Cryopreservation of Babesia bigemina for in vitro cultivation

Abstract

SUMMARY 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 −80C and then rapid transfer to liquid N 2 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% O 2 , 5% CO 2 , and 93% to 90% N 2 .