Direct Negative Staining Method for Detection of Virus Particles in Fresh Preparations from Infected Plant Tissues

Abstract

A rapid method designated “direct negative staining method”, has been developed to detect plant virus particles in fresh preparations. It is a combination of dipping and negative staining methods, being in principle similar to that reported recently by Hitchborn and Hills (1965) and Kitajima (1965). The procedure is as follows: A drop of 2% solution of sodium phosphotungstate (adjusted to pH 7.0 with NaOH, and added with small 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, elongated particles could be easily detected in all 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 belong to bean yellow mosaic virus group), carnation mosaic virus (=carnation vein mottle virus), soybean mosaic virus, soil-borne wheat mosaic virus, and barley yellow mosaic virus.As regards spherical or polyhedral 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 readily. Difficulty in revealing spherical or bacilliform 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% formalin for 2-3 hours, and washed in distilled water, in advance of dipping. It was noted that the shape and fine structure of the particles in the preparations of all the foregoing viruses were well-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. Especially it has advantage of simplicity and reliability in detection of latent viruses or virus complex. For instance, a preparation made from a carnation plant (cultivar Peter Fisher) infected with both carnation mosaic virus and carnation mottle virus readily demonstrated elongated particles of the former virus and spherical particles of the latter.