Techniques for Preparing and Staining Phytophthora Oospores in Soybean Rootlets

Nylon mesh tissue carriers were constructed to hold soybean rootlets through fixing, dehydrating and embedding. Mesh pieces three centimeters square were doubled and sealed at each end by heat. Tissue samples were placed inside with an identifying piece of aluminum foil and the carrier sealed. Rootlets were fixed in Karpechenko's solution, dehydrated in an alcohol series and infiltrated with paraffin. They were embedded in paraffin after removal from the carrier, and sectioned on a microtome. Sections were mounted on glass slides and deparaffinized. A new stain was developed to differentiate oospores of Phytophthora megasperma var. sojae formed in these rootlets. The stain was prepared by dissolving 100 mg bromphenol blue in 50 ml of 95% ethanol and adding 3 g silver nitrate. Procedure: 5 sec in 95% ethanol, 30 min in silver stain, tap water rinse, 5 sec in 95% ethanol, 1 sec in saturated methylene blue in ethanol, immediate rinse in tap water, dehydration in absolute ethanol, rinse in tertiary butanol and xylene and mount. Previous clearing of the tissue was not required, and no air bubbles accumulated within the mesh carrier. This low cost, permeable carrier preserved the minute tissue specimens throughout processing, and the simple, progressive stain clearly differentiated oospores from surrounding tissue.