title: Flow cytometric viability assessment and transmission electron microscopic morphological study of bacteria in glycerol.
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 degrees C and 36 degrees 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 degrees C compared with 4 degrees 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.
authors.affiliation: Department of Microbiology, University Hospitals Leuven, Leuven, B-3000, Belgium.
authors.forename: Veroniek S M
authors.initials: VS
authors.lastname: Saegeman
authors.forename: Rita
authors.initials: R
authors.lastname: De Vos
authors.forename: Nilvanira D
authors.initials: ND
authors.lastname: Tebaldi
authors.forename: Jan M
authors.initials: JM
authors.lastname: van der Wolf
authors.forename: Jan H W
authors.initials: JH
authors.lastname: Bergervoet
authors.forename: Jan
authors.initials: J
authors.lastname: Verhaegen
authors.forename: Daniel
authors.initials: D
authors.lastname: Lismont
authors.forename: Bert
authors.initials: B
authors.lastname: Verduyckt
authors.forename: Nadine L
authors.initials: NL
authors.lastname: Ectors
mesh_terms: D001419:Bacteria / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005990:Glycerol / Q000494:pharmacology*; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000254:growth & development / Q000648:ultrastructure; D006094:Gram-Positive Bacteria / Q000187:drug effects / Q000254:growth & development / Q000648:ultrastructure; D050296:Microbial Viability / Q000187:drug effects*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000254:growth & development / Q000648:ultrastructure; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development / Q000648:ultrastructure; D013696:Temperature
chemical_list: D005990:Glycerol