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Mechanism of Resazurin Reduction in Milk of Low Bacterial Content

Division of Animal Science, Dairying and Animal Husbandry, The University of British Columbia, Vancouver, Canada

ABSTRACT

An accurate quantitative method of determining resazurin reduction has been developed. The method depends on the extraction of the dye with n-butanol and the spectrophotometric estimation of the oxidized and reduced forms. The reducing capacity of milk was assessed by adding an excess 2,6-dichlorophenolindophenol having an E¹_o within the Eh range of fresh milk and determining the amount converted to its reduced form. In individual samples of milk the rate of resazurin reduction depended on the concentration of ascorbic acid and the reduction rate decreased with time as the ascorbate concentration decreased. However, milk from different sources differed widely in reduction rates at the same constant ascorbate concentrations. This observation, along with the decreased rate of reduction on dilution of milk, indicated that some factor in milk in addition to ascorbate acted to reduce resazurin. It is visualized that zones of high hydrogen ion activity occur at the surface of bacteria, leucocytes, and fat globules. Resazurin diffusing into these zones in the presence of ascorbate becomes reduced and remains reduced on diffusion back into the milk plasma. Thus, the rate of reduction would depend on the number and pH of the zones and the ambient ascorbate concentration. It was concluded that the zones acted by virtue of their pH and not their Eh.