A SOLVENT-BASED FIXATIVE FOR ELECTRON-MICROSCOPY TO IMPROVE RETENTIONAND VISUALIZATION OF THE INTESTINAL MUCUS BLANKET FOR PROBIOTICS STUDIES

Samples of pig small intestine, cecum, and large intestine were prepar ed for scanning electron-microscopy (SEM), concentrating on mucus blan ket retention and visualization. Samples were fixed using three aqueou s-based fixatives which included a standard glutaraldehyde fixative al one as the control and the standard fixative formulation with either r uthenium red or alcian blue added and using one solvent-based fixative , osmium tetroxide dissolved in FC-72 (a degreasing fluorocarbon solve nt produced by 3M Canada, Inc.), which had been successfully used by S ims et al. [(1991) Biotech. Histochem., 66:173-180] to preserve trache al mucus of nonhuman mammals. Pig intestine samples prepared using the solvent-based fixative retained a contiguous mucus blanket, while the aqueous-based treatments retained only patchy or fibrous remnants to a degree depending on fixative composition and intestinal site. We con clude that preparation of the pig intestinal mucus layer using the sol vent-based fixative suggested by Sims et al. (1991) preserves the mucu s blanket in its entirety and gives superior results to aqueous-based fixatives containing the standard additives ruthenium red and alcian b lue. We recommend that this anhydrous fixation, which requires only a slight modification from standard conditions, be adopted when mucus la yer retention and visualization is important, as in tile field of prob iotics. Overcoming this major technical obstacle will now allow electr on microscopy (EM) to once again provide new in situ information in th is reemerging field.