REDUCTASE TEST USING METHYLENE BLUE

Objective

Checking for microbial growth in a given sample for example checking the quality of milk sample.

Principle

Milk that contains large numbers of actively growing bacteria will have a lowered oxidation-reduction potential due to the exhaustion of dissolved oxygen by microorganisms.
The fact that methylene blue loses its color (becomes reduced) in such an environment is the basis for the reductase test.

Materials

Two (2) sterile test tubes with rubber stoppers for each student raw milk samples of low- and high-quality (samples A and B) water bath set at 35° C methylene blue (1:25,000) 10 ml pipettes 1 ml pipettes gummed labels
Apparatus:
⦁ 2 sterile test tubes with rubber stoppers for each student
⦁ Water-bath set at 35/or any other equipment that can provide constant temperature
Sample:
⦁ Raw milk
Reagents:
Stock solutions:
Working solutions:
⦁ Methylene Blue

Information on chemicals

https://pubchem.ncbi.nlm.nih.gov/compound/6099#section=Top

Procedure:

1. Attach gummed labels with your name and type of milk to two test tubes. Each student will test a good-quality as well as a poor-quality milk.
2. Using separate 10 ml pipettes for each type of milk, transfer 10 ml to each test tube. To the milk in the tubes add 1 ml of methylene blue with a 1 ml pipette. Insert rubber stoppers and gently invert three times to mix. Record your name and the time on the labels and place the tubes in the water bath, which is set at 35° C.
3. After 5 minutes’ incubation, remove the tubes from the bath and invert once to mix. This is the last time they should be mixed.
4. Carefully remove the tubes from the water bath 30 minutes later and every half hour until the end of the laboratory period. When at least four-fifths of the tube has turned white, the end point of reduction has taken place. Record this time on the Laboratory Report.

Results:

The classification of milk quality is as follows:
Class 1: Excellent, not decolorized in 8 hours.
Class 2: Good, decolorized in less than 8 hours, but not less than 6 hours.
Class 3: Fair, decolorized in less than 6 hours, but not less than 2 hours.
Class 4: Poor, decolorized in less than 2 hours.

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