Preparation of microbiological specimens for scanning electron microscopy.

This tutorial paper describes techniques for processing microorganisms for scanning electron microscopy (SEM). For ease of discussion, the subject is divided into two sections: A. General Processing Requirements, and B. Specific Processing Techniques. The objective of the first section is to outline processing requirements, explain their purpose, and point out where variations are possible. The following basic steps are discussed: (1) specimen selection, (2) prefixation treatment, (3) chemical fixation, (4) dehydration, (5) critical-point drying and freeze-drying, (6) coating, and (7) viewing. The second section describes methods of manipulating microorganisms through the processing steps. Instructions will cover microorganisms processed for SEM (1) in suspension, (2) in tissues. (3) in tissue culture, and (4) on agar. We are relying heavily on our own experiences in the laboratory and the results are illustrated by use of bacteria, mycoplasmas, rickettsiae, fungi, free-living protozoa, and parasitic protozoa. This tutorial is intended to be a general reference for processing microorganisms for study with the scanning electron microscope. Although we have described basic requirements and several specific techniques, it must be emphasized that there is a wide range of preparation flexibility permissible, depending upon the objectives of the study.