Methylene Blue Reduction Test

Methylene Blue Reduction Test (MBRT) is the test used for checking the microbiological quality of milk. It is the process where a redox dye called methylene blue is added to milk, and the time taken for disappearance of the blue colour is recorded.

It is based on the activity of microorganisms present in the milk. These microorganisms utilise lactose and oxygen during their metabolic activity, and the dissolved oxygen is removed quickly. When the oxygen level becomes low, the dye is reduced into its colourless form which is called leuco–methylene blue. It is this reduction time that is used as the indicator for the milk quality.

It is observed that good–quality milk having low microbial load will take a longer time for decolourisation whereas highly contaminated milk will get reduced within a very short period. The test is commonly used in milk collection centres, dairy plants and processing units for acceptance or rejection of raw milk. The major basis is that there is a direct relation between the microbial population and the methylene blue reduction time.

Decolourisation is considered complete when the milk becomes colourless except a thin blue ring (around 5 mm) on the top. The milk is then graded depending on reduction time such as very good (5 hours or more), good, fair or poor (less than half hour). It is qualitative in nature since it measures metabolic activity of microorganisms rather than their exact number.

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

The principle of the Methylene Blue Reduction Test is based on the reduction–oxidation behaviour of methylene blue dye in the milk sample. It is the process where the dye remains blue in its oxidised state, but it becomes colourless when it is reduced by the activity of microorganisms. In fresh milk the oxidation–reduction potential is high and the dissolved oxygen keeps the dye in its blue form. When microorganisms are present, they start utilising lactose and oxygen during their metabolic activity and the oxygen level in the milk begins to decrease steadily.

It is the point when the oxygen is depleted that the oxidised dye acts as an artificial electron acceptor. The dehydrogenase enzymes from the active microorganisms transfer electrons and hydrogen to the dye causing the formation of the colourless leuco–methylene blue. This change generally occurs when the reduction potential of milk drops to a lower value. The principle states that the time required for disappearance of the blue colour is inversely related to the microbial load. Milk containing large number of bacteria will reduce the dye quickly, whereas good–quality milk with low microbial population will take a longer time for decolourisation. Thus, the reduction time indicates the metabolic activity of microorganisms rather than their exact number.

Materials

Apparatus Required

Some of the main apparatus used in this test are–

• Test tubes– Thick-walled tubes (5/8” × 6” or 5/8” × 8”) is used. These tubes must be sterile and it is kept chemically clean.
• Stoppers– Sterilized rubber or cork stoppers is required to close the test tubes during incubation.
• Measurement tools– A pipette or metal dipper calibrated to deliver 10 ml milk is used. Another pipette or burette is used to measure 1 ml dye solution.
• Water bath / Incubator– It is kept at 37 ± 1°C. The volume of water should be enough so that the milk sample reaches 35°C within 10 minutes of immersion. The bath is provided with a cover so the tubes is protected from light. A test tube stand or wire rack is also used.

Reagents and Solution Standards

• Milk sample– Raw or pasteurized milk is used for this test. It is also suitable for sweet cream when acidity is below 0.21%.
• Methylene blue dye solution– It is a water-soluble redox indicator.
  Some important points are–
  – The dye is prepared freshly every week using distilled water.
  – The standard concentration used in MBRT is 0.005% methylene blue.
  – If tablets are used, one certified tablet is dissolved in 200 ml hot distilled water to make the standard dye solution.
  – For cream samples, a triple-strength solution (3 tablets in 200 ml) is used because it gives deeper original color and better reading.

Key Conditions During the Procedure

• Sterility– The whole procedure is carried out in sterile condition. It is important because outside contamination will change the reduction time.
• Mixing ratio– The standard mixture contains 10 ml milk and 1 ml dye solution. Sometimes 10 ml milk with 0.5 ml dye is used in extended MBRT studies.
• Temperature control– Incubation is done at 37 ± 1°C. If temperature increases, the reduction time becomes shorter because bacterial activity is increased.
• Light exclusion– The tubes are kept away from direct light since light accelerates the reduction of dye.
• Inversion of tubes– When the sample reaches around 36°C (96.8°F), the tubes is gently inverted to allow uniform creaming. For better accuracy, the tubes is inverted after each observation so the bacteria do not accumulate in the top fat layer.

Factors Affecting Accuracy

• Oxygen content– The dissolved oxygen must be consumed first before decolorization begins. If milk contains more oxygen due to cooling, shaking or excess aeration during milking, the reduction time becomes longer.
• Dye concentration– A higher concentration of methylene blue increases the reduction time, so the dye strength must be uniform.
• Interfering substances– Presence of antibiotics, hydrogen peroxide, or natural inhibitors like lysozyme and lactoferrin can delay reduction. High somatic cell count in mastitic milk also affects the reaction.

Methylene Blue Reduction Test Procedure

Phase 1: Preparation and Reagent Setup

1. Ensure all apparatus is sterile. It is done by keeping the glassware and stoppers chemically clean and sterilized (autoclaving or boiling water).
2. The dye solution is prepared by using the standard 0.005% methylene blue solution. One certified tablet is dissolved in 200 ml hot distilled water and kept in a light-protected container.

Phase 2: Sample Preparation

3. Measure 10 ml milk sample and transfer it into a sterile MBRT test tube.
4. Add 1 ml standardized methylene blue dye solution to the milk.
5. Stopper the tube with a sterilized rubber stopper. The tube can be kept in a refrigerator (0–4°C) for a short time before incubation if required.

Phase 3: Incubation and Monitoring

6. Place the tubes in a stand and immerse them in a water bath kept at 37±1°C. The bath must warm cold samples to 35°C within 10 minutes.
7. When the sample temperature reaches about 36°C (96.8°F), the tubes is gently inverted for uniform mixing. This time is noted as the beginning of incubation.
8. The tubes is protected from light by covering the bath because light accelerates dye reduction.
9. After 30 minutes, the tubes is checked for loss of color. If decolorization occurs, the reading is noted as MBRT – 30 minutes.
10. Further readings are taken at hourly intervals. After each observation, the tubes that still show color is gently inverted so the bacteria do not collect in the fat layer.

Phase 4: Endpoint Determination and Recording

11. The end point is reached when the main milk portion becomes colorless and only a faint blue ring (about 5 mm) remains near the top, or when four-fifths of the blue color disappears.
12. The final reduction time is recorded in whole hours between the last inversion and the time the sample becomes colorless. Faster decolorization indicates poorer microbial quality of the milk.

Interpretation and Result of Methylene Blue Reduction Test

I. Interpretation of Reduction Time and Microbial Load

1. The test is based on an inverse relation between reduction time and microbial activity. When the blue color disappears quickly, it means the microbial load is high and the milk quality becomes poor.
2. The dye is reduced because microorganisms consume oxygen and release reducing substances by dehydrogenase enzymes. It is the metabolic rate that is measured, not the exact number of bacterial cells.
3. The end point is taken when the main body of milk becomes colorless and only a faint blue ring (about 5 mm) is seen at the top, or when about four-fifths of the blue color has vanished. The reduction time is written in whole hours from the last inversion to the moment of decolorization.

II. Correlation with Standard Plate Count (SPC)

– There is a strong relationship between MBRT time and bacterial count obtained by SPC.
– A high coefficient of determination (r² ≈ 0.91) is reported between MBRT duration and log CFU/ml.
– Because of this correlation, MBRT is used as an economical alternative to costly plate count methods.

III. Standard Grading System

Some of the common grades based on reduction time are–

• Very Good / Excellent
  Reduction time: not decolorized in 8 hours or more, or 5 hours and above.
  Basis: very low microbial load.
• Good
  Reduction time: less than 8 hours but not less than 6 hours (or 6½–7½ hours), or 3–4 hours.
  Basis: low contamination risk.
• Fair / Medium
  Reduction time: less than 6 hours but not less than 2 hours (or 2½–6 hours), or 1–2 hours.
  Basis: marginal quality but usable for processing.
• Poor
  Reduction time: less than 2 hours or less than ½ hour.
  Basis: high microbial load; milk should not be used unless cooked.

A very rapid decolorization within 30 minutes is taken as very poor and unfavorable quality.

IV. Interpretation of Extended MBRT (MBRTe)

The extended test uses 24-hour incubation and classifies milk by clot formation type.

• MBRTe–I (Homogeneous solid or liquid clot)
  Quality: good.
  Correlation: about 94% samples show first quality (SCC < 310,000 SC/ml and TBC < 300,000 CFU/ml).
• MBRTe–II (Lumpy heterogeneous clot)
  Quality: intermediate to good.
  Correlation: 94% in first-quality TBC range but SCC above limit, suggesting preclinical or clinical mastitis.
• MBRTe–III (Gaseous heterogeneous clot)
  Quality: poor to intermediate.
  Correlation: acceptable SCC but TBC above limit, showing poor hygiene and possible coliform contamination.
• MBRTe–IV (Lumpy + gaseous clot)
  Quality: poor.
  Correlation: both TBC and SCC exceed limits, indicating mastitis and poor management. Consumption not advised.

V. Factors Affecting Interpretation and Accuracy

• Type of organisms
  Coliforms reduce dye very fast. Lactococcus lactis is also rapid. Psychrotrophs reduce dye very slowly or sometimes not at all, causing overestimation of quality in cold-stored milk.
• Somatic cells and mastitis
  High SCC adds endogenous reductase activity which may reduce dye, giving a false-positive fast reduction.
• Chemical inhibitors
  Antibiotics, hydrogen peroxide and natural inhibitors can give incorrect readings.
• Physical factors
  Increased dissolved oxygen from pouring, cooling or aeration delays reduction. Light accelerates reduction, so samples must be kept protected from light.

Grading of milk in MBR test

1. Principle of Grading

• The grading is based on the inverse relation between the reduction time and the microbial quality of milk.
• When the dye becomes colorless very fast, it shows high metabolic activity of microorganisms and the milk becomes poor in quality.
• The reduction time is taken as the main basis for acceptance or rejection at dairy collection points.

2. Grading Based on BIS 1479 (Part 3): 1977

• Very Good
  Reduction time: 5 hours and above.
• Good
  Reduction time: 3 to 4 hours.
• Fair
  Reduction time: 1 to 2 hours.
• Poor
  Reduction time: less than ½ hour.
  Milk decolorizing within 30 minutes is considered very poor and unfavorable.

3. General / Best Practice Grading (8-Hour Standard)

• Excellent
  Reduction time: over 8 hours.
  Basis: very low contamination risk.
• Good (High Quality)
  Reduction time: 6 to 8 hours (or 6½–7½ hours).
• Fair (Medium Quality)
  Reduction time: 2 to 6 hours (or 2½–6 hours).
  Basis: moderate risk; common in properly collected raw milk.
• Poor
  Reduction time: less than 2 hours.
  Basis: high microbial load and should be heated before use.

4. Basis for Interpretation

• The grading is supported by strong correlation between MBRT reduction time and microbial load counted as CFU/ml.
• A coefficient of determination (r² around 0.91) is reported, which shows MBRT can act as an economical substitute to plate count test.

5. Extended MBRT (MBRTe) Classification

This method uses 24-hour incubation at 37°C and the grading depends on clot characteristics.

• MBRTe–I
  Clot: homogeneous solid or liquid; slight acidic odor.
  Quality: good.
  Implication: about 95% show first-quality TBC and SCC.
• MBRTe–II
  Clot: lumpy heterogeneous, abnormal coloration.
  Quality: intermediate to good.
  Implication: TBC in first-quality range but SCC exceeds limit, showing mastitis tendencies.
• MBRTe–III
  Clot: gaseous heterogeneous with bubbles and grooves.
  Quality: poor to intermediate.
  Implication: SCC acceptable but TBC high, often due to coliform contamination.
• MBRTe–IV
  Clot: lumpy plus gaseous characteristics.
  Quality: poor.
  Implication: both TBC and SCC exceed limits, indicating severe mastitis and management problems.

Factors Affecting on MBR test

1. Microbial Load (TBC)– It is the major factor because the reduction time is inversely related with the number of bacteria. When the bacteria are more, the dye is reduced very fast.
2. Type of Microorganisms– Some organisms reduce the dye very quickly. Coliforms are the most rapid reducers and followed by Lactococcus lactis ssp. lactis and some Streptococci.
3. Psychrotrophic Bacteria– These organisms (like Pseudomonas species) is not active at 37°C. So the dye reduction is very slow and the milk may look good although spoilage has already started.
4. Dye Concentration– If the methylene blue concentration is increased then more time is needed for the complete reduction, so the reduction time becomes longer.
5. Antimicrobial Residues– Antibiotics (penicillin) and hydrogen peroxide inhibit the growth of bacteria. This process results in increased reduction time and the milk may appear of good quality when it is not.
6. Natural Inhibitors in Milk– Milk contains some enzymes and antibacterial substances. These are contributing to rapid reduction even when bacterial load is low. High somatic cell count (SCC) in mastitic milk also affects the reduction due to presence of enzymes like xanthine oxidase.
7. Oxygen Content of Milk– The oxygen must be consumed first before dye reduction start. Cold milk holds more dissolved oxygen and pouring milk between containers increases oxygen. High oxygen makes the reduction time longer.
8. Temperature of Incubation– It is carried out at 37±1°C. When the temperature is higher the bacterial activity increases and reduction time is shortened.
9. Light Exposure– Light helps in faster reduction of methylene blue. Tests are carried out in dim light or kept covered.
10. Creaming Effect– Fat globules rise and carry microorganisms to the top. It creates uneven distribution. Inverting the tube during incubation gives more accurate reduction time.
11. Heat Treatment of Sample– Some samples are heated at 82°C for 5 minutes to destroy natural inhibitors. It helps to avoid false positive rapid reduction that is not due to microbial load.

Advantage of Methylene Blue Reduction Test

• It is a simple and low-cost method. The test needs very few materials and simple glass tubes. It is economical for routine milk quality checking and the procedure is easy to perform even in small dairy units.
• It requires minimum equipment A thermostatic water bath, methylene blue solution and general laboratory tubes is enough for carrying out the test.
• It is useful for rapid screening of milk. The reduction time gives a quick idea about the microbiological quality of raw or pasteurized milk, so it is used at milk chilling centers and receiving docks for acceptance or rejection of milk.
• It acts as an alternative to long microbiological tests. The test can be used in place of Standard Plate Count because it is much faster and less labor-intensive.
• It correlates well with microbial load. Reduction time shows good relation with bacterial count. It reflects the metabolic activity of bacteria present in the sample.
• It measures bacterial activity. In this test the dye is reduced by the combined activity of bacteria, so it detects the active microorganisms instead of only counting colony forming units.
• It is suitable for field conditions. The test can be performed in rural milk collection centers where advanced facilities are not available.
• Extended MBRT (MBRTe) helps in herd health assessment. The extended type shows relation with total bacterial count and somatic cell count, so it helps in identifying mastitic milk.
• It helps to detect management problems. Different clot types in MBRTe indicate high somatic cells or poor milking, handling, and storage conditions.
• It supports improvement of milk quality. Milk from individual farmers can be tested so that the producers not following good practices can be identified, and proper corrective measures can be taken.

Limitations of Methylene Blue Reduction Test

• It is an indirect qualitative test. The test measures the metabolic activity of microorganisms only. It cannot give the actual bacterial number because the dye reduction depends on oxygen removal and electron release.
• Lack of precision in detailed analysis. Although it shows correlation with bacterial count, the results sometimes do not match the precision needed for accurate quality assessment of milk.
• Long time needed for good-quality samples. Highly contaminated milk reduces the dye fast, but excellent-grade milk requires 5–8 hours or more, so continuous observation is needed.
• It cannot detect psychrotrophic bacteria properly. Psychrotrophs like Pseudomonas species grow at low temperature and reduce the dye very slowly at 37°C, so spoiled cold-stored milk may appear of good quality.
• It may overestimate the quality of refrigerated milk. This test is not suitable when psychrotrophic load is high because the reduction time becomes misleading and the milk appears acceptable.
• It is not suitable for low-count pasteurized milk. Pasteurized milk contains very few organisms, so dye reduction is extremely slow and the test becomes unreliable.
• Inhibitory substances give false good results. Antibiotics and hydrogen peroxide inhibit bacterial growth. The reduction time is increased and poor milk may appear to be good.
• Natural inhibitors give false positive rapid reduction. Some components of milk reduce the dye without microbial activity. High somatic cell count in mastitic milk or enzymes like xanthine oxidase cause quick decolorization.
• Heat treatment is sometimes needed to remove natural enzyme interference. Heating at around 82°C for few minutes is used to inactivate natural reductases, which otherwise cause rapid reduction.
• Oxygen content affects the reduction time. Cold milk holds more oxygen and pouring milk into containers introduces more dissolved oxygen. High oxygen delays reduction.
• Creaming results in uneven bacterial distribution. Fat globules rise and carry microorganisms upward. This makes the reduction time inconsistent unless the tube is inverted periodically.
• Light and temperature variations influence the reaction. Light speeds up the reduction, while change in incubation temperature alters microbial activity and reduction time.
• Dye concentration must be uniform. If dye concentration is high the reduction takes longer. Small variations produce different results.
• It cannot identify the type of microorganisms present. The test only shows the reduction time. It cannot differentiate between spoilage, pathogenic, or harmless bacteria.
• Different organisms reduce dye at different rates. Coliforms reduce the dye very fast but psychrotrophs are slow, so the reduction time varies according to species present.
• It has been replaced by more accurate modern methods. Flow cytometry and PCR-based tests are now used as they give quick and precise information about microbial load and specific contaminants.

Uses

• It is used for assessing the microbiological quality of milk. The reduction time gives an idea of the level of microbial contamination. When the dye disappears quickly the milk is of poor quality, and when the color remains for long the milk is of better quality.
• It helps in quick screening of raw and pasteurized milk. The test is used as a rapid indirect method to evaluate the milk at different stages of handling. The reduction time shows the metabolic activity of viable bacteria..It is used as an acceptance or rejection test at dairy units
• Cooling centers, reception docks and factories use this method to decide whether the milk should be accepted for processing or rejected. It is suitable for on-site screening in remote areas
• The test can be carried out easily where laboratory facilities are not available, especially in small farmer associations.
• It is used for grading of milk. The milk is divided into different grades based on the reduction time. Very Good, Good, Fair or Poor quality is given depending on the hours taken for decolorization.
• It is useful for grading of cream also. The method was used earlier for sweet cream and found to be a satisfactory index of the bacterial content.
• It helps in monitoring hygiene and handling practices. The reduction time reflects the milking hygiene, storage conditions and cooling practice on the farm. Poor sanitation results in fast reduction.
• It is used in improvement programs of milk quality. Dairy farms use this test to find out faults in milking and cleaning practices so that the overall quality of milk or cream can be improved.
• It acts as an economical substitute for laboratory tests. Due to its strong correlation with Standard Plate Count it can be used as an alternative screening method where costly tests are not possible.
• It is used in research for estimating metabolically active biomass. In some studies the test was optimized to show rapid reduction and gave correlation with colony forming units of aerobic organisms.
• Extended MBRT (MBRTe) is used for detecting subclinical mastitis. The 24-hour extended test correlates with somatic cell count and bacterial count. It helps in identifying mastitic milk in dairy herds.
• It helps in identifying clot types for quality management. The clot forms after the extended test (MBRTe-I to MBRTe-IV) indicate the hygienic condition, possible industrial uses and expected market value.
• It is useful for detecting individual problematic producers. Milk from different farmers can be tested to identify those with mastitis issues or poor handling practices so that corrective steps can be taken.

What biochemical action causes methylene blue decolorization?

It is the reduction of the dye caused by metabolically active bacteria present in the milk. In raw milk, the dissolved oxygen keeps methylene blue in its oxidized blue form. When bacterial cells begin to respire, the oxygen is rapidly utilized and the redox potential of the milk starts to fall. It is at this stage that the dye becomes sensitive to reduction.

In this process, microbial dehydrogenase enzymes are active on the cell surface or inside the membrane. These enzymes release electrons and hydrogen ions during the breakdown of lactose. The released reducing substances convert the oxidized methylene blue into its colorless reduced form. This reduced form is referred to as leuco–methylene blue (LMB). It is the formation of this compound that causes the loss of blue colour in the test.

In this step, methylene blue acts as an artificial electron acceptor. When the redox potential decreases to a low level, the dye accepts electrons coming from the dehydrogenase system of bacteria. This reaction is the main reason for the decolorization. The higher the metabolic activity of bacteria, the faster the dye is reduced.

Some natural enzymes in milk such as xanthine oxidase can also reduce the dye. These enzymes consume oxygen and produce reducing substances even without bacterial contamination, so this may create a false-positive result.

Thus, the decolorization is the result of a reduction reaction where the dye is converted into its leuco form due to oxygen removal and the production of reducing compounds by active bacteria.

What natural milk enzyme causes false-positive MBRT results?

In raw milk, the enzyme that is mainly responsible for giving false-positive results in the Methylene Blue Reduction Test is Xanthine Oxidase (XO). It is the enzyme that has natural reductase activity, and it is capable of reducing the dye even when the bacterial count is not high.

Xanthine oxidase is present in fresh bovine milk, and it is converted from its dehydrogenase form. It is the process where the enzyme consumes oxygen from the milk and forms reducing substances. These reducing substances act on the methylene blue dye. It is reduced to the colorless leuco–methylene blue form, so the test shows faster decolorization. This is referred to as a false-positive result because it is not due to microbial metabolism.

The activity of this enzyme is also supported by the lactoperoxidase system (LPO–SCN⁻). These components are part of the natural immune factors of milk. They also help in lowering the oxygen and increasing the reducing action. It is in this condition that the dye becomes reduced very quickly.

Some of the milk samples that have high somatic cell count (SCC) show faster reduction of the dye. These cells contain enzymes and other reducing materials that enhance the reduction rate.

This enzyme is heat-labile, and it is inactivated by heating the milk about 82°C for 5 minutes. When the rapid reduction disappears after heating, it shows that the earlier result was caused by natural reductase enzymes like xanthine oxidase.

FAQ on MBR test