-34- 



detection by light microscopy were described in the first 

 chapter. In addition, immunofluorescence microscopy and 

 immunogold labelling were used in revealing the specific 

 protein composition of viral inclusions. 



Materials and Methods 

 Leaf Dips 



For CyMV and ORSV, vanadyl molybdate-phosphotungstate 

 (VaMo) was used for negative staining (Boothroyd and 

 Israel, 1980). The VaMo staining solution consisted of 1 

 part vanadyl molybdate (a mixture of 1 part of 1% vanadyl 

 sulphate and 4 parts of 1% ammonium heptamolybdate) , 3 

 parts of 2% sodium phosphotungstate and 4 parts of 0.025% 

 bacitracin. One to 3 cuts were made into virus-infected 

 tissue ( ca . 3x4 mm) in the freshly prepared staining 

 solution. A drop of this suspension consisting of 

 diffusing plant sap and staining solution was transferred 

 to a grid such that the droplet covered about one-third of 

 the grid. After 30 seconds the excess liquid was removed 

 using a piece of filter paper. The grid was then examined 

 with an electron microscope. The droplet edge was located 

 on the grid at low magnification and then enlarged to 

 locate virus particles accumulated there. The 

 phosphotungstic acid (PTA) staining was processed in the 

 same manner as VaMo staining. 



