This document provides instructions for performing the methylene blue reductase test to determine the quality of milk samples. The test works by adding methylene blue dye to milk samples and observing how quickly the dye loses its color, indicating reduction by microorganisms present in the milk. Faster color loss corresponds to higher microbial load and poorer milk quality. The document outlines the materials, procedure, interpretation of results, and important points about factors that influence the reduction rate.

1. Microbiology Lab
Prepared by: Kazhaleh Mohammadi
Knowledge University
2022-2023
1
Methylene Blue Reductase Test

2. Methylene Blue Reductase Test
• Milk is a good medium for the growth of microorganisms. A variety of
microorganisms 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 the transmission of diseases from animals to humans.

3. • 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 the udder, and adjacent areas. Unsterilized dairy utensils such as
milking machines, and 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 milk.
Methylene Blue Reductase Test

4. • The principle of the 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 loses its color in 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.
Methylene Blue Reductase Test

5. Indications of Milk spoilage
• Milk contains energy sources such as lactose (sugar), and nitrogenous
compounds such as proteins, amino acids, ammonia, urea, etc. for the
growth of microorganisms. Acid fermentation by bacteria is common under
ordinary conditions.
• Lactic acid fermentation is common in raw milk at room temperature. At
temperatures from 10 to 37 oC souring is mainly due to Streptococcus
lactis, Enterococci, Lactobacilli, and other coliform bacteria. At
temperatures from 37 to 50 oC, the most common contaminants of milk are
S. faecalis and S. thermophilus. Thermophilic bacteria such as L.
thermophilus can grow in milk at higher temperatures. Pasteurization is an
important process to kill most acid-producing microorganisms.

6. Principle
• This reductase test is based on the oxidation-reduction activities of the
bacteria present in the milk sample.
• The indicator used in the reaction is methylene blue which is color sensitive
to oxygen concentration. The indicator is blue in the oxidized state and
leuco or white in the reduced conditions. The speed of color disappearance
of methylene blue is proportional to the microbial load in the milk sample.
The more the bacteria present the faster will be the reduction.

7. Materials Required
• Milk samples to be analyzed
• Screw-cap test tubes
• Test tube rack
• Pipettes(10 ml and 1ml)
• Water bath (37o C)
• Bunsen Burner
Screw-cap test tubes

8. Procedure
• Transfer 10 ml of each milk sample into an appropriately labeled test tube.
• Add 1 ml of redox indicator, methylene blue to each test tube containing
the milk sample.
• Tighten the test tube mouth with stoppers. Gently invert the tubes at
about four or five times to ensure proper mixing of the methylene blue
solution.
• Keep the tubes in the water bath at 37 o C
• Note the incubation time. That is the time elapsed for the color to turn a
whitish appearance.
• Stabilize the tubes for 5 minutes.

9. Result Interpretation
• Reduction within 30 minutes: Very poor quality
• Reduction occurring between 30 minutes and 2
hours : Poor quality
• Reduction occurring between 2 and 6 hours: Fair
quality
• Reduction occurring between 6 and 8 hours:
Good quality
• Not reduced in 8 hours: Excellent quality

10. Important points
• The 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 the
milking process, it has more oxygen content than the other cases.
• The rate of reduction depends on the nature of the organism present in the milk.
The rate of reduction by different microorganisms is arranged in the 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 a uniform concentration of methylene blue dye in all test samples. Addition
of more methylene blue dye will result in more reduction time.