Bacterial envelope profiles revealed by freeze-substitution

Abstract

Traditional methods of processing bacteria for thin section electron microscopy rely on chemical fixation and dehydration under conditions which maximize specimen deterioration. Cryotechniques, however, use physical fixation (rapid freezing) and are slowly being recognized as a superior alternative to the more conventional methods. Freeze-substitution is a cryotechnique which combines cryofixation with a gentle chemical fixation and dehydration regimen, yielding specimens amenable to standard embedment procedures and ultramicrotomy. Previous study has shown that freeze-substitution retains the molecular composition of eubacteria better than conventional methods of processing. In this study we extend our observations and show that a simple freeze-substitution protocol reliably preserves the ultrastructure of a diverse range of microorganisms including archaeobacteria and anaerobic eubacteria.