TEM analysis was performed on specimens carried by two volunteers in order to visualize the ultrastructure of the biofilm. After removal of the splints, all the four different samples followed a rigorous protocol according to the methods in Nobre et al. [19 (link)]. Except that in the present experiment, the following additional procedure was performed only for titanium and ceramics samples: after polymerization, titanium and ceramics were removed by treatment with hydrofluoric acid (5%) during 48 h, and the specimens were re-embedded in Araldite. The enamel was decalcified due to exposure in 0.1M HCl for 4 h, and the specimens were re-embedded in Araldite.
As the final step, the specimens were cut in ultra-thin sections in an ultramicrotome with a diamond knife (Leica EM UC7, Germany) and mounted on Pioloform-coated copper grids and contrasted with aqueous solutions of uranyl acetate and lead citrate at room temperature. After intensive washing with distilled water, biofilms were analyzed with a TEM Tecnai 12 Biotwin (FEI, Eindhoven, The Netherlands) under a magnification up to 100,000-fold.
As the final step, the specimens were cut in ultra-thin sections in an ultramicrotome with a diamond knife (Leica EM UC7, Germany) and mounted on Pioloform-coated copper grids and contrasted with aqueous solutions of uranyl acetate and lead citrate at room temperature. After intensive washing with distilled water, biofilms were analyzed with a TEM Tecnai 12 Biotwin (FEI, Eindhoven, The Netherlands) under a magnification up to 100,000-fold.
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