Permanent gregarine specimen techniques

Working with parasitic protozoans requires a set of standardized techniques for making permanent specimens, especially for biodiversity discovery and taxonomic studies. The techniques presented here have been developed and refined over the last 20 years to enable us to make permanent museum specimens of gregarines. The techniques are adapted for use in the field and the laboratory, depending upon the source of insect hosts. Briefly, we make smears of host intestinal contents on clean 22mm coverslips, fix the specimens by flotation on hot AFA, and then store them in 80% ethanol in columbia jars. When we're in the field, we parafilm the columbia jars and do the final staining and mounting after returning to the lab. We use acetocarmine stain for routine work and depend upon DIC microscopy to elucidate details of epimerite morphology. It's a fast, easy, effective approach (see the banner image above). However, hematoxylin staining is particularly useful for elucidating details of very young trophozoites, epimerite morphology, and the structure of very small gregarine species. We use damar balsam as a mountant for all of our specimens. We find it preferable to canada balsam because damar balsam is more predictable by resin lot, less expensive, less acidic, and less prone to darkening over time than is canada balsam. Unlike permount and other synthetic resins, damar balsam is stable over time. We have a lot of specimens mounted in damar balsam over 20 years ago and there is no sign of deterioration or stain fade.

Use the links at right for details of our techniques or download the complete PDF.



Live gregarine culture techniques

Empirical and life cycle work with live gregarines requires the constant development of new techniques and the adaptation of old techniques to new genera or species. We've developed a fairly robust set of "gregarine farming" techniques over the years. Some are suited for laboratory and empirical work, others are designed for field use. We've published all of these techniques at one time or another, but somehow folks find that it just takes more detailed information to actually replicate the techniques than is readily gleaned from a "Materials and Methods" section.

The links at right provide elaborate detail and illustration of some of our more common techniques.

"Oocyst cultivation" explains how we gather and incubate gametocysts for oocyst production. There are two variants: the "lab" variant is designed to produce oocysts from colony material or for empirical work. The "field" variant is designed for a few gametocysts collected during field survey for gregarine taxonomic work.