Methylene Blue Reduction Test

Aim: To determine the quality of milk by Methylene blue reductase test

Principle: Methylene Blue Dye Reduction Test, commonly known as MBRT test is a quick method to assess the microbiological quality of milk. This test is based on the fact that the methylene blue dye solution added to the milk get decolorized when the oxygen present in the milk get exhausted due to microbial activity. The sooner the decolorization, it is assumed that the more inferior is the bacteriological quality of milk.

Methylene blue is a redox indicator, that lose its color under the absence of oxygen and is thought to be reduced to colorless leucomethylene blue. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism causes the color to disappear. The greater the number of bacteria in milk, the quicker will the oxygen be consumed, and in turn the sooner will the color disappear. Thus, the time of reduction is taken as a measure of the number of organisms in milk.

Milk is a good medium for the growth of microorganism.  A variety of microorganism can be found in both raw milk and pasteurized milk. Normally the milk is contaminated with microorganisms such as Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Enterobacter spp., Bacillus spp., etc.  Contaminated milk is one of the important sources for transmission of diseases from animals to humans. The main reason for this contamination is the un-proper handling of milk. Normally milk is contaminated during the milking process by the microorganisms present in the exterior surface of the animals, pipelines such as udder and adjacent areas. Unsterilized dairy utensils such as milking machines, milk cans are also a good source of contamination by the microorganism.

Methylene Blue Dye Reduction Test is widely used at the dairy reception area, processing units and milk chilling centres where it is followed as acceptance/rejection criteria for the raw and processed milk

Material Required

1.      Milk samples (raw, boiled, pasteurized)

2.      Reagent: Methylene Blue dye (1:25000)

3.      Sterile screw capped tubes

4.      Water bath

5.      Bunsen burner

Procedure

1.     1 mg of Methylene Blue dye was dissolved in 250 ml of sterile distilled water

2.     Each of the milk samples were thoroughly mixed

3.    3 ml of Methylene Blue dye was added to appropriately labelled MBRT tubes.

        Using different  pipettes, transfer about 30 ml of each type of milk into the above tubes and they were closed with stopper. 

6.     The contents of the tubes were mixed gently by inverting each test tube.

7.     The tubes were incubated in water bath at 37^oC for 5-6 hours

Observation

Milk samples observed for Methylene Blue reduction every five minutes for the first 30 minutes and then every 30 minutes for the rest of the time. Reduction is demonstrated by decolorization of the dye.

Result

The tubes which contained the samples of packet milk showed colour change from blue to white within 30 minutes, showing Methylene Blue dye reduction test positive. Raw milk showed Methylene Blue dye reduction within 5 minutes indicating very poor quality of the milk. Disappearance of colour within the limited time indicates the absence of oxygen in milk. Hence it shows packet milk and raw milk are of poor quality. Pasteurized milk showed Methylene blue reduction within 130 minutes, indicating fair quality of the sample. Boiled milk showed Methylene blue reduction in 450 minutes indicating good quality. 

Observations (left hand side)

Quality of milk sample by Methylene Blue Dye Reduction Test

Milk sample	Methylene Blue Dye Reduction Time	Quality
Packet milk	Within 30 minutes	Very poor
Raw milk	Within 30 minutes	Very poor
Pasteurized milk	130 minutes	Fair
Boiled milk	450 minutes	Good

 

Classification of Milk Quality

Methylene Blue Dye Reduction Time	Quality of milk	Approximate number of Bacteria
Within 30 minutes	Very poor quality	>2,00,00,000
31-120 minutes	Poor quality	>40,00,000 to 2,00,00,000
121-360 minutes	Fair quality	>5,00,000 to 40,00,000
361-450 minutes	Good quality	Below 500000