Bioassay to measure effects of cooling and warming rates and protection by dimethyl sulphoxide on survival of frozen Babesia rodhaini

The percentages of Babesia rodhaini parasites that survived different rates of cooling to −79 °C were determined by titrating infectivity in CBA mice before freezing and after thawing. The cryoprotective effect of DMSO and the effect of warming rate were also assessed.When parasitized blood containing 1.5 DMSO was cooled at nominal rates of 2.5 °, 265 °, and 2785 °C/min and warmed at 4320 °C/min, the respective survival rates were 0.075, 4.9, and 0.1%, indicating the existence of an optimal cooling rate. Blood without DMSO cooled and warmed under the same conditions was over 1000 times less infective. When parasitized blood containing DMSO was cooled at 2785 °C/min and warmed at 4320 °, 24.5 °, and 1.84 °C/ min, infectivity decreased progressively with the warming rate. The degrees of haemolysis in frozen and thawed blood indicated that cooling rate was more important than an intact host cell to survival of the parasite.The growth rate of B. rodhaini in CBA mice, estimated to be one binary fission in 8.5 hr, was not affected by the addition of DMSO followed by freezing and thawing.