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Tomato fruit infected with the vulgare strain of tobacco mosaic virus were used for the isolation of protoplasts infected with tobacco mosaic virus (Gregory & Cocking, 1965). Protoplasts were fixed in glutaraldehyde, post-fixed in osmium tetroxide and embedded in a butyl methacrylate+styrene mixture (70:30, v/v) as previously described (Mohr & Cocking, 1968). Two different staining procedures were employed. The first (method A) involved incubation of protoplasts during dehydration in a freshly prepared, Millipore-filtered 1% solution of uranyl acetate in absolute ethanol for 1 hr at 18°. In the second procedure (method B) incubation was for 15 hr at 25° in a freshly prepared, Millipore-filtered 1% solution of uranyl acetate in 70% ethanol. With both staining procedures sections were post-stained for ½ hr with lead citrate (Reynolds, 1963). Electron micrographs were made with an A.E.I. EM6B at 60 kv using a 25 µ objective aperture.

Appearance of Tobacco Mosaic Virus in Thin Section Using Different Staining Procedures