Safe specimen preparation for electron microscopy of pathogenic fungi by freeze-substitution after glutaraldehyde...

Area of Science:

• Mycology
• Microscopy
• Cell Biology

Background:

• Observing the ultrastructure of highly infectious fungi presents safety challenges.
• Traditional methods may not preserve the natural state of fungal cells.
• High-resolution imaging is crucial for understanding fungal biology and pathogenicity.

Purpose of the Study:

• To describe a safe and effective method for examining the ultrastructure of highly infectious fungi using electron microscopy.
• To provide a reliable technique for high-resolution imaging of fungal cells in a near-natural state.

Main Methods:

• Chemical fixation of fungal cells using glutaraldehyde.
• Rapid freezing of samples using propane slush cooled by liquid nitrogen.
• Freeze-substitution in acetone with 2% osmium tetroxide.

Main Results:

• The described method yields clear, high-resolution images of fungal ultrastructure.
• The preserved cell images closely resemble those of rapidly frozen, freeze-substituted living cells.
• The technique was successfully demonstrated with Exophiala dermatitidis and Cryptococcus neoformans.

Conclusions:

• This electron microscopy technique offers a safe way to study infectious fungi.
• The method preserves cellular integrity, providing near-natural ultrastructural details.
• The technique is applicable to other highly infectious fungi, including Coccidioides immitis.