A toluidine blue-membrane filter method for the quantitative staining of bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.