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The application of safranin-O for staining virus-vector trichodorid nematodes for electron and confocal laser scanning microscopy

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For more content, see Nematologica.

Post-fixation with osmium tetroxide (OsO4), prior to examination by electron microscopy, enables nematodes to be located in epoxy-resin blocks, and improves contrast of ultrastructural features. For specimen visibility, alternatives to OsO4 were examined as it can inhibit antigenicity, thus preventing application of serological techniques. Basic fuchsin stain does not affect antigenicity and when applied to trichodorids enabled specimens to be readily located in the block, but had several disadvantages such as causing substantial alteration of the nematode structure. Safranin-O stain enabled Paratrichodorus anemones and P. pachydermus specimens to be located in resin blocks, different parts of the nematode body to be distinguished, and did not affect specimen ultrastructure. Also, with viruliferous specimens it allowed immunogold labelling techniques to be applied for identifying tobacco rattle virus particles at the site of retention in the nematodes. Safranin-O is fluorescent and this feature was used to examine sections from the spicula region of a male P. pachydermus specimen under a confocal laser scanning microscope.

La post-fixation au tétroxyde d’osmium (OsO4) avant l’observation en microscopie électronique empêche la localisation des nématodes dans les blocs de résine epoxy et augmente le contraste des caractéristiques ultrastructurales. Pour la localisation des spécimens, des solutions alternatives au OsO4, qui peut inhiber l’antigénicité et donc empêcher l’application de techniques sérologiques, ont été examinées. Le coloration à la fuschine basique n’affecte pas l’antigénicité et permet de situer les spécimens dans les blocs, mais elle présente de nombreux désavantages telle l’altération substantielle des structures du nématode. La safranine-O permet la localisation de Paratrichodorus anemones et de P. pachydermus, les différentes parties du corps du nématode étant alors identifiables, et n’affecte en rien l’ultrastructure du nématode. Chez les spécimens portant des virus, elle permet également l’utilisation de techniques de marquage immunologique à l’or pour identifier les particules du virus du tobacco rattle dans les sites de rétention chez le nématode. La safranine-O est fluorescente et cette particularité a été utilisée pour observer des sections de la région des spicules chez un mâle de P. pachydermus en microscopie confocale à balayage.


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