Sorogena stoianovitchae Bradbury and Olive

1. When the Sorogena have sufficiently reduced the number of prey Colpoda in the culture, their ability to aggregate and form sorocarps is diminished, and both predator and prey form cysts.  To revive an encysted culture, the liquid overlay should be aseptically removed and replaced with fresh medium, which will stimulate new bacterial growth and encourage excystment of Colpoda and then Sorogena.
2. Incubate the culture at 20-25°C under a 14 hour light (~50 µEinsteins/m²/s irradiance)/10 hour dark cycle.  Active trophozoites (ciliates) of both Colpoda and Sorogena should be observed within 2-3 days. Sorogena sorocarps (cysts) should form just above the air-liquid interface in 5-7 days.
3. To encourage faster proliferation of Sorogena, additional Colpoda sp. may be added to the culture from a separate culture of the prey organism kept in parallel (i.e., ATCC^® 30920™ or similar, not provided).
4. The Sorogena may be passaged to a new petri plate or T-25 flask by gently rubbing the agar surface with a spread bar to dislodge attached sorocarps and/or cysts, then transferring 0.5 to 2 mL to a fresh petri plate or T-25 flask containing a bed of agar medium and 10-15 mL total liquid overlay (ATCC medium 1330).
5. Incubate the culture at 20-25°C under a 14 hour light (~50 µEinsteins/m²/s irradiance)/10 hour dark cycle.  Active trophozoites (ciliates) of both Colpoda and Sorogena should be observed within 2-3 days. Sorogena sorocarps (cysts) should form just above the air-liquid interface in 5-7 days.  Several cycles of growth and encystment may be required in order for the Sorogena culture to produce fruiting bodies.

1. Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.
2. Harvest Sorogena and Colpoda cysts from a culture that has recently passed peak density by centrifugation at 1000 x g for 5 min.
3. Adjust the concentration of cells to at least 2 x 10⁴/mL in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions by adding the cryoprotective solution to the cell suspension in 3 equal aliquots at 2 min. intervals.
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 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. 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. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place the vial in a 35°C water bath. Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial. Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and transfer to a petri plate or T-25 tissue culture flask containing a bed of agar medium and 10-15 mL total liquid overlay (ATCC medium 1330).
9. Optionally, aseptically transfer 0.2-0.5 mL from a growing culture of Colpoda sp. to the petri plate or T-25 flask (See section on Culture Maintenance).
10. Incubate the culture at 20-25°C under a 14 hour light (~50 µEinsteins/m²/s irradiance)/10 hour dark cycle.  Active trophozoites (ciliates) of both Colpoda and Sorogena should be observed within 2-3 days.  Once the culture is established, follow the protocol for maintenance of culture.

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