Flow cytometric viability assessment and transmission electron microscopic morphological study of Bacteria in Glycerol

V.S.M. Saegeman, R. de Vos, N.D. Tebaldi, J.M. van der Wolf, J.H.W. Bergervoet, J. Verhaegen, D. Lismont, B. Verduyckt, N.L. Ectors

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    14 Citations (Scopus)

    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.
    Original languageEnglish
    Pages (from-to)18-29
    JournalMicroscopy and Microanalysis
    Volume13
    Issue number1
    DOIs
    Publication statusPublished - 2007

    Keywords

    • escherichia-coli
    • donor skin
    • survival
    • water
    • microbiology
    • populations
    • cells

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