Abstract
Discussion and Conclusions
The lack of a uniform and reproducible plaque assay for rhinoviruses (now totaling about one hundred types) has handicapped characterization of these important human pathogenic agents. Different procedures involving variations in the optimal cell culture, composition of the overlay medium and incorporation of anti-inhibitor and/or enhancing substances (e.g., DEAE dextran and Mg++ ions) have been described by various workers. In an attempt to develop a simple and reproducible procedure for plaquing a substantial number of rhinoviruses, we found the following conditions to be optimal for plaque formation by all 16 rhinoviruses that have been tested: (1) fully grown WI-38 cell monolayers in prescription bottles; (2) an overlay medium consisting of HCAEM containing 10% newborn calf serum and 0.7% Ionagar No. 2; (3) double agar overlay technique with the second overlay (containing neutral red) added 6 days after the first; and (4) incorporation of 25 μg/ml of DEAE dextran to the first overlay medium as an additional means to improve plaque formation.
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