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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
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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 Applied Microbiology , February 2007 , pp. 18 - 29
Copyright
2007 Microscopy Society of America

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