CYTOCHEMICAL-DEMONSTRATION AND HISTOCHEMICAL-DEMONSTRATION OF LIGNINSIN PLANT-CELL WALLS - AN EVALUATION OF THE CHLORINE WATER ETHANOLAMINE SILVER-NITRATE METHOD OF COPPICK AND FOWLER
Ba. Fineran, CYTOCHEMICAL-DEMONSTRATION AND HISTOCHEMICAL-DEMONSTRATION OF LIGNINSIN PLANT-CELL WALLS - AN EVALUATION OF THE CHLORINE WATER ETHANOLAMINE SILVER-NITRATE METHOD OF COPPICK AND FOWLER, Protoplasma, 198(3-4), 1997, pp. 186-201
The chlorine water/ethanolamine-silver nitrate method introduced by Co
ppick and Fowler for the detection of lignins was evaluated for cyto-a
nd histochemical work using different reagents and fixatives for speci
mens embedded in epoxy resin. Fixation schedules tested included ethan
ol, glutaraldehyde, and glutaraldehyde followed by OsO4 as a post-fixa
tive. Chlorine water, sodium hypochlorite, and calcium hypochlorite we
re the oxidising agents evaluated for their efficacy as part of the Co
ppick and Fowler procedure. The Coppick and Fowler method was tested a
gainst stem woody tissue of Lophomyrtus obcordata, and haustorial xyle
m tissue of the sucker of its attached dwarf mistletoe Korthalsella li
ndsayi. The presence of lignins in walls of these cells was indicated
in thin sections for transmission electron microscopy by fine electron
-dense deposits. Post-staining thin sections did not affect the lignin
reaction, but tended to mask its effect due to increased wall contras
t. In histological preparations lignified walls stained orange/brown.
Counterstaining in methylene blue/azur B caused lignified walls to app
ear dark green/brown and non-lignified walls blue. Fixation in either
ethanol or glutaraldehyde produced identical staining for lignins. Pen
etration by chlorine water was sometimes irregular, more se with gluta
raldehyde fixation, with parts of tissues consequently not responding
to the lignin reaction. Post-fixation in osmium tetroxide following pr
imary fixation in glutaraldehyde slightly improved penetration of chlo
rine water. However, osmium caused greater amounts of extraneous stain
deposits compared with other fixative regimes. Chlorine water was con
firmed as the most effective oxidising agent for reacting with groups
in lignins to produce reducing residues in the Coppick and Fowler meth
od. Sodium hypochlorite caused no reaction. Calcium hypochlorite exhib
ited limited oxidative capacity resulting in slight staining for ligni
ns. The Coppick and Fowler procedure was concluded to be a suitable me
thod for demonstrating lignins in cyto- and histochemical preparations
using material fixed in either ethanol or glutaraldehyde, and with em
bedding in epoxy resin.