Enumeration of enterovirus particles by scanning electron microscopy.

Enumeration of virus particles requires relatively concentrated and uniformly dispersed virus preparations, which is difficult to achieve by the usual methods of negative staining and transmission electron microscopy. We have developed an electrophoretic method that concentrates enteroviruses onto a polycarbonate membrane for examination by high-resolution scanning electron microscopy. The electrophoretic apparatus comprises three chambers in electrical series, each containing 3.5 ml of dilute buffer. The center chamber is inoculated with virus. A 15-nm porosity membrane, which does not pass virus, separates the center from the side chambers. A constant current is applied, and chilled buffer is pumped past the electrodes for 2 h. The virus suspension is recovered, and changes in titer (or radioactivity if labeled virus is used) due to electrophoresis are measured. Buffer pH, relative to the viral isoelectric points, determines the direction of virus migration. Particle counts are calculated from the mean of 25 randomly chosen fields photographed at 35-60,000 X magnification and related to titers measured by plaque assay.