Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity.
AUTOR(ES)
Nir, R
RESUMO
We describe a novel method for quantitative measurement of beta-galactosidase (beta-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 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-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 beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=185080Documentos Relacionados
- Analysis of Escherichia coli beta-galactosidase expression in transgenic mice by flow cytometry of sperm.
- Concentrated beta-galactosidase and cell permeabilization from Saccharomyces fragilis IZ 275 for beta-galactosidase activity in the hydrolysis of lactose
- Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.
- Beta-galactosidase activity in single differentiating bacterial cells.
- Transgalactosylation activity of ebg beta-galactosidase synthesizes allolactose from lactose.