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. 1996 Nov;62(11):3939–3947. doi: 10.1128/aem.62.11.3939-3947.1996

Bacterial plasmolysis as a physical indicator of viability.

D R Korber 1, A Choi 1, G M Wolfaardt 1, D E Caldwell 1
PMCID: PMC168211  PMID: 8899980

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.

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Selected References

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  1. Bottomley P. J., Maggard S. P. Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Appl Environ Microbiol. 1990 Feb;56(2):533–540. doi: 10.1128/aem.56.2.533-540.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Geesey G. G., Morita R. Y. Relationship of cell envelope stability to substrate capture in a marine psychrophilic bacterium. Appl Environ Microbiol. 1981 Sep;42(3):533–540. doi: 10.1128/aem.42.3.533-540.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hua S. S., Tsai V. Y., Lichens G. M., Noma A. T. Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity. Appl Environ Microbiol. 1982 Jul;44(1):135–140. doi: 10.1128/aem.44.1.135-140.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Killham K., Firestone M. K. Salt stress control of intracellular solutes in streptomycetes indigenous to saline soils. Appl Environ Microbiol. 1984 Feb;47(2):301–306. doi: 10.1128/aem.47.2.301-306.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Koch A. L. Shrinkage of growing Escherichia coli cells by osmotic challenge. J Bacteriol. 1984 Sep;159(3):919–924. doi: 10.1128/jb.159.3.919-924.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Korber D. R., James G. A., Costerton J. W. Evaluation of Fleroxacin Activity against Established Pseudomonas fluorescens Biofilms. Appl Environ Microbiol. 1994 May;60(5):1663–1669. doi: 10.1128/aem.60.5.1663-1669.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kurath G., Morita R. Y. Starvation-Survival Physiological Studies of a Marine Pseudomonas sp. Appl Environ Microbiol. 1983 Apr;45(4):1206–1211. doi: 10.1128/aem.45.4.1206-1211.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lawrence J. R., Korber D. R., Caldwell D. E. Behavioral analysis of Vibrio parahaemolyticus variants in high- and low-viscosity microenvironments by use of digital image processing. J Bacteriol. 1992 Sep;174(17):5732–5739. doi: 10.1128/jb.174.17.5732-5739.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. MAGER J., KUCZYNSKI M., SCHATZBERG G., AVI-DOR Y. Turbidity changes in bacterial suspensions in relation to osmotic pressure. J Gen Microbiol. 1956 Feb;14(1):69–75. doi: 10.1099/00221287-14-1-69. [DOI] [PubMed] [Google Scholar]
  10. Marquis R. E. Salt-induced contraction of bacterial cell walls. J Bacteriol. 1968 Mar;95(3):775–781. doi: 10.1128/jb.95.3.775-781.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nix P. G., Daykin M. M. Resazurin reduction tests as an estimate of coliform and heterotrophic bacterial numbers in environmental samples. Bull Environ Contam Toxicol. 1992 Sep;49(3):354–360. doi: 10.1007/BF01239637. [DOI] [PubMed] [Google Scholar]
  12. POSTGATE J. R., CRUMPTON J. E., HUNTER J. R. The measurement of bacterial viabilities by slide culture. J Gen Microbiol. 1961 Jan;24:15–24. doi: 10.1099/00221287-24-1-15. [DOI] [PubMed] [Google Scholar]
  13. Rodriguez G. G., Phipps D., Ishiguro K., Ridgway H. F. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol. 1992 Jun;58(6):1801–1808. doi: 10.1128/aem.58.6.1801-1808.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Roszak D. B., Colwell R. R. Survival strategies of bacteria in the natural environment. Microbiol Rev. 1987 Sep;51(3):365–379. doi: 10.1128/mr.51.3.365-379.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schall B. F., Marathe G. V., Ghosh B. K. Stereological analysis of plasmolysis in logarithmic-phase Bacillus licheniformis. J Bacteriol. 1981 Apr;146(1):391–397. doi: 10.1128/jb.146.1.391-397.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Scheie P. O. Plasmolysis of Escherichia coli B-r with sucrose. J Bacteriol. 1969 May;98(2):335–340. doi: 10.1128/jb.98.2.335-340.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tabor P. S., Neihof R. A. Improved method for determination of respiring individual microorganisms in natural waters. Appl Environ Microbiol. 1982 Jun;43(6):1249–1255. doi: 10.1128/aem.43.6.1249-1255.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ward D. M., Weller R., Bateson M. M. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature. 1990 May 3;345(6270):63–65. doi: 10.1038/345063a0. [DOI] [PubMed] [Google Scholar]
  19. Yu F. P., McFeters G. A. Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes. J Microbiol Methods. 1994;20:1–10. doi: 10.1016/0167-7012(94)90058-2. [DOI] [PubMed] [Google Scholar]

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