Successful cryopreservation of adult male Onchocerca gutturosa in liquid nitrogen.
Experiments involved the use of methanol and ethanediol (ED) as potential cryoprotectants for the storage of adult male Onchocerca gutturosa at low temperatures. Worms were incubated with or without 20% (v/v) methanol for 15 min at 0 degrees C before cooling to -10 degrees, -25 degrees or -79 degrees C. Results of trials of storage from 1 to 14 days showed that prolonged survival could only be obtained by storing at -79 degrees C without the addition of cryoprotectant. However, worms were also successfully cryopreserved in liquid nitrogen (-196 degrees C) using a simple 2-step procedure in which ED was used as an essential cryoprotectant. This schedule involved an initial incubation of parasites in serum free media with 10% (v/v) ED at 37 degrees C for 15 min, followed by incubation in 40% (v/v) ED for 10 min at 0 degrees C on crushed ice. Worms were then transferred individually into 20 microliter drops of 40% (v/v) ethanediol on glass coverslips at 0 degrees C and immediately plunged into liquid nitrogen. A rapid thaw of the parasites resulted in normal motility and survival levels equivalent to unfrozen controls. This technique will be useful for the long-term storage and transportation of this and possibly other species of Onchocerca parasites.
D000818:Animals; D003451:Cryoprotective Agents; D005026:Ethylene Glycols; D005615:Freezing; D008297:Male; D000432:Methanol; D009584:Nitrogen; D009854:Onchocerca; D011309:Preservation, Biological
D003451:Cryoprotective Agents; D005026:Ethylene Glycols; D009584:Nitrogen; D000432:Methanol