Negative Staining of Thinly Spread Cells and Associated Virus

The first demonstration of intracytoplasmic virus by negative staining was that of Horne and Nagington (9) who subjected poliovirus-infected cells to freezing and thawing and negatively stained a suspension of the fragments. Dales (5) examined the attachment of some viruses to the cell surface by the negative staining of whole mounts of cells subjected to osmotic shock. Whittaker and Horne (15) examined normal cell components by the negative staining of fractions of tissue homogenates separated by centrifugation. Methods of negative staining of thin sections of frozen or frozen-dried tissue have been developed by Fern(tndez-Mor~tn (7). Almeida and Howatson (1, 2) have recently developed a method of studying viruses and virus-cell relationships by negative staining of frozen sections. The technique of spreading the cytoplasmic structures of cells on a water surface, for electron microscope examination, was originated by Fern~ndez-Mor~m (6). A needle was inserted into the tissue and then dipped into distilled water or saline. Surface tension drew the ceils off the needle and spread the cytoplasmic contents into a very thin film. Nuclei were unaffected. In the method to be described, cells are spread by a similar method and then treated with potassium phosphotungstate (PTA). The negative contrast