Voges- Proskauer (VP) Test (Non – acid end product test)

Objective:

    To determine the ability of microorganism produce non- acidic or neutral end products from glucose.

Principal:

            Some bacteria digest glucose to form key intermediate pyruvic acid which can further be metabolized to produce acetoin (acetyl methyl carbinol) as an intermediate and this acetoin further reduced to form 2,3-butanediol. The detection of 2,3, butanediol is difficult, so the test detects the intermediate acetoin by addition of 40 % potassium hydroxide (KOH) and 5 % alpha napthol solution (Barritt’s reagent) . The presence of acetoin and reagent , develops crimson red color.

           

Materials Required:
            -Bacterial Culture (Klebsiella sp., and E. coli)

            -MR-VP broth

           -Barritt’s Reagent : (40 % potassium hydroxide (KOH) , 5 % alpha napthol solution and 0.5% creatinine)

Procedure:
1.      Inoculate loopful of bacterial culture into MR-VP test tubes 
2.      Test tubes are incubating at 37° C for 24 hrs.     
3.   The next day add 0.6 ml of alpha napthol (Barritt’s reagent A)to the tubes and mix thoroughly. 
4.      Add 0.2 ml of 40 % KOH (Barritt’s reagent B) to the culture tube and shake vigorously.
5.      After 15 min. examine the color formation in the culture tubes.

Observation:  
- Pink color formation and turns crimson red color formation within few minutes in Klebsiella sp., tube.
 -Yellow or copper color formation in E.coli tube.

Result: 
-Klebsiella sp.,  is VP positive (present of acetoin)
-E. coli is VP negative ( acetoin absent)

Interpretation:

          If the culture produces acetoin, it is spontaneously oxidized to diacetyl in the presence of oxygen and potassium hydroxide (alpha napthol is used to increase the sensitivity of the reaction). Diacetyl then reacts with creatinine to form a pink colored complex and within few minutes it forms crimson red color.

VP Positive Organisms :

         Klebsiella sp., Enterobacter

MR/VP medium Composition:

Peptone    5g

Dipotssium hydrogen phosphate    5g

Glucose (10% solution)         50 ml

Distilled water 1000ml           

Barritt’s Reagent:

Solution A:    40 % potassium + 0.5 g creatine

Solution B:   5 % solution of α-napthol in absolute alcohol