Microanalysis of lactose in tissue culture medium using an enzymatic-fluorometric method

A fluoroenzymatic microdetermination of lactose in tissue culture medium is described. The method is based on the conversion of lactose to galactonic acid by the enzymes galactosidase and galactose dehydrogenase. The resultant reduction of NAD to NADH is measured fluorometrically. Before assaying for lactose the high background fluorescence of tissue culture medium is reduced by deproteinization and adsorption of fluorescent substances with either Florisil or dextran-charcoal. As little as 0.05 nmol of lactose in medium with or without phenol red can be detected after this treatment, whereas for untreated medium the limit of sensitivity of the assay is 1.0 nmol. This method should prove useful for the detection of lactose produced by small numbers of mammary epithelial cells in culture.