Abstract
Bacterial plasmolytic response to osmotic stress was evaluated as a physical indicator of membrane integrity and hence cellular viability. Digital image analysis and either low-magnification dark-field, high-magnification phase-contrast, or confocal laser microscopy, in conjunction with pulse application of a 1.5 M NaCl solution, were used as a rapid, growth-independent method for quantifying the viability of attached biofilm bacteria. Bacteria were considered viable if they were capable of plasmolysis, as quantified by changes in cell area or light scattering. When viable Salmonella enteritidis biofilm cells were exposed to 1.5 M NaCl, an approximately 50% reduction in cell protoplast area (as determined by high-magnification phase-contrast microscopy) was observed. In contrast, heat- and formalin-killed S. enteritidis cells were unresponsive to NaCl treatment. Furthermore, the mean dark-field cell area of a viable, sessile population of Pseudomonas fluorescens cells (approximately 1,100 cells) increased by 50% as a result of salt stress, from 1,035 +/- 162 to 1,588 +/- 284 microns2, because of increased light scattering of the condensed, plasmolyzed cell protoplast. Light scattering of ethanol-killed control biofilm cells underwent little change following salt stress. When the results obtained with scanning confocal laser microscopy and a fluorescent viability probe were compared with the accuracy of plasmolysis as a viability indicator, it was found that the two methods were in close agreement. Used alone or in conjunction with fluorochemical probes, physical indicators of membrane integrity provided a rapid, direct, growth-independent method for determining the viability of biofilm bacteria known to undergo plasmolysis, and this method may have value during efficacy testing of biocides and other antimicrobial agents when nondestructive time course analyses are required.