# Methylene Blue Reduction Test (MBRT) – Principle, Procedure and Results

> The Methylene Blue Reduction Test (MBRT) is an indirect microbiological quality assessment method used for testing milk. It is based on the reduction of...

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The Methylene Blue Reduction Test (MBRT) is an indirect microbiological quality assessment method used for testing milk. It is based on the reduction of methylene blue dye due to the metabolic activity of bacteria present in milk. The blue colour disappears when the dye is reduced.

Milk containing higher bacterial activity generally takes less time for dye reduction, indicating poorer microbiological quality. The test is used for examining both raw and pasteurized milk. It does not directly count or identify the bacteria present in milk. The microbiological quality is assessed from the reduction time.

## What Is MBRT?

The Methylene Blue Reduction Test (MBRT) is an indirect microbiological test used to examine the bacteriological quality of milk. It is commonly used as a preliminary screening method to check the microbial condition of raw and [pasteurized milk](https://biologynotesonline.com/milk-pasteurization/).

The test is based on the reduction of methylene blue dye due to the metabolic activity of bacteria present in milk. During this process, the blue dye is converted into its colourless reduced form. The time required for the disappearance of blue colour is called the reduction time. Milk containing higher bacterial activity generally shows faster dye reduction.

MBRT does not give the exact number of bacteria present in milk. It provides an indirect indication of microbial activity rather than a direct bacterial count. The test also cannot identify the bacterial species present in the sample.

## Objectives of Methylene Blue Reduction Test

- To assess the bacteriological quality of milk by measuring the time required for reduction of methylene blue dye.

- To obtain an indirect estimate of the bacterial load and metabolic activity present in milk. It does not give the exact bacterial count.

- To classify milk samples into different quality grades based on their reduction time.

- To identify milk with poor microbial quality during collection and before it is mixed with bulk milk for processing.

- To get an indication of the hygienic conditions maintained during milking, handling, transportation and storage.

- To help assess the possible keeping quality of milk, as milk containing higher bacterial activity generally undergoes faster spoilage.

## Principle of Methylene Blue Reduction Test

![Diagram of bacterial oxygen consumption in milk, reduced redox potential and conversion of blue methylene blue to colourless leucomethylene blue.](https://biologynotesonline.com/wp-content/uploads/2026/01/Principle-of-Methylene-Blue-Reduction-Test-MBRT-1024x576.webp)Principle of MBRT showing how microbial activity produces reducing conditions in milk, resulting in the decolourization of methylene blue.

The principle of Methylene Blue Reduction Test (MBRT) is based on the oxidation-reduction reaction taking place in milk due to the metabolic activity of bacteria. Methylene blue is used as a redox indicator. It remains blue in its oxidized state and becomes colourless when reduced.

The bacteria present in milk consume dissolved oxygen during their metabolic activities. As the oxygen is used up, the oxidation-reduction potential of milk decreases. Under these reducing conditions, methylene blue accepts electrons and is converted into its colourless reduced form, known as leucomethylene blue. The blue colour disappears.

Milk containing greater microbial reducing activity generally shows faster decolourization. The bacteria consume oxygen more rapidly and the reducing conditions develop within a shorter period. The time required for reduction also depends on the types and metabolic activity of bacteria present in milk.

## Materials Required for MBRT

The following materials and equipment are required for performing the Methylene Blue Reduction Test (MBRT).

- Milk Samples- Raw or pasteurized milk samples collected in clean, sterile containers for examination.

- Sterile Test Tubes- Sterile MBRT test tubes with sufficient capacity to hold 10 ml of milk and 1 ml of methylene blue solution.

- Rubber Stoppers- Sterilized rubber stoppers or screw caps are used to close the test tubes and prevent the entry of external air during incubation.

- Graduated Pipettes- Sterile 10 ml and 1 ml pipettes are required for measuring the milk samples and methylene blue solution. A pipette filler is used for transferring the liquids.

- Methylene Blue Solution- A 0.005% aqueous solution of methylene blue is used as an oxidation-reduction indicator.

- Water Bath- A thermostatically controlled water bath maintained at 37 ± 1°C for incubation of milk samples.

- Thermometer- Used to check and maintain the required temperature of the water bath.

- Stopwatch or Timer- To record the time taken for the disappearance of blue colour from the milk sample.

- Test Tube Rack- Used to hold the test tubes in an upright position during preparation and incubation.

- Other Materials- Sterile sample bottles, test tube marker, analytical balance, volumetric flask, distilled water, amber-coloured reagent bottle and necessary equipment for [aseptic handling](https://biologynotesonline.com/different-aseptic-techniques-in-microbiology-laboratory/).

### Preparation of Methylene Blue Solution

According to the Dairy Knowledge Portal MBRT protocol, which refers to IS 1479 (Part 3):1977, a 0.005% methylene blue solution is used for testing milk.

- Weigh 0.005 g (5 mg) of methylene blue dye using an analytical balance.

- Dissolve the dye in a small quantity of sterile distilled water.

- Transfer the solution into a 100 ml volumetric flask and make up the volume to 100 ml with sterile distilled water.

- Mix the solution properly to obtain a uniform 0.005% (w/v) methylene blue solution.

- Store the prepared dye solution in a clean, tightly stoppered amber-coloured bottle protected from direct sunlight.

For MBRT, 1 ml of the prepared indicator solution is added to 10 ml of milk. The dye is used to detect the reducing activity produced during bacterial metabolism.

The MBRT indicator solution is different from methylene blue solutions used for [staining microorganisms](https://biologynotesonline.com/simple-staining-procedure-principle-result/). Methylene blue staining solutions may contain a much higher concentration of dye (e.g. 0.3%) and are used for colouring bacterial cells during microscopic examination. These staining solutions should not be used in place of the standard MBRT reagent.

## Procedure of Methylene Blue Reduction Test

![MBRT laboratory workflow showing 10 mL milk, addition of 1 mL methylene blue dye, gentle mixing, incubation at 37°C and observation of decolourization.](https://biologynotesonline.com/wp-content/uploads/2026/01/Methylene-Blue-Reduction-Test-Procedure-for-Milk-1024x576.webp)Procedure of MBRT showing the preparation, incubation and observation of milk samples to determine methylene blue reduction time.

The following are the steps involved in the Methylene Blue Reduction Test (MBRT).

- At first, collect the representative milk samples in clean and sterile sample bottles. The milk is mixed properly before sampling. Label each bottle with the sample details.

- Take clean and sterilized MBRT test tubes and arrange them in a test tube rack. The test tubes are labelled according to the milk samples.

- Transfer 10 ml of milk from each sample into a separate sterile test tube using a graduated pipette.

- Now, add 1 ml of 0.005% methylene blue solution to each test tube containing milk. A sterile pipette is used to avoid contamination of the dye solution.

- The test tubes are closed with sterilized rubber stoppers. Gently invert each tube twice to mix the milk and methylene blue solution. Avoid vigorous shaking and unnecessary aeration.

- Place the test tubes in a water bath maintained at 37 ± 1°C. The tubes are kept in an upright position. Record the time when the tubes are placed in the water bath.

- After 30 minutes, examine the test tubes for the disappearance of blue colour. The tubes showing complete decolourization are removed. If the blue colour is still present, continue incubation. The remaining tubes are observed at hourly intervals after the first 30-minute reading.

- The test is considered complete when the blue colour disappears from the milk, except for a faint blue ring of about 5 mm at the top. The tubes in which decolourization has started should not be shaken or inverted. However, the tubes showing no decolourization may be gently inverted after each observation.

- Record the reduction time from the beginning of incubation until the specified endpoint is reached. If decolourization occurs at the first 30-minute observation, the reduction time is recorded as 30 minutes. The subsequent results are recorded in whole hours according to the standard observation schedule.

- During the test, the temperature of the water bath should be maintained properly and sterile glassware must be used. Avoid unnecessary disturbance of the test tubes, entry of air and delay in recording the observations.

## Results and Interpretation of MBRT

The results of the Methylene Blue Reduction Test (MBRT) are recorded as the time required for the disappearance of blue colour from the milk sample. This is known as the methylene blue reduction time. The reduction time is used to assess the bacteriological quality of milk. Milk containing greater microbial reducing activity generally shows faster decolourization.

![Comparison of methylene blue reduction in three milk samples after 1, 3 and 5 hours, corresponding to fair, good and very good quality grades.](https://biologynotesonline.com/wp-content/uploads/2026/01/MBRT-Results-and-Interpretation-of-Milk-Quality-1024x576.webp)Interpretation of MBRT results using reduction time. In the selected grading system, milk samples reducing the dye after 1, 3 and 5 hours are classified as fair, good and very good, respectively.

### Observation of Colour Change

The milk sample appears blue after the addition of methylene blue solution. During incubation, the blue colour gradually disappears due to the reducing activity of microorganisms present in milk.

According to IS 1479 (Part 3):1977, complete decolourization is recorded when the milk becomes colourless, except for a faint blue ring of about 5 mm at the surface.

The observations are interpreted as follows.

- Rapid Reduction- The blue colour disappears within a short period. It generally indicates higher microbial reducing activity and poorer bacteriological quality of milk.

- Delayed Reduction- The milk retains its blue colour for a longer period before decolourization. This generally indicates lower microbial reducing activity and better bacteriological quality.

- No Reduction During Observation- The blue colour remains unchanged up to the last observation. The result is recorded as "not decolourized" within the observed period. It does not indicate the complete absence of bacteria in milk.

### Calculation of Methylene Blue Reduction Time

The reduction time is calculated from the beginning of incubation to the observation at which the specified decolourization endpoint is reached.

Reduction Time = Time of Decolourization Observation − Starting Time of Incubation

For example, if incubation starts at 9:00 AM and the endpoint is observed at 12:30 PM, the elapsed time is 3 hours 30 minutes.

According to the reporting schedule of IS 1479 (Part 3):1977, the first positive reading at 30 minutes is recorded as MBRT 30 minutes. Later observations are recorded in whole hours. A sample becoming decolourized between the 2.5-hour and 3.5-hour readings is reported as MBRT 3 hours.

The actual moment of dye reduction may occur between two observations.

### Grading of Milk Quality by MBRT

The grading of raw milk is based on the methylene blue reduction time. According to IS 1479 (Part 3):1977, as reproduced by the National Dairy Development Board's [Dairy Knowledge Portal](https://www.dairyknowledge.in/dkp/content/methylene-blue-dye-reduction-test-assessing-raw-milk-quality), the following grades are used.

Methylene blue reduction timeQuality of milkLess than 30 minutesPoor1–2 hoursFair3–4 hoursGood5 hours and aboveVery good

The milk samples showing reduction within a shorter period generally contain greater bacterial reducing activity. A longer reduction time indicates comparatively lower microbial activity.

The published grading table specifies "less than 30 minutes" for poor quality, while the same protocol records decolourization at the first 30-minute reading as MBRT 30 minutes. The exact 30-minute boundary is not separately classified in this table. Other published MBRT methods also use different grading ranges, including an eight-hour grading system.

### Example of an MBRT Observation Table

The following table contains hypothetical observations of five milk samples. All samples are assumed to have been placed in the water bath at 9:00 AM.

Milk sampleInitial dye colourObservation timeReduction time (reported)InterpretationSample ABlue9:30 AM30 minutesRapid reduction; exact grade boundary unspecifiedSample BBlue10:30 AM1 hourFairSample CBlue12:30 PM3 hoursGoodSample DBlue2:30 PM5 hoursVery goodSample EBlue2:30 PM, still blueGreater than 5.5 hours, endpoint not reachedNo decolourization observed up to 5.5 hours

The reported reduction times in Samples B, C and D follow the whole-hour recording convention of the selected protocol. Sample E has no observed endpoint within the stated observation period.

## Factors Affecting MBRT Results

The results of the Methylene Blue Reduction Test (MBRT) are affected by several factors which influence the rate of dye reduction in milk. Some of the important factors are as follows.

- Bacterial Load- The number of bacteria present in milk affects the reduction time. Milk containing a large number of metabolically active bacteria generally shows faster decolourization due to increased oxygen consumption and reducing activity.

- Types of Bacteria- Different bacteria have different capacities to reduce methylene blue. [Coliform bacteria](https://biologynotesonline.com/coliform-bacteria/) and certain lactic acid bacteria are relatively strong reducers. Some [psychrotrophic and thermoduric bacteria](https://biologynotesonline.com/foodborne-microorganism-bacteria/) may reduce the dye slowly, even when present in large numbers.

- Dissolved Oxygen- The oxygen content of milk affects the time required for dye reduction. Milk containing more dissolved oxygen generally takes a longer time to decolourize. Cold milk can hold more oxygen than warm milk. Excessive shaking or aeration during handling may also increase the oxygen content.

- Incubation Temperature- The rate of bacterial metabolism depends on temperature. An increase in incubation temperature within a suitable range may increase bacterial activity and shorten the reduction time. The water bath temperature should be maintained at 37 ± 1°C according to the selected MBRT protocol.

- Concentration of Methylene Blue- The concentration of the dye should be uniform in all test samples. A higher dye concentration may increase the reduction time, particularly when the concentration is considerably above the recommended level. The age and quality of the dye solution may also affect the results.

- Exposure to Light- Light can increase the rate of methylene blue reduction. The test tubes should be protected from direct light during incubation.

- Fat Content and Cream Formation- During incubation, the fat globules rise towards the surface of milk and form a cream layer. Some bacteria are carried along with the rising fat, resulting in an uneven distribution of bacteria within the milk. This may cause variation in the reduction time.

- Presence of Leucocytes- Milk containing a large number of leucocytes (white blood cells), such as milk from animals with mastitis, may show changes in reduction time. The reducing activity of these cells can also influence the dye reduction reaction.

- Antibacterial Substances- The presence of antibiotics, antiseptics or other substances that inhibit bacterial activity may delay methylene blue reduction. The dye may remain blue for a longer period even when bacteria are present in the milk.

- Storage and Handling of Milk- The temperature and duration of milk storage before testing can affect the bacterial population. Milk kept under conditions favourable for bacterial growth may show a shorter reduction time. Refrigeration slows down bacterial multiplication.

- Mixing and Observation- Improper mixing may cause uneven distribution of methylene blue in the milk sample. The tubes should not be shaken after decolourization has started. Differences in observation intervals or the selected decolourization endpoint may also affect the recorded reduction time.

## Applications of MBRT

- Used to assess the bacteriological quality of milk by measuring the time required for methylene blue reduction.

- It is used for preliminary screening of raw milk at dairy collection centres and milk processing plants.

- Used to classify milk into different quality grades based on its reduction time.

- The test helps in assessing the hygienic conditions maintained during milking, handling, transportation and storage.

- Used to obtain an indirect indication of the bacterial load and microbial activity present in milk.

- It is also used to estimate the possible keeping quality of milk. Milk showing faster dye reduction generally has a greater risk of [microbial spoilage](https://biologynotesonline.com/preservation-and-spoilage-of-milk-and-milk-products/).

## Advantages of MBRT

- It is a simple test and does not require complicated laboratory procedures.

- It requires only basic laboratory equipment such as test tubes, methylene blue solution and a water bath.

- The test is inexpensive compared to conventional bacterial counting methods.

- It provides a relatively quick indication of the bacteriological quality of milk, often within a few hours.

- It does not require the cultivation of bacteria or [counting bacterial colonies](https://biologynotesonline.com/pour-plate-method-principle-procedure/).

- Several milk samples can be tested at the same time and classified into different quality grades based on their reduction time.

- It is useful for preliminary assessment of the hygienic condition and possible keeping quality of raw milk.

## Limitations of MBRT

- The test does not measure the exact number of bacteria present in milk. The reduction time cannot be directly converted into a reliable bacterial count.

- Different bacteria have different rates of methylene blue reduction. Some psychrotrophic and thermoduric bacteria reduce the dye slowly. They may be present in large numbers, but the reduction of methylene blue remains slow.

- MBRT cannot identify the bacterial species or detect specific pathogenic microorganisms present in milk.

- The dissolved oxygen content of milk affects the test result. Excessive shaking or aeration of the milk sample may delay the reduction of methylene blue.

- Leucocytes present in milk can also influence dye reduction, particularly in milk obtained from animals suffering from mastitis.

- The presence of antibiotic residues or other inhibitory substances may suppress the bacterial activity. This can increase the reduction time.

- The incubation temperature must be maintained properly. The condition of the methylene blue solution also affects the reaction. Any improper temperature control or changes in the dye solution may produce inaccurate results.

- During incubation, the fat globules rise towards the cream layer carrying some bacteria along with them. This may affect the reduction time.

- It may take several hours to obtain the result, especially when the milk contains low bacterial reducing activity.

- The test is less reliable for heat-treated milk and does not always show a good correlation with the actual viable bacterial count.

## References

- Anderson, E. B., & Wilson, G. S. (1945). 317. The resazurin and methylene-blue tests as a measure of the keeping quality of milk. Journal of Dairy Research, 14(1–2), 21–27. [https://doi.org/10.1017/S0022029900032271](https://doi.org/10.1017/S0022029900032271)

- Barkworth, H., & Hosking, Z. (1952). Experiments with the methylene blue reduction test for milk. Proceedings of the Society for Applied Bacteriology, 15(1), 116–125. [https://doi.org/10.1111/j.1365-2672.1952.tb00014.x](https://doi.org/10.1111/j.1365-2672.1952.tb00014.x)

- Brazis, A. R., & Black, L. A. (1966). Some factors affecting the correlation of methylene blue reduction times and standard plate counts of raw milk. Journal of Milk and Food Technology, 29(8), 255–259. [https://doi.org/10.4315/0022-2747-29.8.255](https://doi.org/10.4315/0022-2747-29.8.255)

- Chalmers, C. H. (1938). 204. The methylene blue reduction test and the keeping quality of milk. Journal of Dairy Research, 9(3), 351–355. [https://doi.org/10.1017/S0022029900002636](https://doi.org/10.1017/S0022029900002636)

- Davis, R. N., & Lines, K. W. (1939). The physiological types of bacteria in milk in relation to reduction of methylene blue. Journal of Dairy Science, 22(4), 209–218. [https://doi.org/10.3168/jds.S0022-0302(39)92877-8](https://doi.org/10.3168/jds.S0022-0302(39)92877-8)

- Hastings, E. G., Davenport, A., & Wright, W. H. (1922). The influence of certain factors on the methylene blue reduction test for determining the number of bacteria in milk. Journal of Dairy Science, 5(5), 438–454. [https://doi.org/10.3168/jds.S0022-0302(22)94173-6](https://doi.org/10.3168/jds.S0022-0302(22)94173-6)

- Homhual, S., & Jindal, V. K. (2001). Simple tests for rapid assessment of the quality of raw milk. Journal of Food Protection, 64(12), 1996–2000. [https://doi.org/10.4315/0362-028X-64.12.1996](https://doi.org/10.4315/0362-028X-64.12.1996)

- Jackson, C. J. (1936). Factors in the reduction of methylene blue in milk. Journal of Dairy Research, 7(1), 31–40. [https://doi.org/10.1017/S002202990000162X](https://doi.org/10.1017/S002202990000162X)

- Johns, C. K. (1938). Concerning the accuracy of the methylene blue reduction test. Journal of Dairy Science, 21(4), 227–237. [https://doi.org/10.3168/jds.S0022-0302(38)95639-5](https://doi.org/10.3168/jds.S0022-0302(38)95639-5)

- Nichols, A. A., & Jackson, C. J. (1938). A comparison between the methylene blue reduction test and the plate count with changing conditions of milk production and with changing conditions of storage of the milk samples before testing. Proceedings of the Society of Applied Bacteriologists, 1(1), 22–25. [https://doi.org/10.1111/j.1365-2672.1938.tb03894.x](https://doi.org/10.1111/j.1365-2672.1938.tb03894.x)

- Robinson, R. K. (Ed.). (2002). Dairy microbiology handbook: The microbiology of milk and milk products (3rd ed.). John Wiley & Sons. [https://doi.org/10.1002/0471723959](https://doi.org/10.1002/0471723959)

- Thornton, H. R., & Hastings, E. G. (1929). Studies on oxidation-reduction in milk. I. Oxidation-reduction potentials and the mechanism of reduction. Journal of Bacteriology, 18(5), 293–318. [https://doi.org/10.1128/jb.18.5.293-318.1929](https://doi.org/10.1128/jb.18.5.293-318.1929)

- Thornton, H. R., & Hastings, E. G. (1930). Studies on oxidation-reduction in milk: The methylene blue reduction test. Journal of Dairy Science, 13(3), 221–245. [https://doi.org/10.3168/jds.S0022-0302(30)93520-5](https://doi.org/10.3168/jds.S0022-0302(30)93520-5)

- Thornton, H. R., & Sandin, R. B. (1935). Standardization of the methylene blue reduction test by the use of methylene blue thiocyanate. American Journal of Public Health and the Nation's Health, 25(10), 1114–1117. [https://doi.org/10.2105/AJPH.25.10.1114](https://doi.org/10.2105/AJPH.25.10.1114)

- Wehr, H. M., & Frank, J. F. (Eds.). (2004). Standard methods for the examination of dairy products (17th ed.). American Public Health Association. [https://books.google.com/books?id=i4uuzh0r-9MC](https://books.google.com/books?id=i4uuzh0r-9MC)

- Whitehead, H. R. (1930). The reduction of methylene blue in milk: The influence of light. Biochemical Journal, 24(3), 579–584. [https://doi.org/10.1042/bj0240579](https://doi.org/10.1042/bj0240579)