A modification of Mover's method has been found satisfactory for the quantitative measurement of resazurin reduction in biological systems. For convenience, the reducing systems were studied separately and the contribution of each system to total dye reduction was determined. The rapid reduction of resazurin in milk due to sun-light was found to be a photochemical reaction initiated by the excitation of the dye by light of a wavelength of approximately 600 mμ. It was found that the main

more »

... system in normal raw milk was ascorbic acid dependent, whereas in mastitic milk the main reducing system was leucocytes. Physical fractionation of mastitic milk, sonic treatment, and the use of metabolic inhibitors strongly indicated that there was an intimate relationship between the presence of leucocytes and the reducing activities of mastitic milk. Studies with cell-free extracts of leucocytes and bacteria showed that the reducing systems were essentially the same, and involved reduced pyridine nucleotides as electron donors. The mechanisms of resazurin reduction have been confirmed by studies involving the use of metabolic inhibitors, respirometry, variation of the energy source and kinetic studies. Finally, the successful reconstruction of several artificial resazurin reducing systems further confirmed the validity of this mechanism.