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 48C and 368C with transmission electron microscopy~TEM! and flow cytometry using viability stains indicating membrane damage~SYTO9, propidium iodide! or esterase activity~carboxyfluores-cein 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 368C compared with 48C. 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.

Glycerol and the skin: holistic approach to its origin and functions

“…Glycerol possesses a keratolytic effect (revealed by desmosome digestion), 3 anti‐irritant (AI) properties, 4,5 plasticizes SC, 6 reduces light scattering in skin, 7 and accelerates the process of wound healing 8,9 . Among its diverse influences on the skin, even an antimicrobial effect was described 10 . Recent studies showed that endogenous glycerol maintains skin physiological and biochemical properties.…”

“…These findings were correlated to morphological changes of the bacteria, suggesting different damaging mechanisms. Moreover, Gram‐negative species were more susceptible than the Gram‐positives, probably due to the difference in the bacterial wall structure between the groups 10 …”

“…Moreover, Gram-negative species were more susceptible than the Gram-positives, probably due to the difference in the bacterial wall structure between the groups. 10 It was demonstrated that glycerol possesses a virucidal effect. 84 The virucidal action was tested against different pathogenic agents (i.e.…”

“…8,9 Among its diverse influences on the skin, even an antimicrobial effect was described. 10 Recent studies showed that endogenous glycerol maintains skin physiological and biochemical properties. This review summarizes the accumulated knowledge on the biological and biophysical effects of glycerol on the skin.…”

Pharmacological Targeting of the Epidermal Barrier

“…Barrier damage leads to changes in skin flora [87]; therefore, patients with irritant or atopic dermatitis may need protection against bacterial colonization. Glycerol in 85% concentration was found to have antimicrobial effects [88]. Additionally, glycerol possesses virucidal properties [89].…”