Alkaline Phosphatase Test to Check Efficiency of Pasteurized in Liquid Milk



Pasteurization is a critical process in the production of milk, ensuring it is safe for consumption by eliminating harmful pathogens. One key indicator of proper pasteurization is the presence of Alkaline Phosphatase, an enzyme naturally found in raw milk but destroyed at pasteurization temperatures. The Alkaline Phosphatase test is used to determine whether milk has been adequately pasteurized or if it has been contaminated with raw milk after the pasteurization process. This article presents a detailed laboratory procedure for conducting the Alkaline Phosphatase test


Apparatus Required:

  1. Water-Bath: A thermostatically controlled water-bath, maintained at 37±1⁰C, for incubation purposes.
  2. Comparator: Equipped with special discs of standard colour glasses calibrated in µg p-nitro phenol per ml milk and two 25 mm cells for colour comparison.
  3. Test Tubes: Size 16 x 1.50 mm with rubber stoppers to fit.
  4. Pipettes: 1 ml, 5 ml, and 10 ml pipettes for precise measurements.
  5. Filter Paper: Use what man No. 2 or equivalent filter paper.
  6. Litmus Paper: For checking the pH level of the samples.



  1. Sodium Carbonate-Bicarbonate Buffer: Dissolve 3.5 g of anhydrous sodium carbonate and 1.5 g of sodium bicarbonate in one litter of distilled water.
  2. Buffer Substrate: Dissolve 1.5 g of disodium p-nitro phenyl phosphate in one litter of sodium carbonate-bicarbonate buffer. Store this solution at 4°C or below for up to one month, but always conduct a colour control test on stored solutions.



  1. Prepare the Test Samples: Pipette 5 ml of buffer substrate into a clean and dry test tube. Add 1 ml of the milk sample to be tested. Stopper the tube securely, mix the contents by inversion, and place it in the water-bath.


  1. Prepare the Control Sample: Simultaneously, place a control tube in the water-bath. This control tube should contain 5 ml of the buffer substrate and 1 ml of boiled milk of the same kind as the sample under test, pasteurized, homogenized, and low-fat.
  2. Incubation: Allow both the test and control tubes to incubate in the water-bath for exactly 2 hours.
  3. Reading the Results: After 2 hours, remove the tubes from the water-bath, invert each tube, and read the colour developed using the comparator and the special colour discs. Place the tube containing the boiled milk control on the left of the comparator stand and the tube with the sample under test on the right. Record the readings that lie between two standard colour discs by adding a plus (+) or minus (-) sign to the figure of the nearest standard.

Important Note:

When taking readings, ensure you use an approved ‘daylight’ source of illumination if artificial light is required for accurate colour comparison.



The Alkaline Phosphatase test is a vital quality control measure for assessing the adequacy of pasteurization in milk production. By following the detailed laboratory procedure outlined in this article, milk processors can ensure that their products are safe, free from pathogens, and maintain the desired quality standards. Implementing this test helps maintain consumer confidence and safeguards public health, making it an indispensable step in the production of safe and nutritious milk.