The grid‐cell‐culture technique: the direct examination of virus‐infected cells and progeny viruses

We describe a method for the structural analysis and identification of viruses, without purification or concentration steps which could alter virus morphology. Virus-infected cells grown on carbon-Parlodion-coated electron microscope grids release large numbers of progeny viruses which adsorb to the surface of the grid and are revealed by negative staining. The technique is rapid, sensitive and can be used at three levels. Negative staining of whole cell preparations revealed both extracellular and intracellular viruses or nucleocapsids beneath the plasma membrane; non-ionic detergent extraction of cells infected with certain viruses reveals cytoskeleton-associated, virus-specific structures normally only observed after thin sectioning; cultures prepared by either procedure are suitable for colloidal gold immunological studies. Extracellular and cytoskeletal-associated viruses were heavily and specifically labelled with gold. The results indicate that the technique may be used to rapidly identify unknown viruses on the basis of size, topography, morphology and mode of maturation from the infected cell, as well as the presence of characteristic intracellular cytoskeletal-associated structures. The technique also has potential use in the sero-grouping and sero-typing of viruses with specific monoclonal antibodies.