-35- 



Thin Sectioning 



After staining with either the 0/G combination or 

 Azure A (see Chapter 2), ORSV-inf ected tissues containing 

 inclusions were rinsed with 50% ethanol to remove Euparal 

 and then fixed with 5% glutaraldehyde and postfixed with 

 1-2% osmium tetroxide. Tissues were then dehydrated with 

 acidified 2, 2-dimethoxypropane as described by Muller and 

 Jacks (1975), and embedded in Spurr's low-viscosity medium 

 (Spurr, 1969). Sections were made with either a glass or 

 a diamond knife and stained with potassium permanganate, 

 uranyl acetate, and lead citrate ( Ko and Chen, 1982). 



The 0/G and Azure A stains were used to select tissues 

 infected with ORSV, CyMV, CMV, BYMV and rhabdoviruses to 

 be processed for embedding. Semi thin ( . 5-1 . /am) 

 sections were cut with a glass knife mounted on a Sorvall 

 Porter-Blum MT2-B ultramicrotome . Sections were then 

 placed on a glass microscope slide and stained with 1% 

 toluidine blue ( w/v dissolved in 1% sodium borate 

 solution) for 10-30 seconds on a hot plate at 55-60 C. 

 The semithin sections were then examined by light 

 microscopy. When inclusion-containing areas were located, 

 ultrathin sections were made from the same block still 

 mounted on the microtome. Resulting ultrathin sections 

 were stained in a conventional manner as described above 

 and examined with an electron microscope. 



