ダイレクト・メガティブ染色法による感染植物組織からのウイルス粒子の検出

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A rapid method designated "direct negative staining method", has been deve1oped to detect plant virus particles in fresh preparations. It is a combination of dipping and negative staining methods, being in principle similar to tha treported recently by Hitchborn and Hi11s (1965) and Kitajima (1965). The procedure is as fo11ows: A drop of 2% so1ution of sodium phosphotungstate (adjusted to pH 7.O with NaOH, and added with sma11 amount of neutral detergent as a spreader) was placed on a carbon-coated grid. The drop of stain, in which a fresh cut-end of an infected tissue piece was dipped for 2-3 seconds, was air-dried. The preparation was immediately examined under the electron microscope. By this method, e1ongated particles could be easily detected in a11 the tested 13 viruses, viz., tobacco mosaic virus, sugarcane mosaic virus, potato viruses X, Y and S, cowpea aphid-borne mosaic virus and azuki-bean mosaic virus (both be1ong to bean ye11ow mosaic virus group), carnation mosaic virus (=carnation vein mottle virus), soybean mosaic virus, soi1-borne wheat mosaic virus, and barley ye11ow mosaic virus. As regards spherica1 or po1yhedral viruses, particles of a virus isolated from Chenopodium album, carnation mottle virus, rice dwarf virus, rice black-streaked dwarf virus and tobacco ringspot virus were identified readi1y. Dimculty in revea1ing spherica1 or baci11iform particles in cucumber mosaic virus and alfalfa mosaic virus was considered to be due to breakdown of the particles during the treatment. In these viruses, however, a number of particles became discernible when the tissue piece to be examined was fixed by 5-10% forma1in for 2-3 hours, and washed in disti11ed water, in advance of dipping. It was noted that the shape and fine structure of the particles in the preparations of a11 the foregoing viruses were we11-preserved and the dimensions measured were comparable with those of previous workers. The direct negative staining method described here seems to be useful for detection of plant viruses. Especia11y it has advantage of simp1icity and re1iabi1ity in detection of latent viruses or virus complex. For instance, apreparation made from a carnation plant (cultivar Peter Fisher) infected with both carnation mosaic virus and carnation mottle virus readily demonstrated e1ongated particles of the former virus and spherical particles of the latter.
日本植物病理学会の論文
1969-06-30

ダイレクト・メガティブ染色法による感染植物組織からのウイルス粒子の検出

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