Suspended Cell Tissue Cultures for Study of Virus Growth Kinetics. ∗

The embryonated hen's egg, the deembry-onated egg, and, more recently, tissue cultures using the chick embryo chorioallantoic membrane have been widely used in the study of the growth of influenza virus (1-13). The present investigation was undertaken to appraise the suspended cell tissue culture for the quantitative determination of the multiplication of this agent. Attention has been focused on early virus production as measured by titration of infectivity in tissue culture flasks.

Materials and methods. Virus. The PR8 strain of influenza A virus propagated in chick chorioallantoic membrane tissue culture has been used in all experiments. This agent was received from Dr. Frank Horsfall 15 years ago and has been maintained since then by allantoic passage in the chick embryo. The stock virus consisted of the fluid removed from cultures inoculated 36 to 48 hours previously with 0.1 ml of infected allantoic fluid diluted 10^-3. It was stored at −70°C in glass-sealed ampules.

“Titration” flasks. For titration of viral infectivity at various intervals tissue culture flasks such as those used by Enders and his coworkers (14,15) were prepared with scissor-minced chorioallantoic membrane tissue from 9-11 day chick embryos. Three ml of a mixture consisting of 3 parts Hanks' balanced salt solution and one part Simms' ox serum ultra-filtrate was added to each 25 ml Erlenmeyer flask. The medium also contained 50 units of penicillin and 50 μg of streptomycin per ml. A 50% suspension of the minced tissue was prepared by centrifuging the fragments at 1000 rpm for 5 minutes and adding the required volume of Hanks-Simms' solution. An equal number of drops (2 or 3) of the well agitated suspension was delivered into each flask from a large bore capillary pipette avoiding the first and the last few drops in the pipette.