Methyl Red-Voges-Proskauer test(MR-VP)/ Acetoin Production Assay

Methyl Red-Voges-Proskauer test(MR-VP)/ Acetoin Production Assay

Basic principle:

Methyl red test

The methyl-red test is used to detect the organism’s ability to produce sufficient acid from glucose fermentation and maintenance of the acidic environment (pH < 4.5) as shown by a colour change of the methyl-red indicator that is added at end of incubation of the sample.

Voges-Proskauer test

This test is usually done in conjunction with the MR test since the production of acetyl methyl carbinol or butylene glycol usually results in insufficient acid accumulating during fermentation to give a MR positive reaction. All VP positive bacteria produce acid first and hence may give a positive MR test in the early stages of the incubation period. If the incubation period is short, positive VP test may or may not be given. On longer incubation period, MR test may become negative and VP test positive.

Materials:

  1. Glucose phosphate peptone water (GPPW)/ Buffered peptone-glucose broth (commercially available as MR-VP broth)
  2. Bacterial culture in broth
  3. VP reagents : Barritt’s reagent A: 5% (wt/vol) a-naphthol in absolute ethanol,Barritt’s reagent B: 40% (wt/vol) KOH in deionized water (this might be replaced by a 40% (wt/vol) NaOH solution).Reagents must be prepared fresh. Reagents are also referred to as VP-1 and VP-2 or VP-A and VP-B.
  4. Methyl red solution: Completely dissolve 0.1 g of methyl red in 300 ml of ethanol (95%). Add 200 ml of deionized water to make 500 ml of a 0.05% (wt/vol) solution in 60% (vol/vol) ethanol. Store the prepared methyl red solution at 4°C.
  5. Other essentials:
  • Sterile culture tubes containing 5 ml of MR-VP broth
  • Sterile culture tubes
  • Test and control bacterial strains (commonly used controls are Escherichia coli and Enterobacter aerogenes) from trypticase soy agar or broth
  • Inoculation loop (disposable or have additional equipment available for sterilization of the inoculation loop)
  • Transfer pipettes

Procedure

  1. Prepare 5 mL  MR-VP Broth(2.5 for MR+2.5 for VP) as directed by the manufacturer. Prior to use, allow the medium to come to room temperature.
  2. Inoculate a tube of MR-VP broth (5ml) with light inoculum of pure fresh bacterial culture and incubate the tube for at least 24 hours at 37 o

N.B: For comparision it is suggested that Escherichia coli (MR+, VP-) and Enterobacter aerogenes (MR-, VP+) be used as control cultures.

Methyl red test

  1. Transfer 2.5 ml of culture into a new sterile culture tube.
  2. Add 5 drops of the methyl red reagent.
  3. Compare the test organism to the control cultures to immediately interpret the result, MR positive (e.g., E. coli) (Fig. 1A) or MR negative (e.g., E. aerogenes)

Observations:

If the colour changes to distinct red it indicates positive test. Whereas if the reagent turns yellow or yellowish orange, it indicates negative test.

Voges-Proskauer test:

  1. Transfer 2.5 ml of culture into a new sterile culture tube
  2. Add 12 drops of 5% alpha napthol (Barritt’s reagent A) and 4drops of 20% aqueous potassium hydroxide (Barritt’s reagent B) solutions in the tube of 2.5mL MR-VP culture.
  3. Carefully shake the tube for 30 seconds to 1 minute to expose the medium to atmospheric oxygen (necessary for oxidation of acetoin to obtain a colour reaction).
  4. Allow the tube to stand for at least 30 minutes.
  5. Within 1 hour, compare the test result to control cultures

Observations:

Positive test is indicated by the development of a bright cherry red colour after 5 to 15 minutes or longer, No change in colour indicates negative test.