Dairy MicrobiologyMicrobiology

Determination Of Phosphatase Activity Of Milk

The milk samples are diluted with buffer at pH 10.2 and incubated at a temperature 37ºC for 2 hours. Any alkaline phosphatase present in the milk sample will liberate p-nitrophenol from artificially added disodium p-nitrophenyl phosphate which can be compared with the standard colour discs.


  • Water bath
  • Comparater
  • Milk samples
  • Test tubes
  • Pipettes
  • Litmus paper
  • Sodium carbonate-bicarbonate buffer*
  • Buffer substrate

*Sodium carbonate-bicarbonate buffer

Dissolve 3.5 g of anhydrous sodium carbonate and 1.5 g of sodium bicarbonate in 1 litre of distilled water.

*Buffer substrate

Dissolve 1.5 g of disodium p-nitrophenyl phosphate in 1 litre of sodium carbonate-bicarbonate buffer.


  1. Collect 1 ml sample of boiled milk in a test tube and 5 ml of buffer substrate into another
  2. tube.
  3. Keep both the tubes in water bath for 2 hours.
  4. Take 5 ml of buffer substrate and 1 ml of milk to be tested in another tube. Stopper the tube and keep in the water bath as above.
  5. After 2 hours, remove the tubes from the water bath, invert them and compare the colour development in the tube containing boiled milk and sample under test.


Record the readings which lie between the two standard colour discs. Make index of the colour with a plus (+) or minus (−) sign in the figure of nearest standard.


Dr. R. C. Dubey – Practical Microbiology

Gaurav Singh

Editor in Chief Medical Microbiology & RDT Labs - RDT Labs Magazine | BSc Medical Microbiology | MSc Microbiology

Leave a Reply