Successful cryopreservation of Brugia pahangi third-stage larvae in liquid nitrogen

Experiments are described which lead to the retention of infectivity of Brugia pahangi third-stage larvae after cooling to -196 degrees C. Methanol, a well documented cryoprotectant was used at a concentration of 20% (v/v). The schedule consisted of a 5 degrees C min-1 cool to an intermediate temperature of -21 degrees C and a subsequent rapid cool into liquid nitrogen. A rapid thaw of the parasites led to approximately 34% of motile cryopreserved larvae developing in multimammate rats (Mastomys natalensis) compared to unfrozen control larvae. Cooling rate and intermediate temperature were both found to be crucial variables affecting survival levels of the larvae.