Plaquing of Viruses in the Absence of a Carbon Dioxide Atmosphere

Extensive plaquing studies performed in this laboratory with respiratory syncytial (RS) and parainfluenza (types 2 and 3) viruses have made it necessary to employ an overlay medium that does not require a CO2 atmosphere for the maintenance of the tissue culture film or for virus growth. Since the experiments being undertaken involve different incubation temperatures ranging from 26 to 36 C, the use of multiple CO2 incubators becomes both impractical and expensive. To facilitate the plaquing of these viruses either for cloning or for determining PFU titers in the absence of a CO2 atmosphere, medium L-15 (A. Leibovitz, Am. J. Hyg. 78:173, 1963) is being employed routinely. This medium, as prepared by Flow Laboratories Inc., is supplied as a 2 X concentrate containing 1 x tyrosine and 4 mg of phenol red per liter (this concentration of phenol red facilitates subsequent staining of films with neutral red) together with a separate but equal volume of 1 x tyrosine dissolved in distilled water. The latter solution is used to prepare the desired concentration of 2X agar or methyl cellulose (4,000 centipoise) and is autoclavable to insure sterility. We routinely have employed a 1%