The preservation of mucus and surface‐associated microorganisms using acrolein vapour fixation

Abstract

SUMMARY Three fixation schedules were devised and compared in terms of their influence on the preservation of mucus and surface‐associated microorganisms contained within it. Different mucus‐secreting epithelial tissues from normal and spoiled oysters and normal rats were examined by scanning and transmission electron microscopy and light microscopy. On all tissues, mucus was best preserved in specimens fixed by 10% acrolein vapour for 1 h then immersed in 3% glutaraldehyde—3% formaldehyde fixative containing 0·05% ruthenium red, cacodylate buffer pH 7·4, for at least 3 h. This fixation schedule also greatly increased the preservation of microorganisms in mucus in specimens from spoiled oysters and normal rats. In contrast, the retention of mucus and surface‐associated microorganisms was poor in tissues fixed either by 1% OsO4 vapour for 1 h followed by immersion in combined aldehyde fixative, or by direct immersion. The quality of preservation of the mucus layer, epithelium and sub‐epithelium was also noted by transmission electron microscopy in tissues prepared by the different fixation schedules. Cellular preservation was satisfactory in directly immersed tissues but poor in vapour fixed specimens.

Morphology and function of the skin epithelium covering the giant keyhole limpet Megathura crenulata

“…We also saw no evidence of reduction in total thickness of the mucous layer, such as has been reported as an artifact of tissue processing in several vertebrate systems (Garland et al. ; Strugala et al. ; Cohen et al.…”

“…This differs from tissues, such as those in fish gills, in which standard fixation procedures cause a loss of the mucous layer (Lumsden et al 1994). We also saw no evidence of reduction in total thickness of the mucous layer, such as has been reported as an artifact of tissue processing in several vertebrate systems (Garland et al 1982;Strugala et al 2003;Cohen et al 2012). Nowles & Boucher (2002) discuss the ability of mucins lining the mammalian airway to bind a great range of chemically distinct particles.…”

“…; Minuth & Denk ); and (4) fixation by acrolein vapor for 1 h, followed by routine fixation (Garland et al. ).…”

“…Procedures to stabilize the mucous coating for examination by TEM included the following modifications of the standard fixation procedure: (1) adding 1% Alcian blue 8GX to the primary fixative (Flood & Fiala-Medioni 1981;Powell et al1992; respectively); (2) adding 1% lanthanum nitrate to the primary fix, then rinsing in phosphate buffer at 4°C, followed by postfixation with OsO 4 containing 1% LaNO 3 in cacodylate buffer (Shaklai & Tavassoli 1977); (3) following an overnight fixation with standard primary fixative, washing the sample for 1 h in ice-cold 0.1 mol L À1 sodium cacodylate pH 7.4 with 0.1% ruthenium red, with postfix containing the dye as well (Vitkov et al 2001;Tucker et al 2011;Minuth & Denk 2014); and (4) fixation by acrolein vapor for 1 h, followed by routine fixation (Garland et al 1982).…”

Patchy distribution of mucosal lesions in ileal Crohnʼs disease is not linked to differences in the dominant mucosa-associated bacteria

“…The mucus is composed of glycoproteins (mucins) and constitutes a physical barrier between the epithelium and aggressive agents in the lumen. [1][2][3][4] The etiology of Crohn's disease (CD) is still unknown, but the intestinal microbiota seems to play a crucial role in the onset and recurrence of the disease. [5][6][7] In most studies the mucosa-associated bacteria (MAB) differ from the luminal bacteria.…”