Contributions to Investigation of Oral Biofilms Using Low-Vacuum Scanning Electron Microscopy

摘要

A number of 8 periodontally-involved extracted teeth and 3 titanium implants terminally affected by periimplantitis were evaluated of the morphology of the biofilm of the periodontal pockets with a modified protocol involving a simplified histologic sample preparation and a low-vacuum SEM examination method. The samples were carefully washed with saline solution, underwent fixation in modified Karnovsky solution (2.5% glutaraldehyde, 4% paraformaldehyde, 0.1M sodium cacodilate, pH7.2-7.4) and were dehydrated in alcohol series (70%, 95%, 100%). The samples were immediately examined under the low-vacuum SEM (INSPECT S, FEI, Hillsboro, OR, USA), at 80-250 Pa and 15kV. The low-vacuum inspection resulted in electrical charge-free images of the non-conductive or heavily-contaminated biofilm-covered surfaces. The low-vacuum SEM method avoids the sputtering of samples, the destruction of the biologic samples and the electrostatic charges. The low-vacuum SEM is a simple and cost-saving method, resulting in excellent quality biofilm images in contradistinction to conventional SEM that requires a complicate procedure, consisting in fixation of samples in glutaraldehyde and/or osmium tetroxide, followed by dehydration using either critical point drying with liquid C02 or an alcohol series, and coating (sputtering) of biofilm with conductive metallic material (Au, Pa or Carbon). The microorganisms described by the SEM investigation were mostly coccoid forms, followed by rare rods and filaments. No spirilar shapes and spirochetes were observed during investigation.