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

Post-fixation with osmium tetroxide (OsO4), prior to examination by electro n microscopy, enables nematodes to be located in epoxy-resin blocks, and im proves contrast of ultrastructural features. For specimen visibility, alter natives to OsO4 were examined as it can inhibit antigenicity, thus preventi ng application of serological techniques. Basic fuchsin stain does not affe ct antigenicity and when applied to trichodorids enabled specimens to be re adily located in the block, but had several disadvantages such as causing s ubstantial alteration of the nematode structure. Safranin-O stain enabled P aratrichodorus 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 al lowed 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 sp icula region of a male P. pachydermus specimen under a confocal laser scann ing microscope.