14 Fine-structural Analysis of Mycorrhizal Fungi and Root Systems: Negative Staining and Cryoelectron Microscopic Techniques

Abstract

Publisher Summary This chapter looks at several different approaches that can be used for fine-structural analysis. These are the negative staining technique, not commonly used in mycorrhiza research; the preparation of frozen-hydrated samples for high resolution transmission electron microscopy; the freeze substitution of material subsequently cut for transmission electron microscopy; the preparation of ultra-thin cryosections; and immunoelectron microscopic techniques. Negative staining is appropriate for very small cells, such as bacteria and for cell components of sizes down to macromolecules, such as enzyme complexes. However, the negative staining technique does not lend itself to good structural preservation due to the air-drying which constitutes the final step of the procedure prior to electron microscopic examination. Another approach not commonly used in mycorrhiza research is the preparation and analysis of frozen-hydrated samples; this technique avoids air-drying. It can be applied for the visualization of ultrastructural details at the macromolecular level and up to dimensions of viruses, membrane vesicles, isolated cell components, cells, and tissues. The preparation procedure appears to be simple; however, in practice difficulties are encountered when good quality micrographs are needed. The reasons for this are two-fold: first, freezing (vitrification) may not proceed without complications which may lead to the appearance of artefacts and second, this kind of sample requires adequate instrumentation and careful treatment (that is low dose electron microscopy) during screening and image recording; focusing especially is a matter of experience, and usually the primary contrast obtained by imaging is low.