A Negative Staining Method for Cell-associated Virus

The virus host-cell relationship has been studied in the electron microscope almost entirely by the thin sectioning technique. This method has given information about the sites of virus production within the cell and the appearance of virus particles at various stages in the growth cycle (1, 2). I t does not, however, permit high resolution studies on the substructure of individual particles or the manner in which this substructure may change during the development of the virus. For high resolution studies on individual particles it is more effective to use the negative staining method (3), and much has been learned about the architecture of virus particles since the introduction of this technique (4). With two exceptions (5, 6), this method has been applied to purified or semipurified preparations in which the relationship of the virus to the host cell is lost. The following method (7) has been developed in an at tempt to combine the advantages of these two techniques, that is, sectioning and negative staining, and thus enable a study to be made of fine structure of virus particles and virus precursor stages as they occur in the cell. Virus-infected cells are centrifuged for 5 minutes in a clinical centrifuge and the resulting pellet is resuspended in 0.5 ml of a 1:5 dilution of culture medium with distilled water. This hypotonic solution results in swelling and disruption of the cells without excessive disruption of the cell components and also avoids difficulties that will occur when the tonicity is too high in subsequent freezing. The cell suspension is then frozen into a block of size and shape suitable for sectioning. This can be done conveniently by making a cupshaped mold in the following way. An approximately 1 inch square of plastic film (in this laboratory, Parafilm was used) is pressed over the end of a solid cylindrical object, such as a pencil, and the plastic secured with a small strip of adhesive