Methylene Blue Milk Test


Use this test to check the bacteria contamination in a sample of milk. It will visually indicate whether bacteria are present in the milk sample and give an approximate level of milk quality based on the level of viable bacteria in the milk.


The test relies on the fact that methylene blue solution is blue in the presence of oxygen, but will lose color as oxygen is depleted. Bacteria in milk ferment lactose (milk sugar) to form lactic acid. During this process oxygen is used up and electrons are released, which react with methylene blue. One effect is to decolorize the methylene blue. More bacteria present in the milk leads to faster reduction (taking on electrons) of methylene blue and increased rate of color loss.

What you need.

  1. Methylene blue solution, 1% aqueous.
  2. Milk sample.
  3. Test tube.
  4. Test tube stopper.
  5. Pipet.
  6. Water bath.

What to do

  • Mix the milk sample well to distribute any bacteria it may contain. Pour 10ml into a clean, sterilized test tube.
  • Using a pipet, add 1ml of Methylene blue solution to the milk sample in the test tube.
  • Stopper the test tube and gently invert it to mix the contents.
  • Place the test tube in a water bath at 37°C (99°F) for 30 minutes making sure water level is high enough to cover most of the test tube. Cover bath with lid to so test tube sample is in the dark.
  • After 30 min. examine the sample, checking for decoloration.


A positive test results when viable bacteria are able to decolorize the milk within 30 minutes. The milk is considered unsatisfactory.

A negative test results when the milk is not decolorized within 30 minutes. The milk is considered of good quality.

Four levels of quality can be distinguished...

  • Excellent - when no reduction of blue color for up to 8 hours
  • Good - no decoloration in 6-1/2 to 7-1/2 hours
  • Fair - decoloration in 2-1/2 to 6 hours
  • Poor - decoloration in less than 2 hours