A Useful Quantitative Semimicromethod for Viral Plaque Assay

This paper presents a general method for rapid, easy, and convenient quantitation of virus titers which we have utilized in applications varying from measurement of virus distribution in infection in vivo to assay of neutralizing antibody or antiviral agents. Basically, this technique utilizes disposable multicompartmental plastic plates with 1-ml volume flat bottomed wells for cell culture, together with a carboxycellulose (CMC) overlay and a stain which permanently fixes the cell sheet. This then retains the quantitation inherent in plaque assays as conducted in larger plates, but introduces an increased ease and speed of handling because of the bulk processing possible. As well a sharp reduction in cost is introduced because of savings in reagents and plasticware. Materials and Methods. Viruses and cells. Viruses in current use in the Divisions of Cell Pathology and Communicable Diseases were used in these studies. Primary and continuous cell lines were used as listed in Table I. Growth medium. L-15 (Leibowitz) medium was supplemented with 10% tryptose phosphate broth (TPB). In addition penicillin (100 U/ml) and streptomycin (100 U/ml) were used in all experiments. MEM (minimal Eagle's medium) or BME (basal medium Eagles) were also used with certain cells as indicated. Plates. Disposable plastic plates and covers (Linbro Chemical Co., New Haven, CT, Disposotray ∗96CV-TC and ∗912, cover, sterile) containing 96 wells, diameter 1.6 cm (Fig. 1). Before use plates and covers were sterilized by exposure for 30 min to ultraviolet light. Normally, monolayers were prepared in a horizontal laminar-flow cabinet. A covered cabinet with a glass barrier between the operator's face and the plates was used for virus challenge. Preparation of cell monolayers. One milliliter of an appropriate cell concentration was distributed into each well. Plates were then put in closed polyethylene bags and incubated at 37°.