Methylene Blue Reduction Test for Milk

Methylene blue reduction test for milk post we briefly summarise about: principle, reagents requirements, methylene blue test for milk procedure, result, application and limitations of methylene blue test for milk.

Methylene Blue Reduction Test for Milk

Methylene blue test for milk, or MBRT test. This methylene blue test for milk is an qualitative milk test that is used to determine the quality of raw and pasteurized milk. In a basic aqueous solution, a reducing sugar is a carbohydrate that is reduced by a weak oxidizing agent. 

Reducing sugars have the property of generating one or more compounds containing an aldehyde group in an aqueous solution. For example, α-D-glucose contains a hemiacetal group and thus reacts with water to produce an open-chain form with an aldehyde group.


Sugars with a free aldehyde or ketone group are known as reducing sugars. When heated under alkaline circumstances, the colour of methylene blue test for milk blue solution changes from blue to colourless due to the oxidation of free aldehyde and ketone groups (in reducing sugars) to carboxyclic acids.  Oxygen, for example, converts glucose to gluconate in methylene blue test for milk.

methylene blue test

Methylene Blue Test for Milk



1. Dropper

2. Boiling Water bath

3. Test tube

4. Test tube stand


1. Methylene blue (0.1%)

2. 2N Sodium hydroxide


1. Methylene blue (0.1%): Dissolve 0.1g methylene blue in 100 ml distilled water (0.1% MB stock solution).

2. 2N Sodium hydroxide: To make 2 N solution, dissolve 80.00 g of sodium hydroxide in water to make volume 1 liter.


1. Mix the milk sample thoroughly to distribute the fat uniformly. Add 10ml of milk sample in a test tube. Then add 1ml of standard methylene blue solution in this test tube and invert the test tube to mix it properly.

2. After that, place the test tube in a water bath at 37°C (99°F) for 30 minutes, and Cover the bath with a lid. After 30 minutes of incubation observe the sample and check for discoloration, and make subsequent readings at hourly intervals thereafter.

3. After each reading, remove decolorized tubes and then slowly make one complete inversion of remaining tubes. Record reduction time in whole hours between last inversion and decolorization. 

4. For example, if the sample were still blue after 5 hours 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. 

Methylene Blue Test for Milk

Test Interpretation

Positive Result: If the viable bacteria decolorize the milk within 30 minutes, the milk is considered unsatisfactory.

Negative Result: If the milk is not decolorized within 30 minutes, the milk is considered as good quality.

Further Readings