Improved development of Brugia microfilariae following cryopreservation in liquid nitrogen using a technique suitable...

A technique for improved cryopreservation at -196 degrees C of Brugia spp. microfilariae has been developed by modifications of a procedure previously used with Onchocerca spp. A double incubation in ethanediol (ED) solutions, firstly at 37 degrees C in 10% (v/v) ED for 15 min and secondly at 0 degrees C in 40% (v/v) ED for 45 sec followed by plunging into liquid nitrogen, resulted in over 90% of the microfilariae of B. malayi exhibiting normal motility. When used with B. pahangi microfilariae, followed by blood-feeding to Aedes aegypti (Bels New strain) a third-stage larvae yield of 4.04 per fly was obtained, compared with 5.16 in the unfrozen group. This represents an infectivity level of 78% compared with unfrozen controls. The technique also has the benefits of being suitable for field conditions, requiring no sophisticated equipment, merely some ice, ethanediol and liquid nitrogen.