Saturday , 1 April 2023

Methylene Blue Test Procedure & Protocol

This post will explain the methylene blue test. It is also sometimes called methylene reduction blue test.


Methylene blue test is based on the work done by Wilson (1935) and Milk Regulations 1963. This test is used to check the contamination of bacteria in the sample of milk. It tells us about the viable count of bacteria that may be present in the milk. So, by this particular test, we can have a clue about the quality of the milk. We can check that whether it is invaded by bacteria or not and in which quantity these are present.

Principle of Methylene Blue Test

Methylene blue test works on a simple principle. The principle is that if viable bacteria are present in the milk, they will reduce the methylene blue dye. The reduced dye will decolourize the sample if kept for sometime in a dark place. The methylene blue is reduced due to depletion of oxygen in the milk as it is consumed by bacteria.

Chemistry of Methylene Blue Stain

Methylene Blue is a dye used in methylene blue test. It is an aromatic chemical compound having the molecular formula C16H18N3SCl. Its molar mass is 319.85 g/mol. It is a dark green powder at room temperature and when it is dissolved in water, it forms a blue solution.

Methylene blue dye formula
Methylene blue dye formula

The bacteria in the milk ferment lactose to lactic acid. During this process electrons are released, which are taken up by Methylene Blue and results in its reduction and ultimately decoloration. So, more bacteria will produce more electrons, in turn it would be faster reduction and rapid discoloration.

MB + 2(H) = MBH2

(Blue) (Colorless)

Requirements of Methylene Blue Test

Following apparatus and reagents are required for the Methylene Blue Test.

  • Methylene Blue Solution
  • Milk Sample
  • Test Tube
  • Pipette
  • Rubber Stopper
  • Water Bath


Methylene blue test can be performed by a simple procedure given below.

  • Mix the sample well that has to be analyzed and then pour 10 ml of that milk into a clean and sterilized test tube.
  • With the help of pipette add 1 ml of Methylene Blue Solution into the milk sample.
  • The test tube is then closed with a rubber stopper and invered slowly so that all the contents have been mixed.
  • Then this test tube is places in a water bath at 37 C for half an hour making sure that water level is upto sufficient height of the test tube. Covered with lid.
  • Then the milk is examined after 30 minutes for checking the decoloration.


If the viable bacteria decolourize the milk within 30 minutes then the test is positive and milk is of unsatisfactory quality.

If the milk is not decolourized within 30 minutes then the test is negative or milk is of good quality.

Methylene blue test
Color change showing contamination

To interpret further, we can have four levels of quality,

1. Quality of milk is Excellent if no reduction of blue color upto 8 hours.

2. Quality of milk is Good if we have decolouration in 6.5-7.5 hours.

3. Quality of milk is Fair if we have decolouration in 2.5-6 hours.

4. Quality of milk is Poor if we have decolouration in less than 2 hours.

For more information about MBRT visit this link.

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