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Methylene Blue Reductase Test




To check the quality of the given milk sample based on the difference in the microbial load milk sample provided.




Milk is a good medium for the growth of microorganism.  A variety of microorganism can be found in both raw milk and pasteurized milk. These actively growing microorganisms reduce the oxidation reduction potential of the milk medium due to the exhausted oxygen by the microorganism.  Normally the milk is contaminated with microorganisms such as Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeroginosa, Enterobacter spp., Bacillus spp., Paenibacillus 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. The formation of Methylene blue reductase is thus becoming a popular tool for determining the quality of the milk. 


The principle of methylene blue reduction test depends on the fact that the color imparted to the milk by adding a dye such as methylene blue will disappear more or less quickly, which depends on the quality of the milk sample to be examined. Methylene blue is a redox indicator, that lose its color under the absence of oxygen and is thought to be reduced. The depletion of oxygen in the milk is due to the production of reducing substances in the milk due to the enhanced rate of bacterial metabolism. The dye reduction time refers to the microbial load in the milk and the total metabolic reactions of the microorganism.


Indications of Milk spoilage


The milk contains energy sources such as lactose (sugar), nitrogenous compounds such as proteins, amino acids, ammonia, urea etc. for the growth of microorganism. Acid fermentation by the bacteria is common under ordinary conditions. Souring of milk indicates the milk is spoiled. Acid formation in the milk is indicated by the sour flavor, coagulation of milk to give a jelly like curd appearance or clear whey nature. Lactic acid fermentation is common in the raw milk at the room temperature. At temperature from 10 to 37 o C souring is mainly due to Streptococcus lactis, Enterococci, Lactobacilli and other coliform bacteria. At temperatures from 37 to 50 o C the most common contaminants of milk are S. faecalis and S. thermophilus. Thermophilic bacteria such as L. thermophilus can grow in the milk at higher temperatures. Pasteurization is an important process to kill most acid producing microorganisms while permitting the growth of heat resistant microorganisms such as Streptococcus thermophilus and Lactobacilli. Other acid producing microorganisms are Micrococcus species, Bacillus species (mainly lactic acid) and Clostridium species (mainly Butyric acid). Microorganisms such as Clostridium spp. and Bacillus spp. can also produce gases such as hydrogen and carbon dioxide which can be indicated by the formation of foaming at the top of the milk suspension. The other way of milk contamination is the hydrolysis of milk proteins by the growing microorganisms. The release of peptides in the milk leads to a bitter flavor to the milk. Ropiness, sliminess in the milk, is caused by the release of slimy capsular material from the cells. Enterobacter aerogenes, E. coli, Micrococcus freudenreichii are examples of microorganisms that can cause ropiness in the milk. Oxidation of unsaturated fatty acids can also lead to change in odor and taste of milk. Production of alkaline products such as ammonia, urea, carbonates etc can also produce off flavors to milk.

 Important points 


  • Disappearance of color with limited time indicates the absence of oxygen in milk.
  • Refrigerated milk contains more oxygen than warm milk. At the time of milking process, it has more oxygen content than the other cases.
  • Rate of reduction depends on the nature of the organism present in the milk. The rate of reduction by different microorganism is arranged in order below.


E.coli (coliforms) > Streptococcus lactis > faecal Streptococci > micrococci> Thermoduric organisms > psychrotrophic organisms

  • The presence of light fastens the reduction rate; hence the test tube under observation should be tightened properly.
  • Use uniform concentration of methylene blue dye in all test samples. Addition of more methylene blue dye will result in more reduction time.
  • Increased incubation time reduces the reduction time since the activity of some organism increases with increased incubation temperature.
  • Periodically invert the tubes at regular intervals during incubation time to improve the accuracy of the test result. Otherwise microorganisms may not be evenly distributed in the milk sample leading to wrong result interpretations.


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