CYTOCHEMICAL-DEMONSTRATION AND HISTOCHEMICAL-DEMONSTRATION OF LIGNINSIN PLANT-CELL WALLS - AN EVALUATION OF THE CHLORINE WATER ETHANOLAMINE SILVER-NITRATE METHOD OF COPPICK AND FOWLER

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.