Chapter 5 Isolation of Euglena Flagella

Abstract

Publisher Summary Euglena flagellum has its surface uniformly coated with a glycoprotein layer termed xyloglycorein and the flagellum is partially enveloped with a stable array of filamentous mastigonemes. The Euglena locomotory flagellum extends from a basal body at the base of the reservoir through a narrow canal and finally to the outside of the cell. Flagellar beat is planar in the reservoir-canal region and progressively more helical outside of the cell. Summarized in this chapter are procedures for the cultivation of Euglena and protocols for flagellar isolation. It is required that all media and culturing glassware should be autoclaved and standard sterile techniques should be used when culturing Euglena, as it can be easily overgrown by bacteria and fungi. Cells of E. gracilis strain Z are routinely grown in acetate-containing medium of Cramer and Myers under continuous fluorescent illumination at room temperature. For biochemical and molecular assays, larger quantities of flagella are needed and these can be isolated from a 15-liter batch culture, whereas 500-ml cultures are adequate for smaller-scale experiments such as light and electron microscopy. Harvesting 15 liters of a 5-day Euglena culture at a density of approximately 1x l0 6 cells/ml should yield∼15 g of cells from which 3-6 mg of flagellar proteins can be collected after deflagellation. This chapter describes the harvesting of cells, deflagellation by cold shock, pH shock, mechanical shearing, and by a combined Ca 2+ and cold shock. The protocol using a combined Ca 2+ and cold shock has the advantage of yielding a fraction of flagella containing the putative photoreceptor. To date, the most consistent and reliable method is cold shock, which generates intact Euglena flagella containing most of the axoneme, paraxonemal rod, and flagellar surface.