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LABORATORY PROCEDURES FOR PLANT CELL VIRUSES

Appendix

PVC/1998/2.02 Appendix 5


ELECTRON MICROSCOPY (EM) - LEAF DIP ASSAY

 

INTRODUCTION

All plant materials suspicious for a virus infection or sent to the collection for deposit are subjected to an electron microscopical examination for a preliminary evaluation of the virus(es) present in the sample. Although certain viruses e.g. tospoviruses, geminiviruses, ilarviruses are not reliably detected, electron microscopy is used to indicate for possible contaminating viruses. EM visualisation and evaluation of particle morphology (length, form, structure) allows preliminary grouping of the virus and detection of mixed virus infections.

 

PROCEDURE

Sample buffer

0.1 M Na2H/KH2PO4 buffer pH 7.0

2% (w/v) polyvinyl pyrollidone (PVP, MW 11.000)

0.2 % (w/v) Na2SO3

For leaf dip assay, a small piece of symptomatic tissue is placed on a glass slide and a few drops of sample buffer are added. Using a glass rod, the leave material is macerated in the buffer and the extruding plant sap is used for adsorption preparates.

400 mesh size pioloform coated Cu or Ni grids, used in electron microscopy, are incubated for 5 min with a drop of homogenates prepared from virus infected plant samples and subsequently washed with a gentle stream of approximately 40 drops of deionised water (Milli Q) to remove buffer salts. For negative staining contrasting -, 3-5 drops of a 1% uranyl acetate solution in sterile deionised water are applied after which the grids are dried by gently tapping on a piece of Whatman filter paper and examined in the electron microscope.


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