The use of ethylenediamine in softening hard plant structures for paraffin sectioning

Ethylenediamine has been used as an agent for softening very hard woods prior to sectioning on a sliding microtome. The use of ethylenediamine is recommended for two additional uses: for preparing 1) soft woods in which wide, thin-walled tracheids or vessels tend to collapse during sliding microtome sectioning and 2) plant tissues with sclerenchyma mixed with soft-walled cells (bark, leaves, fruits, etc.) which frequently fail to section well. After softening in ethylenediamine, material is washed, infiltrated, and embedded in paraffin. Preliminary sections are made with a rotary microtome, just exposing the cut surface of the material; this exposed surface is soaked overnight in water. Sectioning is then continued. Sections produced in this fashion are considerably improved. The wood and pith of Podocarpus ustus, a parasitic conifer from New Caledonia, is used as an object to demonstrate improvements in sectioning by the ethylenediamine-paraffin method. Thinner sections with minimal tearing, cell collapse, and unevenness are produced. Sections can be handled easily and stained more effectively than unmounted sections. Variations in timing and in treatment are recommended to suit different materials. Ethylenediamine, used with reasonable caution, is much less hazardous than hydrofluoric acid and is more effective in softening plant material. The ethylenediamine method may be used routinely on any material difficult to section because of hardness.