Bacterial plasmolysis as a physical indicator of viability

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 ϳ50% 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 (ϳ1,100 cells) increased by 50% as a result of salt stress, from 1,035 ؎ 162 to 1,588 ؎ 284 m 2 , 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.

Salt Stress Tolerance of Methylotrophic Bacteria Methylophilus sp. and Methylobacterium sp. Isolated from Coal Mine Spoils

“…di ered in their preferred antioxidant for growth revival under deleterious salt. e intracellular level of solutes was increased to regulate the cellular osmotic balance, but in extreme extracellular salt led to damage the cell wall resulting in drastic leakage of solutes (Korber et al, 1996). Electrolyte and organic solute leakage were more prominent in 500 mM NaCl due to membrane instability and lipid peroxidation in the methylotrophs (Table I).…”

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

“…The lipid bilayer of the outer membrane is closely attached to the peptidoglycan through lipoproteins present in the inner leaflet, and non‐covalent interactions with other outer membrane proteins (Mulder and Woldringh, 1993; Schwarz and Koch, 1995; Korber et al ., 1996). The poles, however, are ‘free’ of peptidoglycan and can present a point of cell weakness.…”

Metabolic Rearrangements Causing Elevated Proline and Polyhydroxybutyrate Accumulation During the Osmotic Adaptation Response of Bacillus megaterium

“…Fluctuation in water potential is one of the typical issues with whom bacterial cells have to cope in both their natural environment, during drought and rainy periods, and in industrial bioreactors in which fed substrate, expected product and sideproduct concentrations are particularly high (Schweder et al, 1999). In the latter case, increased medium osmolarity generates a substantial water efflux from cells, which eventually leads to a reduction of overall bacterial performance through cell dehydration and related impaired metabolism and growth (Korber et al, 1996;Kempf and Bremer, 1998). As water is essential for almost every cellular process from gene translation to protein folding processing, bacteria must be able to restore the cellular water homeostasis in order to survive swiftly.…”