Amoeboaphelidium protococcarum Gromov and Mamkaeva

1. For a plate culture, transfer cells with an inoculating loop to a plate of fresh agar medium from a growing culture at or near peak density.  For a broth culture, inoculate a tube of fresh broth medium with 0.3 to 0.5 mL from a growing culture at or near peak density. 
2. Incubate at 25°C under a 14 hour light (~50 µEinsteins/m²/s irradiance)/10 hour dark cycle.  In the case of a broth culture, screw tube cap on loosely (loosened one-half turn) and incubate on a 15° horizontal slant.
3. Subculture as necessary (typically every 1-2 wks).

1. Allow amoebae to encyst.  Harvest cysts from a culture that has recently passed peak density by centrifugation at 800 x g for 5 min.
2. Adjust the concentration of cysts to 2 x 10⁶ - 2 x 10⁷/mL in fresh medium.
3. While cysts are centrifuging prepare a 20% (v/v) solution of sterile DMSO in fresh ATCC medium 1323 (Page's Balanced Saline).
4. Mix the cell preparation and the 20% DMSO solution in equal portions. Thus, the final concentration will be 10⁶ - 10⁷ cells/mL and 10% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO cryoprotective solution to the beginning of the freezing process should be no less than 15 min and no greater than 60 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.  At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.  Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.  (The cooling rate in this apparatus is approximately -1°C/min.)  
7. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
9. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and add to the surface of an ATCC medium 5 agar plate containing a growing culture of Scenedesmus obliquus.  Alternatively, transfer the thawed contents to a 16 x 125 mm screw-capped test tube containing a growing culture of Scenedesmus obliquus in 5 mL of ATCC medium 5 broth.
10. Incubate the culture at 50-100 µEinsteins/m²/s irradiance at 25°C.  Maintain under a 14/10h light-dark photoperiod.

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