Abstract
Many marine biology studies depend on field work on ships or remote sampling locations where sophisticated sample preservation techniques (e.g., high-pressure freezing) are often limited or unavailable. Our aim was to optimize the ultrastructural preservation of marine invertebrates, especially when working in the field. To achieve chemically-fixed material of the highest quality, we compared the resulting ultrastructure of gill tissue of the mussel Mytilus edulis when fixed with differently buffered EM fixatives for marine specimens (seawater, cacodylate and phosphate buffer) and a new fixative formulation with the non-toxic PHEM buffer (PIPES, HEPES, EGTA and MgCl2). All buffers were adapted for immersion fixation to form an isotonic fixative in combination with 2.5% glutaraldehyde. We showed that PHEM buffer based fixatives resulted in equal or better ultrastructure preservation when directly compared to routine standard fixatives. These results were also reproducible when extending the PHEM buffered fixative to the fixation of additional different marine invertebrate species, which also displayed excellent ultrastructural detail. We highly recommend the usage of PHEM-buffered fixation for the fixation of marine invertebrates.
Highlights
Marine research is often dependent upon species sampling from off-shore research stations, marine vessels and submersibles
PHEM buffer in its 1X concentration was only 219 mOsm, we increased the buffer concentration to 1.5X, resulting in an osmolarity of 323 mOsm, to be comparable with the other two buffers
This study compares the influence of different buffers on the resulting ultrastructural morphology preservation and demonstrates the effectiveness of the isosmotic, non-toxic PHEM buffer in combination with aldehydes when applied as an immersion fixative
Summary
Marine research is often dependent upon species sampling from off-shore research stations, marine vessels and submersibles. As highlighted by the Census of Marine Life, many species remain undiscovered, while the complex details about many others remain unknown (Census of Marine Life, 2010). One of the routine techniques of marine biology research is to preserve sample specimens for light and electron microscopy, for example for the formal description of a new species. Ultrastructural research is especially valuable for elucidating details on the symbiotic relationships between larger metazoan and prokaryotic organisms, like the mussels of the genus Bathymodiolus. These mussels live at hydrothermal vents and cold seeps in the deep sea (reviewed in Dubilier, Bergin & Lott, 2008). The mussels harbor chemoautotrophic bacterial symbionts in their gills which exploit the fluid chemistry at these sites to fix carbon and sustain their host (reviewed in Petersen & Dubilier, 2009)
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