Hostname: page-component-586b7cd67f-rdxmf Total loading time: 0 Render date: 2024-11-27T19:06:55.720Z Has data issue: false hasContentIssue false
  • English
  • Français

Flow Cytometric Viability Assessment and Transmission Electron Microscopic Morphological Study of Bacteria in Glycerol

Published online by Cambridge University Press:  18 January 2007

Veroniek S.M. Saegeman ,
Rita De Vos ,
Nilvanira D. Tebaldi ,
Jan M. van der Wolf ,
Jan H.W. Bergervoet ,
Jan Verhaegen ,
Daniel Lismont ,
Bert Verduyckt  and
Nadine L. Ectors
Veroniek S.M. Saegeman
Affiliation:
Department of Microbiology, University Hospitals Leuven, Leuven, B-3000, Belgium Tissue Banks, University Hospitals Leuven, Leuven, B-3000, Belgium
Rita De Vos
Affiliation:
Department of Pathology, University Hospitals Leuven, Leuven, B-3000, Belgium
Nilvanira D. Tebaldi
Affiliation:
Plant Research International, 6708 PD Wageningen, The Netherlands
Jan M. van der Wolf
Affiliation:
Plant Research International, 6708 PD Wageningen, The Netherlands
Jan H.W. Bergervoet
Affiliation:
Plant Research International, 6708 PD Wageningen, The Netherlands
Jan Verhaegen
Affiliation:
Department of Microbiology, University Hospitals Leuven, Leuven, B-3000, Belgium
Daniel Lismont
Affiliation:
Tissue Banks, University Hospitals Leuven, Leuven, B-3000, Belgium
Bert Verduyckt
Affiliation:
Tissue Banks, University Hospitals Leuven, Leuven, B-3000, Belgium
Nadine L. Ectors
Affiliation:
Tissue Banks, University Hospitals Leuven, Leuven, B-3000, Belgium Department of Pathology, University Hospitals Leuven, Leuven, B-3000, Belgium
Get access

Abstract

Human cadaveric skin allografts are used in the treatment of burns and can be preserved in glycerol at high concentrations. Previously, glycerol has been attributed some antimicrobial effect. In an experimental set-up, we aimed at investigating this effect of prolonged incubation of bacteria in 85% glycerol. Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis were incubated in 85% glycerol. The influence of duration of incubation and temperature on ultrastructure and viability were investigated. Unstressed cultures served as controls. Survival was studied after 24–36 h and 10 days incubation in 85% glycerol at 4°C and 36°C with transmission electron microscopy (TEM) and flow cytometry using viability stains indicating membrane damage (SYTO9, propidium iodide) or esterase activity (carboxyfluorescein diacetate). TEM clearly demonstrated variability in morphological changes of bacteria suggesting different mechanisms of damage. Viability stains supported these findings with faster declining viable cell populations in 85% glycerol at 36°C compared with 4°C. Both methods demonstrated that Gram-negative species were more susceptible than Gram-positive species. In conclusion, 85% glycerol may have some additional antimicrobial effect. Temperature is an important factor herein and Gram-negatives are most susceptible. The latter finding probably reflects the difference in cell wall composition between Gram-positive and Gram-negative bacteria.

Keywords

antimicrobialflow cytometryglycerolmembrane damageskin allografttransmission electron microscopyviability stain
Type
Research Article
Information
Microscopy and Microanalysis , Volume 13 , Issue 1: Special Issue: Environmental, Industrial, and AppliedMicrobiology , February 2007 , pp. 18 - 29
Copyright
2007 Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Ballesteros, A., Chrife, J. & Bozzini, J.P. (1993). Specific solute effects on Staphylococuus aureus cells subjected to reduced water activity. Int J Food Microbiol 20, 51–66.Google Scholar
Beney, L., Marechal, P.A. & Gervais, P. (2001). Coupling effects of osmotic pressure and temperature on the viability of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 56, 513–516.Google Scholar
Bunthof, C., Bloemen, K., Breeuwer, P., Rombouts, F. & Abee, T. (2001). Flow cytometric assessment of viability of lactic acid bacteria. Appl Environ Microbiol 67, 2326–2335.Google Scholar
Davey, H. & Kell, D. (1996). Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single-cell analyses. Microbiol Rev 60, 641–696.Google Scholar
Fuller, M., Streger, S., Rothmel, R., Mailloux, B., Hall, J., Onstott, T., Frederickson, J., Balkwill, D. & De Flaun, M. (2000). Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments. Appl Environ Microbiol 66, 4486–4496.Google Scholar
Haugland, R.P. (2004). Viability and Cytotoxicity Assay Reagents and LIVE/DEAD® BacLight™ Bacterial Viability Kits L7007 and L7012 Product Information. In Molecular Probes Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes Inc., Ch. 15, section 15.2. Eugene, OR: Invitrogen Detection Technologies. URL: http://www.probes.com/.
Hoefel, D., Warwick, L., Grooby, W.L., Monis, P.T., Andrews, S. & Saint, C.P. (2003). Enumeration of water-borne bacteria using viability assays and flow cytometry: A comparison to culture-based techniques. J Microbiol Methods 55, 585–597.Google Scholar
Kearney, J.N. (1998). Quality issues in skin banking: A review. Burns 24, 299–305.Google Scholar
Lambert, J. & Maillard, P.A. (2004). Mechanisms of action of biocides. In Principles and Practice of Disinfection, Preservation and Sterilization, Russell, H., Ayliffe, W.B. & Fraise, A.P. (Eds.), pp. 139–154. Oxford: Blackwell Publishers.
Lin, E.C.C. (1976). Glycerol dissimilation and its regulation in bacteria. Ann Rev Microbiol 30, 535–578.Google Scholar
Lomas, R.J., Huang, Q., Pegg, D.E. & Kearney, J.N. (2004). Application of a high-level peracetic acid disinfection protocol to re-process antibiotic disinfected skin allografts. Cell Tiss Bank 5, 23–26.Google Scholar
Lopez-Amoros, R., Comas, J. & Vives-Rego, J. (1995). Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol. Appl Environ Microbiol 61, 2521–2526.Google Scholar
Madigan, M.T., Martinko, J.M. & Parker, J. (1991). Effect of temperature on growth and osmotic effects on microbial growth. In Brock Biology of Micro-organisms, Snavely, S.L. (Ed.), pp. 151–159. Upper Saddle River, NJ: Pearson Education International, Prentice Hall.
Marshall, L., Ghosh, M.M., Boyce, S.G., Macneil, S., Freedlander, E. & Kudesia, G. (1995). Effect of glycerol on intracellular virus survival: Implications for the clinical use of glycerol-preserved cadaver skin. Burns 21, 356–361.Google Scholar
McDonnell, G. & Russell, A.D. (1999). Antiseptics and disinfectants: Activity, action, and resistance. Clin Microbiol Rev 12, 147–179.Google Scholar
Mille, Y., Beney, L. & Gervais, P. (2002). Viability of Escherichia coli after combined osmotic and thermal treatment: A plasma membrane implication. Biochim Biophys Acta 1567, 41–48.Google Scholar
Morono, Y., Takano, S., Miyanaga, K., Tanji, Y., Unno, H. & Hori, K. (2004). Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate. Biotechnol Lett 26, 379–383.Google Scholar
Pflug, I.J., Holcomb, R.G. & Gomez, M.M. (2000). Principles of the thermal destruction of microorganisms. In Disinfection, Sterilization, and Preservation, Block S.S. (Ed.), pp. 97–100. Philadelphia: Lippincott Williams and Wilkins.
Porter, J., Deere, D., Hardman, M., Edwards, C. & Pickup, R. (1997). Go with the flow—Use of flow cytometry in environmental microbiology. FEMS Microbiol Ecol 24, 93–101.Google Scholar
Potts, M. (1994). Desiccation tolerance of prokaryotes. Microbiol Rev 58, 755–805.Google Scholar
Rogers, H.J. (1983). Aspects of Microbiology (6): Bacterial Cell Structure. Wokingham, UK: Van Nostrand Reinhold.
Schmid, I., Ferbas, J., Uittenbogaart, C.H. & Giorgi, J.V. (1999). Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability. Cytometry 35, 64–74.Google Scholar
Van Baare, J., Buitenwerf, J., Hoekstra, M.J. & Dupont, J.S. (1994). Virucidal effect of glycerol as used in donor skin preservation. Burns 20, S77–S80.Google Scholar
Van Baare, J., Ligtvoet, E.J. & Middlekoop, E. (1998). Microbiological evaluation of glycerolised cadavaric donor skin. Transplant 65, 966–970.Google Scholar
Van Der Wolf, J.M. & Van Beckhoven, J.R.C.M. (2004). Factors affecting survival of clavibacter michiganensis subsp. sepedonicus in water. J Phytopathol 152, 161–168.Google Scholar
Vives-Rego, J., Lebaron, P. & Nebe-Von Caron, G. (2000). Current and future applications of flow cytometry in aquatic microbiology. FEMS Microbiol Rev 24, 429–448.Google Scholar
Wood, J. (1999). Osmosensing by bacteria: Signals and membrane-based sensors. Microbiol Mol Biol Rev 63, 230–262.Google Scholar