1.? Harvest cells from a culture which is at or near peak density by adding 5 ml filtered artificial seawater and washing cells into suspension.? Rub the surface of the plate with a spread bar to detach adhering amoebae.
2.?? Transfer the liquid medium to a sterile centrifuge tube.
3.? If the cell concentration does not exceed 2 x 106 cells/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 20% (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.?
*NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
5.? Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be at least 106 cells/ml and 10% (v/v) DMSO.? The equilibration time? (the time between addition of DMSO? and the start of? the cooling cycle) should be no less than 15 min and no longer than 60 min.
6.?? Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen.
8.? The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
9.?? To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
10.Immediately after thawing, aseptically remove the contents of the ampule and distribute to the center of a fresh plate of ATCC medium 994.? Distribute the material evenly over the plate using a spread bar.? Incubate at 25°C.
