1. To detach trophozoites from the plate, flush the surface with 5 ml filtered artificial seawater.xa0 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.xa0 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:xa0 Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.xa0 Allow the DMSO to solidify.xa0 Add the required volume of refrigerated filtered artificial seawater.xa0 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 7.5% (v/v) DMSO.xa0 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 30 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 the vials in a controlled-rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate atxa0xa0xa0xa0xa0xa0xa0 -1°C/min through the heat of fusion.xa0 At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.)xa0xa0
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.xa0 Distribute the material evenly over the plate using a spread bar.xa0 Seal the plate with Parafilm, place upright in a moist-chamber, and incubate at 25°C.xa0 Trophozoites should be seen within 2-3 d.

Sawyer TKAn analysis of the principal genera of amoeba in surface waters of Chincoteague Bay, Virginia with descriptions of species Ph.D. thesis, Univ. Maryland, 1973