests to differentiate Escherichia from the Enterobacter-Klebsiella group of Enterobacteriaceae, it seemed that there was an inverse relationship between the outcomes of the two tests: Escherichia was MR+ (positive) and VP- (negative), whereas members of the Enterobacter-Klebsiella group showed the opposite results of being MR-and VP+. The MR-VP test is used in clinical laboratories to distinguish between the two groups of Enterobacteriaceae. Through Barritt’s modification, and the increased accuracy of the test it was observed that some of the Enterobacteriaceae could not be identified using the test paired with MR-VP. The organisms tested positively for both the methyl-red test as and the test Voges Proskauer. In 1939, Vaughn and coworkers studied the effects of temperatures and duration on MR-VP test results for this so-called “intermediate” group”. The group comprises members of the generaEnterobacter, Klebsiella, Hafnia,Proteus and Serratia.
Principle
Pyruvic acid is the primary ingredient in the fermentative degradation of glucose further processed through different metabolic pathways, based on enzyme systems used by various bacteria. One of these pathways results in the production of Acetoin (acetyl carbinol methyl) which is a neutral-reacting final product.
If present, acetylmethylcarbinol can be transformed into diacetyl in presence of a-naphthol and strong alkali (40 percent KOH) and oxygen in the atmosphere. Diacetyl and guanidine-containing substances present in the peptones in the broth are then able to condense and create a pinkish-red polymer.
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
Media
Methyl red-Voges Proskauer (MR/VP) broth (formulated by Clark and Lubs) is used in the Voges-Proskauer tests. The composition of the MR/VP broth can be described as follows:
- Polypeptone – 7g
- Glucose – 5g
- Dipotassium phosphate – 5g
- Distilled water – 1L
- Final pH – 6.9
Reagents
- 5% α-naphthol (5 g/100 mL) in 95% ethyl alcohol: The reagent must be kept at 4-8°C in a dark environment. Shelf life of the reagent is between 2 and 3 weeks.
- 40% potassium hydroxide (KOH): The reagent must be kept at 4-8°C in a dark environment. Shelf life of the reagent is between 2 and 3 weeks.
Quality Control of Voges Proskauer Test
Check the broth for signs of contamination, dehydration and degrading prior to use. Conduct QC on every new batch of media or the reagent prior to using with an organism that is known to show an positive reaction as well as one organism that has an adverse reaction.
Organisms for Quality Control
For quality control of Voges Proskauer Test we can use those organisms;
- Klebsiella pneumoniae ATCC 13883—VP positive (red)
- Escherichia coli ATCC 25922—VP negative (no change)
Protocols of Voges Proskauer Test
Innoculate the broth of MR/VP by using a purified culture from the organism you want to test.
- Incubate for 24 hrs at 35 degrees Celsius.
- After this time, add 1mL of the broth into the clean test tube.
- Add 0.6mL of Naphthol* 5 and then 0.2 milliliters of 40 percent KOH.
- (Note Important that the reagents are added to this sequence.)
- Shake the tube with care to let the media breathe oxygen from the atmosphere and let the tube remain unaffected for 10 to 15 mins.
*The a-naphthol did not form part of the original method but was later discovered to work to intensify color according to Barritt and has to be added prior to.
*Shaking the tubes can increase the reaction of the VP.
Results and Interpretation of Voges Proskauer Test
The positive result of a VP test can be evident by the development of a pinkish-red color on the surface after 15 minutes or longer after the addition of the reagents that indicate that diacetyl is present the product of acetoin’s oxidation. It is not recommended to taken after a period of more than one hour as negative Voges-Proskauer culture can produce a copper-like color which could lead to an incorrect interpretation of the test.
If the test results are negative If the test is negative, the test with glucose or MRVP broth may be kept incubated for upto 48 hours and the test can be repeated.
Reporting results
- The majority of members belonging to the family Enterobacteriaceae have opposing MR as well as VP reaction. However, some organisms, such as H. Alvei, and Proteus mirabilis, can provide both positive MR reaction as well as an positive VP reaction (often delay).
- Streptococcus mitis is a group of organisms that are negative for VP while the other viridans group streptococci are positive for VP, with the exception of Streptococcus vestibularis that is VP variable.
Voges-Proskauer (VP) Positive Organisms of Enterobacteriaceae family are
- Klebsiella species
- Enterobacter species
- Hafnia species
- Serratia species
Limitations
- In the case of long-term time incubation (>3 days) Certain VP-positive organisms could cause an acidity within the medium, resulting in negative reactions that are weak or ineffective, as well as false VP reactions.
- Do not exceed the amount of KOH for every 2 ml of medium. The excess amount of KOH could cause an insignificantly positive reaction that could be obscured by the development of a copper-like color as a result from the reaction between KOH with a-naphthol by itself.
- Do not take the test for more than 1 hour after the addition of the VP agents. The color of copper may change and lead to a false-positive reading.
- Reagents need to add in the designated order. Reversals of the order could result in an unsubstantially positive or false-negative results in VP.
Precautions
- The culture should be incubated for no under 48 hours
- The reagents must be used in the appropriate quantity, i.e. VP-I as well as VP-II in the ratio 3:1
- Aeration is maintained by opening the cotton plugs by shaking tubes
- Pay attention to the color changes.
References
- https://microbeonline.com/voges-proskauer-test-principle-procedure-results/
- https://microbiologyinfo.com/voges-proskauer-vp-test-principle-reagents-procedure-and-result/
- https://en.wikipedia.org/wiki/Voges%E2%80%93Proskauer_test
- https://www.onlinebiologynotes.com/voges-proskauer-test-vp-test-objective-principle-procedure-and-result/
- https://asm.org/getattachment/0c828061-9d6f-4ae7-aea3-66e1a8624aa0/Methyl-Red-and-Voges-Proskauer-Test-Protocols.pdf
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547154/
- http://dspace.vpmthane.org:8080/jspui/bitstream/123456789/7582/1/Voges%20-Proskauer%20%28VP%29%20test%20.pdf
- https://www.sciencedirect.com/topics/immunology-and-microbiology/voges-proskauer-test
