Visualizing Antimicrobial Effects on Bacterial Surfaces by SEM: A Comparative Study of Hexamethyldisilazane (HMDS) and Freeze-Drying.

Abstract

Scanning electron microscope (SEM) can observe bacterial morphology with nanoscale spatial resolution and is therefore used to investigate the action mechanisms of disinfectants. Since the structural changes that show antibacterial effects are extremely subtle, reliable specimen preparation is necessary to maintain and observe these traces. In this study, we demonstrated the fixation and drying conditions necessary to observe the effects of disinfectants on bacteria using SEM. In the double fixation of specimens, osmium tetroxide, which fixes lipids, did not appear to affect the SEM observation of the Escherichia coli surface, mainly composed of lipopolysaccharides, under our experimental conditions. Hexamethyldisilazane (HMDS), used to dry the specimens, retained a fine structure comparable to conventional freeze-drying methods without special equipment. Furthermore, shrinkage deformation due to drying was significantly suppressed in two of the three conditions tested compared to freeze-drying. When E. coli was treated with two types of disinfectant, benzalkonium chloride (BAC) and chlorhexidine gluconate (CHG), for 5 min, the number of viable bacteria fell below the detection limit. When E. coli collected from these bacterial suspensions was dried with HMDS, the two disinfectants roughened the bacterial surface. In some BAC-treated bacteria, membrane fusion was observed between adjacent bacteria. Pit structures were observed in the CHG-treated bacteria. These morphological changes, which indicate the effectiveness of the disinfectants, were consistent with the results of observations of the freeze-dried specimens used as a control test. These results suggest that chemical drying with HMDS can quickly and efficiently provide morphological information to understand how disinfectants cause deformation and affect bacterial function.