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Methylene Blue Reduction Test (MBRT Test) for Milk

Methylene Blue Reduction Test, or MBRT test for milk, is another name for this test. It is a test for the quality of milk. It is used to check the quality of both raw milk and pasteurized milk. The Methylene Blue Reduction Test (MBRT Test) for milk is based on the observation that methylene blue solution takes on a blue hue in the presence of oxygen and loses this color as oxygen is decreased.

Lactose, which is milk sugar, is broken down by bacteria in the milk to make lactic acid. During the fermentation process, oxygen is used up, leaving less oxygen in the milk, and electrons are released. The methylene blue solution changes the way these electrons work. So, it makes the methylene blue lose its colour.

Bacteria are primarily responsible for the oxygen consumption in milk. It is assumed that the greater the number of bacteria in milk, the faster the oxygen will be consumed. The total amount of microorganisms found in milk.

Aim of MBRT Test

It is necessary to conduct this MBRT test for milk  to ensure that milk is free of harmful microorganisms. Milk quality and the presence of bacteria in a sample can be visually assessed in this way.

Principle of MBRT Test

Oxidation reduction potential of a substrate may be defined generally as the chemical process in which the substrate either loses or gains electrons. When an element or compound loses electrons the substrate is said to be oxidized, while a substrate that gains electrons becomes reduced.

The milk in the udder has a sufficiently low redox potential to instantly decrease the methylene blue. Because of the incorporation of atmospheric oxygen, processes such as milking, cooling, and dumping raise the oxidation reduction potential of milk to +0.3V.

At this O-R potential, methylene blue is in its oxidised form. When bacterial cells multiply in milk, they consume dissolved oxygen; as the oxygen is depleted, the dye begins to operate as an electron acceptor in place of oxygen.


Figure 1: Methylene Blue Reduction Test Principle

The methylene blue gets reduced due to the decreases of oxidation-reduction potential from + 0.06 to 0.01 V. When one hydrogen atom is added to the double-bonded nitrogen atom in the Methylene Blue dye, the dye goes from being blue to being colorless. The more microorganisms there are in milk, the more metabolic activity there is, and the faster methylene blue is broken down.

MBRT Test Requirements

1% Methylene blue

Milk sample

15 Ml Test tubes

Test tube stopper

Different Pipets

Water bath

MBRT Test Procedure

  1. To distribute the fat uniformly, thoroughly mix the milk sample. Then, pour 10 milliliters of the milk sample into a test tube.
  2. To mix the standard methylene blue solution properly, add 1 milliliter of the solution to the test tube containing the milk sample, and invert the test tube.
  3. Next, place the test tube in a water bath at 37°C (99°F) and cover the bath with a lid. Allow the mixture to incubate for 30 minutes.
  4. After incubating the mixture for 30 mins, observe the sample for discoloration and record any subsequent readings at hourly intervals.
  5.  When taking readings, remove any tubes that have decolorized and slowly invert the remaining tubes once.
  6. Record the reduction time in whole hours between the last inversion and the point at which the sample decolorizes (when four-fifths of the color has disappeared). 

MBRT Interpretation

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

Negative Result: On the other hand, if the milk is not decolorized within 30 minutes, it is considered to be of good quality.

Grading of milk in MBRT

Quality of milkReduction timeApprox. keeping quality/mlApprox. bacterial count per ml
Good5 hour or more40 hrsBelow 5,00,000
Fair2 to 5 hours30 hrs5,00,000 to 40,00,000
Bad20 minutes to 2 hrs10 hrs40,00,000 to 2,00,00,000
Very bad20 minutes or lessLess than 10 hrsAbove 2,00,00,000

Table 1: The quality of milk will be determined by making the following observations.

mbrt test for milk

Figure 2: MBRT test for milk

Factors Affecting on MBR test

  1. Any change in the amount of oxygen can change the result of the test, because all of the oxygen in milk must be used up before the colour goes away.
  2. Various organisms have different reaction times. By contrast, thermoduric and psychrotrophic bacteria reduce methylene blue relatively slowly, while coliform bacteria are the fastest-acting reducers.
  3. The reduction time of mastitic milk will affect the presence of a large number of leucocytes. Light speeds up the process of reduction, so the MBR tests should be done in low light.
  4. The concentration of the Methylene Blue solution affects the reduction time. A uniform concentration is important, because a higher concentration of dye can lengthen the reduction time.
  5. The activity of bacteria in the milk sample increases with higher incubation temperatures, which leads to a shorter reduction time.
  6. Inverting the milk sample during incubation can improve the accuracy of the test, reduce the reduction time, and make the decolorization more uniform.
  7. Creaming the milk can cause an uneven distribution of bacteria in the sample, leading to a shorter reduction time. This is because creaming removes microorganisms from the milk and brings them to the surface with the rising fat.

Advantages of MBRT 

There are several advantages to using the Methylene Blue Reduction Test (MBRT) to analyze milk samples:

  1. Rapid results: The Methylene Blue Reduction Test (MBRT Test for milk) for milk is a relatively quick test, with results typically available within a few hours.
  2. Simple to perform: The Methylene Blue Reduction Test (MBRT Test for milk) for milk  is easy to carry out and does not require specialized equipment or training.
  3. Inexpensive: The Methylene Blue Reduction Test (MBRT Test for milk) for milk is a cost-effective method for analyzing milk samples.
  4. High sensitivity: The MBRT is highly sensitive and can detect low levels of bacteria in milk samples.
  5. Versatility: The MBRT for milk can be used to analyze a variety of milk samples, including milk, and milk products such as cheese and yogurt.

Limitations of MBRT

There are also some limitations to using the Methylene Blue Reduction Test (MBRT) to analyze milk samples:

  1. Limited specificity: The MBRT is not able to identify specific types of bacteria in the milk sample. It only indicates the presence of bacteria in general.
  2. Can be affected by interference: Certain substances, such as certain types of antibiotics and disinfectants, can interfere with the results of the MBRT.
  3. Can be affected by temperature: The accuracy of the MBRT can be affected by the incubation temperature of the milk sample. It is important to maintain a consistent temperature throughout the test to obtain accurate results.
  4. May not detect all types of bacteria: The MBRT may not be able to detect certain types of bacteria, particularly those that are not active in milk.


FAQs on Methylene Blue Reduction Test (MBRT Test) for milk

No, glycerine and methylene blue reduction test are not similar.

The Methylene Blue Reduction Test (MBRT) is a laboratory test used to determine the presence and activity of bacteria in milk samples.

The Methylene Blue Reduction Test (MBRT) is used to check if bacteria are present in milk samples and how active they are. This is important because bacteria in milk can mean that it has been tainted or has gone bad, which can affect the quality and safety of milk products.

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