ADVANTAGES OF ENVIRONMENTAL SCANNING ELECTRON-MICROSCOPY IN STUDIES OF MICROORGANISMS

Microorganisms, including bacteria, fungi, protozoa, and microalgae, a re composed predominantly of water which prohibits direct observation in a traditional scanning electron microscope (SEM). Preparation for S EM requires that microorganisms be fixed, frozen or dehydrated, and co ated with a conductive film before observation in a high vacuum enviro nment. Sample preparation may mechanically disturb delicate samples, c ompromise morphological information, and introduce other artifacts. Th e environmental scanning electron microscope (ESEM) provides a technol ogy for imaging hydrated or dehydrated biological samples with minimal manipulation and without the need for conductive coatings. Sporulatin g cultures of three fungi, Aspergillus sp., Cunninghamella sp., and Mu cor sp., were imaged in the ESEM to assess usefulness of the instrumen t in the direct observation of delicate, uncoated, biological specimen s. Asexual sporophores showed no evidence of conidial displacement or disruption of sporangia. Uncoated algal cells of Euglena gracilis and Spirogyra sp. were examined using the backscatter electron detector (B SE) and the environmental secondary electron detector (ESD) of the ESE M. BSE images had more clearly defined intracellular structures, where as ESD gave a clearer view of the surface. E. gracilis cells fixed wit h potassium permanganate, Spirogyra sp. stained with Lugol's solution, and Saprolegnia sp. fixed with osmium tetroxide were compared using B SE and ESD to demonstrate that cellular details could be enhanced by t he introduction of heavy metals. The effect of cellular water on signa l quality was evaluated by comparing hydrated to critical point dried specimens. (C) 1993 Wiley-Liss, Inc.