Differentiation of Viable and Dead Escherichia coli O157:H7 Cells on and in Apple Structures and Tissues following Chlorine Treatment

Abstract

Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution. Whole unwaxed Red Delicious cultivar apples at 25°C were inoculated by dipping in a suspension of E. coli O157:H7 (8.48 log10 CFU/ml) at 4°C, followed by treatment in water or chlorine solution at 21°C for 2 min. The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain. Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594). The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined. The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%). The location of viable cells within tissues was dependent on the structure. Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces. Further research is warranted to investigate the efficacy of other chemical sanitizers, as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E. coli O157:H7 lodged in protective structures on the surface and within tissues of apples.