The methylene blue reduction test is a test commonly used to estimate the microbial load of a milk sample and as a result, examine the quality of the milk.
The principle of this test is that methylene blue is a dye which loses its colour in the absence of oxygen. Bacterial metabolism can cause the depletion of oxygen in the milk, therefore the methylene's reduction rate is connected to the presence of microorganisms in the milk.
As a consequence, if the tubes were not sealed tightly, then oxygen will enter the tube and the dye's colour will diminish, giving a false result concerning the presence of microorganisms in the sample.