Flow cytometry sorting of viable bacteria and yeasts according to β-galactosidase activity

Abstract

We describe a novel method for quantitative measurement of β-galactosidase (β-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 μm in diameter and analyzing the β-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-β-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of β-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting β-gal-positive cells from an artificial mixture of β-gal-positive and β-gal-negative E. coli strains.