1. Materials and Apparatus •Liquid Milk samples (raw unpasteurized cow's milk, pasteurized milk, and sterilized milk) • Test tubes with rubber stoppers •Pipette or dipper graduated to deliver 10 ml of milk • Water bath for maintaining the samples at 35° to 37°C. (The bath should contain a volume of water sufficient to heat the samples to 35° C within 10 minutes after the tubes enter the water and should have some means of protecting the samples from light during the incubation period. If a hot-air chamber is used, the samples should be heated to 35° C in a water bath since warm air would heat the milk too slowly).
2. Procedure: (1) Sterilize all glassware and rubber stoppers either in an autoclave or in boiling water. Be sure all glassware is chemically clean.
3. (2) Measure 10 ml of milk and pour into a sterile test tube.
4. (3) Aseptically add 1 ml of the methylene blue solution, and blow out the remaining drop after a lapse of 3 seconds. Cover the tubes with rubber stopper and gently invert twice to ensure complete mixing.
5. (4) Place tubes in 37°C water bath. The water level should be slightly higher than the milk in the tube. Close the lid of the water bath to exclude light. Record this time as the beginning of the incubation period
6. (5) Set up control tubes with each batch of milk tested: a. 10 ml milk + 1 ml methylene blue b. 10 ml milk + 1 ml distilled water *Both tubes should be immersed in boiling water for 3 minutes before placing in the. water bath. Control tube a will show start of decolorization and b is for completion
7. (6) Check samples for decolorization after 30 minutes of incubation. Make subsequent readings at hourly intervals thereafter.
8. (7) After each reading, remove decolorized tubes and then slowly make one complete. inversion of remaining tubes.
9. (8) Record reduction time in whole hours between last inversion and decolorization. For example, if the sample were still blue after 0.5 hour but was decolorized (white) at the 2.5 hour reading, the methylene blue reduction time would be recorded as 2 hours. Decolorization is considered complete when four-fifths of the color has disappeared.
