Chapter VIII-1 - Complementary techniques: Preparations of entomopathogens and diseased specimens for more detailed study using microscopy

Abstract

This chapter provides the researcher with some of the basic techniques and protocols used to study insect pathogens realizing that the guidelines must be tailored for specific needs. The first evidence for the presence of a pathogen is often observed with either a stereoscopic or compound microscope. There are a number of general protocols for the handling of entomopathogens and diseased individuals regardless of the host or pathogen. Chemical fixation is the process of stabilizing the cellular integrity of tissues for detailed histological examinations. The fixative must penetrate quickly to preserve the tissues in a natural state with a minimum of artifacts due to swelling, shrinkage, leaching or other detrimental effects. Following fixation, the tissue must be dehydrated, infiltrated with paraffin and embedded in paraffin prior to sectioning and staining. An alternative to chemical fixation is freeze-substitution. This protocol was developed to avoid the many artifacts associated with conventional chemical fixation. After fixation, tissues must be dehydrated and embedded. Dehydration is achieved by transferring the material through an ascending alcohol or acetone series into absolute alcohol or acetone. It is found that scanning electron microscopy has been used in the study of insect pathology primarily for examining surface morphology of microsporidian spores and fungal conidia and developmental stages.