Advantages of environmental scanning electron microscopy in studies of microorganisms

Abstract

Microorganisms, including bacteria, fungi, protozoa, and microalgae, are composed predominantly of water which prohibits direct observation in a traditional scanning electron microscope (SEM). Preparation for SEM requires that microorganisms be fixed, frozen or dehydrated, and coated with a conductive film before observation in a high vacuum environment. Sample preparation may mechanically disturb delicate samples, compromise morphological information, and introduce other artifacts. The environmental scanning electron microscope (ESEM) provides a technology for imaging hydrated or dehydrated biological samples with minimal manipulation and without the need for conductive coatings. Sporulating cultures of three fungi, Aspergillus sp., Cunninghamella sp., and Mucor sp., were imaged in the ESEM to assess usefulness of the instrument in the direct observation of delicate, uncoated, biological specimens. 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 (BSE) and the environmental secondary electron detector (ESD) of the ESEM. BSE images had more clearly defined intracellular structures, whereas ESD gave a clearer view of the surface. E. gracilis cells fixed with potassium permanganate, Spirogyra sp. stained with Lugol's solution, and Saprolegnia sp. fixed with osmium tetroxide were compared using BSE and ESD to demonstrate that cellular details could be enhanced by the introduction of heavy metals. The effect of cellular water on signal quality was evaluated by comparing hydrated to critical point dried specimens. © 1993 Wiley‐Liss, Inc.

Microorganisms Attack Synthetic Polymers in Items Representing Our Cultural Heritage

“…In the past, both optical microscopy and electron microscopy have been used to monitor biodeterioration of polymeric surfaces (4,44). Environmental scanning electron microscopy and atomic force microscopy might provide high-resolution information on the outermost features of biofilms maintained under hydrated conditions (5,16,31). Spectroscopic techniques are an alternative for monitoring biofilm formation at the molecular level.…”

Manipulating biological samples for environmental scanning electron microscopy observation

“…Therefore, those samples do not require chemical fixation. If prolonged observation is required, fixation may be helpful for samples having less tolerance to the electron beam (Collins et al 1993). At least three different methods were used to fix biological samples chemically.…”

Characterization of polyox ® granules using environmental scanning electron microscopy

“…The hydration capabilities of the ESEM have been used to characterize a variety of samples including polymers (Wight and Zeissler 1993), microorganisms (Collins et al 1993), and biological materials (Egerton-Warburton et al 1993). Within the pharmaceutical industry, the ESEM has been used to observe the dissolution and erosion characteristics of both raw and formulated drug substances (Kelly 1991).…”