Methyl Red and Voges Proskauer Test – Principle, Result

Methyl Red and Voges Proskauer Test

• Methyl Red and Voges Proskauer are two different tests, which are performed to differentiate between two major types of facultative anaerobic enteric bacteria or acid-forming and acetoin forming bacteria.
• For example, Methyl red and Voges Proskauer test or MRVP test helps in differentiation between Escherichia coli ( MR+ and VP-) and Enterobacter aerogenes (MR- and VP+).
• Methyl red is a pH indicator, which is detect the acid-forming bacteria.
• Voges Proskauer test helps in the detection of acetoin (neutral-reacting end products ) producing bacteria when cultivated in specific media.
• Basically, the MRVP test (Methyl red and Voges Proskauer test) is used for the detection of which fermentation pathway is used by the bacteria to utilize glucose. Because there are present two types of pathway by which bacteria utilizes glucose such as; mixed acid fermentation pathway and 2,3 butanediol fermentation pathway.
• In the case of a mixed acid fermentation pathway, glucose is fermented and produces different types of organic acids for example; lactic, acetic, succinic, and formic acids.
• If the mixed acid fermentation pathway continued it will produce a huge amount of organic acid to overcome the phosphate buffer and will result in a pH of below 4.4.
• If methyl red ( pH indicator) added to a culture broth containing the pH below of 4.4, a red color will appear. It indicates that the mixed acid fermentation pathway is conducted within test bacteria.
• If methyl red ( pH indicator) added to a culture broth containing the pH above 6.0, a yellow color will appear. It indicates that the mixed acid fermentation pathway has not been utilized.
• In case of a 2,3 butanediol fermentation pathway, glucose will be fermented and produce a 2,3 butanediol end product instead of organic acids.
•  In these cases to test this pathway, an aliquot of the MR/VP culture is removed and a-naphthol and KOH are added.
• They are shaken together vigorously and set aside in static conditions for about one hour until the results can clearly appear.
• In case of Voges Proskauer test, if detect the presence of acetoin (precursor of 2,3 butanediol) or show positive for acetoin, it will turn “brownish-red to pink”.
• If Voges-Proskauer test show negative for acetoin, it will turn “brownish-green to yellow”.
• Methyl red and Voges Proskauer test or MRVP test helps in differentiation between Escherichia coli ( MR+ and VP-) and Enterobacter aerogenes (MR- and VP+).
• In 1915, Clark and Lubs first discovered the ability of coliform bacteria to produce and maintain acid products when cultivated in specific media. They found that cultures of E. coli show a red color upon addition of methyl red (pH indicator).
• In 1898, Voges and Proskauer, first observed the production of a red color after the addition of potassium hydroxide to cultures grown on specific media.

Why are the Methyl Red and Voges Proskauer test often performed together

• Both, methyl red and Voges Proskauer test are performed simultaneously because they are physiological related and are performed on the same medium methyl red Voges Proskauer broth or MR-VP broth.
• Opposite results are usually obtained for the methyl red and  Voges Proskauer tests.
• For example, Escherichia coli shows positive results in methyl red test and negative results in Voges Proskauer test.

Principle of Methyl Red and Voges Proskauer test

Methyl Red (MR) Test Principle

The pH indicator Methyl red is used for the detection of acidity when an organism ferments glucose. Those bacteria ferment glucose and produce organic acid in the culture medium, they turn in red color upon addition of methyl red, (pH below 4.4 ), which is called MR positive (MR+ ).

Those bacteria use the butylene glycol pathway to metabolize pyruvic acid to neutral end products, they will turn in yellow color upon addition of methyl red, (pH above 6.0), which is called MR negative (MR-).

In methyl red positive (MR+) organisms, they continue to produce more acids and as a result, increased acidity overcomes the phosphate buffer, thus resulting in a low pH and development of a red color.

In methyl red negative (MR-) organisms, they further metabolize the fermentation products by decarboxylation and result in an alkaline reaction (no red color) due to the formation of acetoin which neutralizes the pH and results in no red color development.

Voges-Proskauer test Principle

Voges-proskauer test performs to detect the neutral-reacting end products (acetoin) when cultivated in specific media.

Enteric bacteria that ferment glucose, further metabolize pyruvic acid to end product acetyl-methyl carbinol (acetoin). This end product is converted to diacetyl, in the presence of atmospheric oxygen and 40% potassium hydroxide.

Diacetyl is converted into a red complex, under the catalytic action of alpha-naphthol and creatine. This is a positive Voges-Proskauer (VP) test reaction.

The VP test is used primarily to separate Escherichia coli (VP-negative) from the Klebsiella-Enterobacter groups (VP-positive).

Material Require for Methyl Red and Voges Proskauer test

1. Nutrient broth culture of test organisms (For example, Escherichia coli and Enterobacter aerogenes)
2. MRVP (Methyl Red Voges-Proskauer ) broth.
3. Methyl Red pH indicator.
4. V-P solution I (napthol solution)
5. V-P solution II (40% potassium hydroxide)

*Composition of Methyl Red Voges-Proskauer broth or MRVP broth:

Peptone	7.0 g
Dextrose/Glucose	5.0 g
Potassium phosphate	5.0 g
Distilled water	1000.0ml

*Note: change the amount of the ingredients based on the required amount of MRVP broth.

Procedure of Methyl Red and Voges Proskauer test

Basic Initial Steps For both test

1. Take sterile and clean test tubes (if you want appropriate result then you can do it in double, means take two test tubes for each organism.)
2. Pour MRVP broth to each test tube.
3. Inoculate them using test organisms ( Escherichia coli and Enterobacter aerogenes).
4. Then incubate all these test tubes at 35-degree Centigrade for 48 hours.

Methyl Red Test

1. Add 3-5 drops of methyl red to each test tubes containing test organisms.
2. Observe the color change in medium.

Voges-Proskauer test

1. Add 12 drops of V-P solution I (napthol solution) and 2-3 drop of V-P solution II (40% potassium hydroxide) to each test tubes containing test organism.
2. Cotton plug each test tubes and Shake them for 30 sec.
3. Then expose the medium to oxygen.
4. Keep them in undisturbed condition for 10-15 minutes.
5. Then observe the color change in medium.

Result of Methyl Red and Voges Proskauer test

Methyl Red Test

• The result will be positive, If a stable red color develops on the surface of the medium, after the addition of methyl red indicator.
• The result will be negative, If a yellow color develops on the surface of the medium, after the addition of methyl red indicator.

Voges-Proskauer test

• The result will be positive, If a pink-red color develops on the surface of the medium, within 15 minutes to 1 hour after the addition of methyl red indicator.
• The result will be negative, If a yellow color develops on the surface of the medium, within 15 minutes to 1 hour after the addition of methyl red indicator.

List of Methyl Red Positive and negative Organisms

Organism Name	Result
Escherichia coli	Positive (MR+)
Klebsiella pneumoniae	Negative (MR-)
Shigella species	Positive (MR+)
Enterobacter species	Negative (MR-)
Salmonella species	Positive (MR+)
Hafnia species	Negative (MR-)
Proteus species	Positive (MR+)
Citrobacter species	Positive (MR+)
Serratia marcescens	Negative (MR-)
Yersinia species	Positive (MR+)

List of Voges-Proskauer Positive and negative Organisms

Organism Name	Result
Enterobacter	Positive (VP+)
Citrobacter sp.	Negative (VP-)
Klebsiella	Positive (VP+)
Shigella	Negative (VP-)
Serratia marcescens	Positive (VP+)
Yersinia	Negative (VP-)
Hafnia alvei	Positive (VP+)
Vibrio cholera biotype eltor	Positive (VP+)
Edwardsiella	Negative (VP-)
Salmonella	Negative (VP-)
Vibrio furnissii	Negative (VP-)
Vibrio fluvialis	Negative (VP-)
Vibrio vulnificus	Negative (VP-)
Vibrio parahaemolyticus.	Negative (VP-)
Vibrio alginolyticus	Positive (VP+)

Limitation of Methyl Red and Voges Proskauer test

• The medium has been incubated for a minimum of 48 hours, before the methyl red test. Otherwise, it will show a false-positive interpretation.
• If you incubate the culture medium more than 48 hours or 48-72 hours, then there are present some organisms which are capable of producing acetyl methyl carbinol (acetoin) will show false-negative VP reactions.
• The inoculum should be light, because too heavy inoculum may inhibit  the growth of bacteria and results in invalid test results.
• Organisms that are VP-positive are not necessarily MR-negative. For example, Enterobacter hafnia and Proteus mirabilis shows MR- and VP-positive.
• During VP test, it’s important that the alpha-naphthol be added first and the KOH added second.
• False-positive VP results may occur if the VP test’s result is observed beyond one hour following the addition of reagents.

Also Read: Indole test – Principle, Procedure, Uses.