IMPROVED METHOD FOR COUNTING VIRUS AND VIRUS-LIKE PARTICLES

An improved method for counting virus and virus like particles by elec tron microscopy (EM) was developed. The procedure involves the determi nation of the absolute concentration of pure or semi-pure particles on ce deposited evenly on EM grids using either centrifugation or antibod y capture techniques. The counting of particles was done with a Microf iche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low mag nification (2500 x) by EM. Initially, latex particles of a known conce ntration were counted using this approach, to prove the accuracy of th e technique. The latex particles were deposited evenly on an EM grid u sing centrifugation (Modified Beckmen EM-90 Airfuge technique). Subseq uently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a hovel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventi onal method, showed good agreement. The method is simple, accurate, ra pid, and reproducible when used with either pure particles or with par ticles from crude cell culture extracts.