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The effects of virus input multiplicity and of tissue cell concentration upon the growth of Rift Valley fever virus in L cells (Earle) were determined. The titers obtained in suspension cultures with cells obtained from two separate laboratories were significantly different. With both monolayer culture and suspension culture systems, a virus input multiplicity of 2.5 resulted in the greatest proliferation of virus. Optimal viral yields were obtained in suspension cultures containing 4 X 105 tissue cells per ml of suspension.

Studies were conducted on the interaction of various parameters which affect the storage stability and growth potential of liquid cultures of Pasteurella tularensis live vaccine strain (LVS) and Rift Valley fever virus Van Wyk strain (RVFV). Storage variables studied with LVS included four storage temperatures (4,-20,-65,-175 C), single and multiple freeze-thaw cycles, two freezing and two thawing rates (slow and fast), various inoculum levels (1, 3, 5, and 10%) for the determination of growth potential, and the retention of immunizing potential (mice and guinea pig) after storage. Neither the freezing rate nor the number of freezethaw cycles seriously affected the growth of LVS after storage at-175 C; however, the slow rate of thaw proved deleterious as were all temperatures of storage except-175 C after 1 year of storage, as shown by both criteria of evaluation. RVFV produced in two combinations of cell lines and media (LM cell line-199 peptone medium and LDR cell line-Eagle's minimum essential medium) was stored at three serum levels (10, 20, 40%), three pH values (6.2., 7.0, 7.8), and three temperatures (-20,-65,-175 C). These studies indicated: (i) virus produced in the LDR cell line and Eagle's medium was more stable than that produced in the LM cell line and 199 peptone medium for either shortor long-term storage; (ii) serum levels did not affect stability; and (iii) low pH resulted in losses during long-term storage under all conditions tested. Thus, cryogenic storage is advantageous for stock culture maintenance of bacteria and viruses and for other similar applications.

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