1. Mix the components of the cryoprotective solution in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 400 x g for 5 min.
3. Adjust the concentration of cells to at least 2 x 10⁵/mL in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions.
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.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.) xa0
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.xa0 Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.xa0xa0 Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 mL complete medium bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831™) or Enterobacter aerogenes (ATCC^® 13048™).
9. Incubate at 20-25°C with the cap screwed on tightly.
10. Follow the protocol for maintenance of culture.

Lahr DJ, et al. Comprehensive phylogenetic reconstruction of amoebozoa based on concatenated analyses of SSU-rDNA and actin genes. PLoS One 6(7): e22780, 2011. PubMed: 21829512

Nucleotide (GenBank) : JF694297 Actin

Nucleotide (GenBank) : JF694280 18S rRNA gene