Long Term Maintenance of Cell Cultures under Agar Overlay and Development of Herpes T Plaques

Summary

A method for the long term maintenance of various cell cultures under agar overlay with the use of Temin's Eagle's medium has been demonstrated. Primary cell cultures of chick fibroblast, cat kidney, rabbit kidney, and chorioallantoic membrane were maintained under agar overlay for 53, 32, 24, and 17 days, respectively. Cell lines of palm civit, HeLa, rabbit kidney, and African green monkey kidney were maintained for 25, 24, 21, and 13 days respectively under the same conditions. These long term cultures proved useful to study the plaque forming ability of several viruses (herpes T, herpes simplex, herpes canis, herpes suis, infectious bovine rhinotracheitis, sand rat nuclear inclusion agent, Cebus monkey strain (an adeno virus), another herpes virus (squirrel monkey isolate no. 83), and plaque-purified Newcastle disease virus (Delaware-Hickman strain). In chick fibroblast herpes T virus and Newcastle disease virus formed plaques. The herpes T virus plaques developed to a maximum plaque size of 15 mm in 45 days and remained at this plaque size up to 53 days with recovery of viable virus. The Newcastle disease virus-large plaque reached a maximum plaque size of 5 mm within 10 days. In rabbit kidney primary herpes T, herpes simplex, herpes suis, infectious bovine rhinotracheitis, and sand rat nuclear inclusion agent formed plaques reaching maximum plaque sizes of 10 mm, 7-8 mm, 7 mm, 4 mm, and 2-2.5 mm, respectively within a period of 14 days. This cell culture remained viable up to 24 days with herpes T plaques. The rest of the viruses destroyed the rabbit kidney primary cells within 2-3 weeks.