Electron microscopy of frozen-hydrated bacteria

Abstract

Amorphous, unstained, frozen-hydrated sections of bacteria provide a faithful high-resolution image of prokaryotic cells. Conventinal preparation artifacts due to fixation, staining and dehydration are nonexistent. Freezing damage is avoided by using glucose as a cryoprotectant. Cutting damage on frozen material is severe, but sectioning artifacts, being always related to the cutting direction, can be systematically recognized and thus taken into consideration. Geometry and density distribution of the bacterial envelope can be resolved to about 3 nm. The following main features were observed. In Escherichia coli the inner and outer membranes have an approximately uniform density profile. The distance between the 2 membranes is constant, .apprx. 33 nm. In Staphylococcus aureus the cell wall is .apprx. 40 nm wide. It is bordered on the cytoplasmic side by an asymmetric 5.5-nm-wide bilayer. The bacterial nucleoid, clearly visible with conventional preparation methods, appears in exponentially growing bacteria as an ill-defined central region with approximately the same density as the rest of the cytoplasm. It becomes more clearly visible when bacteria are in the stationary phase, plasmolysed, fixed or stained. Mesosomes, considered to be essential organelles in all prokaryotes, are confirmed as artifacts. They appear in large numbers during osmium fixation.