Fish Microbiology and Quality Assurance (2+2)

3. Voges - Proskauer (VP) Test

In this test, bacteria convert the metabolic intermediate product (pyruvic acid) of glucose into neutral products (acetylmethylcarbinol, acetoin)) and carbon dioxide instead of accumulating acidic products from the fermentation of glucose. The neutral product in the presence of air gets oxidized to diacetyl which reacting with gunidino groups under alkaline condition forms a complex which gives a port wine colour.

Procedure:
The test organism is grown in Glucose Phosphate Peptone (GPP) broth by incubating at 37⁰C for 48 to 96 h. The peptone in the medium serves as a source of guanidino group. After the incubation, a pinch of creatine (to intensify the reaction), 0.6 ml of 5 % α -napthol and 0.2 ml of 40 % potassium hydroxide is added for 1 to 2.5 ml of culture and allowed to stand for 30 minutes to 2 h. A positive reaction is indicated by change of colour from pink to red (port wine colour). , which indicates the presence of acetyl methyl carbinol.

Glucose Phosphate Peptone (GPP) broth

Peptone : 7.0 g
Glucose : 5.0 g
Sodium chloride : 5.0 g
Dipotassium hydrogen phosphate: 5.0 g
Distilled water : 1000.0 ml
pH : 7.1 ± 0.1

Heat to dissolve the ingredients and distribute in test tubes and sterilize at 110⁰C for 10 minutes.

α -napthol (5%)

α – napthol : 5.0 g
Ethanol : 100.0 ml

Dissolve α – napthol in ethanol and store it at 4⁰C.

Potassium hydroxide (40 %)

Potassium hydroxide : 40.0 g
Distilled water : 100.0 ml

Dissolve Potassium hydroxide in distilled water and store it in a polyethylene or paraffin-lined glass reagent bottle at 4⁰C. Instead of adding a pinch of creatine separately, a mixture of 0.3 % creatine and 40 % potassium hydroxide solution can be added.