Influence of Adsorption Time, Rocking, and Soluble Proteins on the Plaque Assay of Monodispersed Poliovirus

Factors that could affect adsorption of monodispersed poliovirus to cell culture monolayers were evaluated. These included varying the virus adsorption period under static and nonstatic (rocked) conditions and altering the rocking rate. The effects of several soluble proteins on plaque formation, enumeration, and size were also evaluated. Rocking involved the mechanical spread of viruses over cell culture monolayers for 1 to 4 h. Rocked cultures exhibited significantly higher (P less than 0.05) plaque counts than corresponding static cultures. Optimal plaque counts were obtained after a 2-h adsorption period with rocking; increasing the period to 4 h did not significantly increase PFU. Optimal counts were not obtained until greater than or equal to 4 h with static adsorption. Plaque counts were not affected by increasing the rocking rate above one oscillation per minute, but a slower rocking rate resulted in a significant decrease in plaques. Adsorption of poliovirus in the presence of 3% solutions of beef and meat extracts, acid-precipitated oyster protein, two brands of skim milk, and 3 and 10% fetal bovine serum was compared with adsorption in protein-free controls. Significant reductions (P less than 0.05) in plaque counts occurred with one brand of skim milk, whereas 3% beef extract yielded highly significant reductions (P less than 0.01) in plaque counts and appreciable decreases in plaque sizes. Salinities of protein-containing virus inocula were high for beef and meat extracts but somewhat below physiological levels for the remaining inocula. Beef extract-associated reductions in PFU were eliminated after the extracts were dialyzed. Plaque reductions were associated with dialyzable components of the beef extract but not with the inoculum salinity.