Centrifugal sedimentation of virus particles for electron microscopic counting

Centrifuge cells with conical chambers were provided by using special inserts for the stainless-steel tubes that fit the Spinco SW-39 rotor. Particulate material, centrifuged in these cells, was collected on carbon-coated glass discs. These discs were exposed to OsO(4) vapor, dehydrated in graded alcohols, air-dried, and metal-shadowed. The metal-shadowed carbon film was floated from the glass, mounted on a grid, and examined. A knowledge of cell geometry and microscope magnification allowed correlation of the number of particles observed to a volume of the original suspension. A precision of +/-6% at the 95% confidence level was attained when counting approximately 100 particles per 10,000 x field. Applications and advantages of the method are discussed.