Fixation of mucus on rainbow trout (Oncorhynchus mykiss Walbaum) gills for light and electron microscopy

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Abstract
Fixation of mucus for the assessment of biofilms and surface associated pathogens often involves complex and expensive techniques. Rainbow trout killed by an overdose of MS 222 had their gills removed and immersion‐fixed gently in buffered glutaraldehyde containing 2% Alcian blue. Control tissues consisted of gills fixed in Alcian blue‐free fixative. Trout were also killed and directly immersed in liquid nitrogen and the gills freeze‐dried then vapour fixed with osmium tetroxide at −50° C. Following fixation gill tissue was processed for light and electron microscopy. A continuous and intact mucous coat was not detected on tissue fixed by conventional methods but the addition of Alcian blue to the fixative preserved an extensive mucous coat trapped between the lamellae and overlying the epithelia. Electron microscopic examination revealed that mucus preservation with the conventional fixative was poor and intermittent whereas the addition of Alcian blue to the fixative greatly enhanced the preservation of the branchial mucous coat. Mucus appeared as interdispersed flocculant material between the epithelial microridges and formed extensive superficial sheets over the epithelium. Freeze‐dried/vapour‐fixed gill tissue also provides excellent preservation of the integrity of branchial mucous coats, the mucus appearing as a continuous sheet over the filament and secondary lamellae. However, freeze‐dried tissue fails to preserve sufficient cellular integrity for this technique to be useful for light or transmission electron microscopy. The potential for use of glutaraldehyde‐Alcian blue fixed‐gill tissue diagnostically and in research are discussed.