Freezing (cryopreservation) and recovery of axenic E. histolytica is always difficult as they are much more sensitive that many other cell types. The critical stage appears to be thawing of vials after removal from LN2. Only a few cells normally survive, so it is important to handle them gently and not to give up too soon!


The most detailed published protocol is Diamond, L. S. (1995) "Cryopreservation and storage of parasitic protozoa in liquid nitrogen. J Euk Microbiol 42(5): 585-590" but the one we have been using recently is based on one by Samarawickrema, N. A., et al. (2001) "A rapid-cooling method for cryopreserving Entamoeba histolytica. Ann. Trop. Med. Parasitol. 95(8): 853-855. "




!!!Pre-chill a cell freezer with isopropanol at -70 °C for at least 24hrs before starting!!!



Using two 16x125 mm borosilicate tubes, fill as follows (This is used even if the cells to be frozen are not axenic):



Volumes depend on the number of tubes being frozen. The protocol below is for 4 cryotubes, derived from 4 tubes of axenic cells:



Reviving of cells:


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