Cryopreservation of Babesia bigemina for in vitro cultivation

Babesia bigemina-infected RBC and merozoites were cryopreserved and used to initiate in vitro cultures in normal bovine RBC; the cryoprotectant was a final 10% polyvinylpyrrolidone in Vega y Martinez solution. A cooling rate of 20 C/min until -80 C and then rapid transfer to liquid N2 storage was satisfactory. Samples for culture initiation were rapidly thawed at 37 C, washed in Vega y Martinez solution and resuspended in complete culture media containing 10% normal bovine RBC. The optimum culture conditions to reestablish cultures were a 24-well plate (16 mm ID), 5 mm in depth, and an atmosphere of 2% to 5% O2, 5% CO2, and 93% to 90% N2.