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facilitate stain penetration in ORSV-inf ected tissues. 

 ORSV, unlike other tobamoviruses , required a longer period 

 of heating to facilitate Azure A stain penetration of 

 virus aggregates . Rhabdoviruses infecting Brassia and 

 Cymbidium required heating at 60 C and treatment in Triton 

 X-100 to facilitate staining inclusions contained within 

 the nucleus. Presently, this is the only practical 

 technique to detect the rhabdovirus infection. Since 

 rhabdoviruses have been considered as important as ORSV 

 and CyMV (Lesemann and Marwitz, 1983), light microscopy 

 could be an important tool for their detection. 



Interpretation of the light microscopy results was 

 facilitated by the development of the electron microscopic 

 techniques improvised for this research. The same 

 inclusions seen in tissues stained with O/G or toluidine 

 blue for light microscopic examination were also observed 

 by electron microscopy. The viral nature of inclusions 

 seen by light microscopy was also confirmed by in situ 

 examination of tissues directly labelled with TRITC- 

 conjugated antisera or indirectly using protein A-gold. 

 Nonspecific antibody binding was eliminated in 

 fluorescence microscopy by pre-incubating TRITC-con jugated 

 antisera with healthy tissue extracts. 



Immunogold labelling has been used to identify the 

 viruses in ultrathin sections (Lin and Langenberg, 1983) 

 and leaf dips (Louro and Lesemann, 1984; Lin, 1984). In 



