USE OF DIGITIZED VIDEO MICROSCOPY WITH A FLUOROGENIC ENZYME-SUBSTRATETO DEMONSTRATE CELL-SPECIFIC AND COMPARTMENT-SPECIFIC GENE-EXPRESSIONIN SALMONELLA-ENTERITIDIS AND BACILLUS-SUBTILIS

A rapid and sensitive method for detection of cell- and compartment-sp ecific gene expression in individual cells of both Gram-negative and G ram-positive microorganisms is described. The method combines the use of gene fusions to lacZ, and a fluorogenic beta-galactosidase substrat e, fluorescein-di-(beta-D-galactopyranoside), with digitized video mic roscopy. All of the reporter constructs tested were successfully detec ted. Secondary staining of the cells with a nucleic acid-specific dye, propidium iodide, allowed cells devoid of nucleic acid to be identifi ed, while cell nucleoid shape and the morphological stage of developme nt could be correlated with the location of beta-galactosidase activit y. The double-staining procedure was used to show that gene expression can be induced in non-culturable cells of Salmonella enteritidis prod uced by carbon/nitrogen starvation. The resolution was sufficient to d istinguish between cells at different morphological stages of sporulat ion in Bacillus subtilis. This highly sensitive and rapid method may h ave many other applications in basic and applied microbiology.