Duration: monoxenic; non-nutrient agar with E. coli
1. Allow the cells to encyst. To detach cysts from the plate flush the surface with 5 ml fresh ATCC® medium 1323 (Page's Balanced Salt Solution). Rub the surface of the plate with a spread bar to detach adhering amoebae.
2. Transfer the cyst suspension to a sterile centrifuge tube.
3. If the cyst concentration does not exceed 2 x 106 cysts/ml adjust the suspension to that concentration. To adjust the concentration, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield 2 x 106.
4. While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath. Allow the DMSO to solidify. Add the required volume of refrigerated medium. Dissolve the DMSO by inverting the tube several times.