Comparison of Two Transmission Electron Microscopy Methods to Visualize Drug-Induced Alterations of Gram-Negative Bacterial Morphology
Nguyen Thi Thu Hang, Lisa A. O’Donovan, Henrietta Venter, Cecilia C. Russell, Adam McCluskey, Stephen W. Page, Darren J. Trott, Abiodun D. Ogunniyi
Abstract
In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.