Electron microscopy of frozen-hydrated bacteria

Area of Science:

• Microbiology
• Electron Microscopy
• Cell Biology

Background:

• Conventional bacterial sample preparation introduces artifacts, obscuring true cellular structures.
• Understanding procaryotic cell morphology requires high-resolution imaging techniques free from preparation-induced distortions.

Purpose of the Study:

• To image procaryotic cells using amorphous, unstained, frozen-hydrated sections.
• To identify and characterize bacterial cell envelope and nucleoid structures at high resolution.
• To investigate the nature of bacterial mesosomes.

Main Methods:

• Cryo-electron microscopy of frozen-hydrated bacterial sections.
• Use of glucose as a cryoprotectant to minimize freezing damage.
• Systematic recognition and consideration of sectioning artifacts.

Main Results:

• Achieved ~3 nm resolution of bacterial envelope geometry and density.
• Observed uniform density profiles for inner and outer membranes in Escherichia coli (33 nm apart).
• Characterized the cell wall (40 nm) and cytoplasmic bilayer (5.5 nm) in Staphylococcus aureus.
• Found bacterial nucleoids appear as ill-defined regions in exponentially growing cells, becoming clearer under specific conditions.
• Confirmed bacterial mesosomes are artifacts of osmium fixation.

Conclusions:

• Amorphous, unstained, frozen-hydrated sections provide faithful high-resolution images of procaryotic cells.
• Cryo-electron microscopy overcomes limitations of conventional preparation methods.
• Bacterial mesosomes are fixation artifacts, not integral cellular components.