Methylene Blue Reduction Test

Methylene Blue Reduction Test also known as mbrt test. It is a qualitative test for milk, it used to check the quality of raw and pasteurized milk.

The Methylene Blue Reduction Test is based on the fact that in the presence of oxygen the methylene blue solution forms blue color, and it will lose the color as the oxygen is depleted. 

The bacteria present in the milk will ferment lactose (milk sugar) to form lactic acid, during this fermentation process the oxygen is used up, which causes in depletion of oxygen in milk, and electrons are released. These electrons react with the methylene blue solution. As a result, it decolorizes the methylene blue.

Mainly bacteria are responsible for the oxygen consumption in milk. It is estimated that assumed that the greater the number of bacteria in milk, the quicker will the oxygen be consumed. The total number of microorganisms in milk.

This test is performed in a dairy reception dock, processing units, and milk chilling centers. There this process is followed as acceptance/rejection criteria for the raw and processed milk.

Aim of Methylene Blue Reduction Test

This test is performed to check the bacteria contamination in milk. It will visually indicate the presence of bacteria in a given milk sample, and it will indicate the level of milk quality.

Methylene Blue Reduction Test Principle

Milk has sufficiently low redox potential which reduces the methylene blue immediately. During the milking, cooling, dumping the oxidation-reduction potential of milk is increased to +0.3V. At this point, the methylene blue remains in an oxidized state. 

When the bacterial cells are started to increase their numbers in milk it consumes more dissolved oxygen from the milk, as a result, the oxygen gets depleted. Then the Methylene Blue starts acting as an electron acceptor instead of oxygen. The methylene blue gets reduced due to the decreases of oxidation-reduction potential from + 0.06 to 0.01 V.

The double-bonded nitrogen atom of  Methylene Blue dye accepts 1 atom of hydrogen as a result the dye is converted into a colorless state. The greater is the number of microorganisms in milk, the greater is the metabolic activity and the faster is the reduction of methylene blue.

Materials

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

Methylene Blue Reduction Test Procedure

1. Mix the milk sample thoroughly to distribute the fat uniformly.
2. Add 10ml of milk sample in a test tube.
3. Then add 1ml of standard methylene blue solution in this test tube and invert the test tube to mix it properly.
4. 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.
5. After 30 minutes of incubation observe the sample and check for discoloration, and make subsequent readings at hourly intervals thereafter.
6. After each reading, remove decolorized tubes and then slowly make one complete inversion of remaining tubes.
7. Record reduction time in whole hours between last inversion and decolorization. For example, if the sample were still blue after L 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.

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.

Grading of milk in MBR test

The quality of milk will be determined by making the following observations;

Quality of milk	Reduction time	Approx. keeping quality/ml	Approx. bacterial count per ml
Good	5 � hour or more	40 hrs	Below 5,00,000
Fair	2 to 5 � hours	30 hrs	5,00,000 to 40,00,000
Bad	20 minutes to 2 hrs	10 hrs	40,00,000 to 2,00,00,000
Very bad	20 minutes or less	Less than 10 hrs	Above 2,00,00,000

Factors Affecting on MBR test

1. Any manipulation in oxygen content can affect the test result, because all the oxygen present in milk must be used up before the color disappears. Cold milk contains more oxygen than warm milk, Pouring milk back and forth from one container to another increases the oxygen, and During milking time much oxygen may be absorbed.
2. The rate of reaction depends on the type of organism. For example, coliform bacteria are the most rapidly reducing organisms and thermoduric and psychrotrophic bacteria reduce methylene blue very slowly.
3. The reduction time of mastitic milk will affect the presence of a large number of leucocytes.
4. Light hastens reduction process and therefore, the MBR tests should be carried out in relatively low light.
5. The time of reduction depends on the Concentration of the Methylene Blue solution. The concentration should be uniform otherwise increased concentration of dye can increase the time of reduction.
6. The activity of the bacteria in the milk sample is increased with the increases of incubation temperature and therefore shortens the reduction time.
7. If the milk sample is rapidly inverted during the incubation the accuracy of the test is increased, reduction time shortened and decolorization more uniform.
8. The Creaming of milk prevents in uniform distribution of bacteria in milk samples which shortens the reduction time. The creaming of milk removes the microorganisms from the milk sample and brings it to the surface with the rising fat.

Advantage of Methylene Blue Reduction Test

• It is a simple and rapid test.
• It is an inexpensive test.
• Only viable cells actively reduce the dye.

Disadvantage of Methylene Blue Reduction Test

• Not all the bacteria reduce the dye equally.
• This test is not applicable to foods containing reducing enzymes.

FAQ on MBR test

References

• https://www.dairyknowledge.in/content/methylene-blue-dye-reduction-test-assessing-raw-milk-quality
• https://www.slideshare.net/RichaKhatiwada1/dye-reduction-test
• http://ecoursesonline.iasri.res.in/mod/resource/view.php?id=5014
• https://www.sciencecompany.com/Methylene-Blue-Milk-Test.aspx