Cryoprotective Solution
DMSO, 1.5 ml
Fresh growth medium w/o bacteria, 8.5 ml

Note: Care must be taken to maintain this organism at 4°C at all times.  Media and cell suspension should be kept on ice during entire process.

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.  Chill to 4°C.
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 800 x g for 5 min.
3. Adjust the concentration of cells at least 2 x 10⁶/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.  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.  Do not leave in water bath after thawing. Aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 ml chilled ATCC medium 2417 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^®  700831).
9. Incubate at 4°C with the cap screwed on tightly.
10. Once the culture is established, vigorously agitate the flask and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 2417.
11. Follow the protocol for maintenance of culture.

Wainright PO, et al. Monophyletic origins of the metazoa: an evolutionary link with fungi. Science 260: 340-342, 1993. PubMed: 8469985

Leadbeater BSC, et al. A new genus, Helgoeca gen. nov., for a nudiform choanoflagellate. Eur. J. Protistol. 44:227-237, 2008. PubMed: 18403187

Carr M, et al. Molecular phylogeny of choanoflagellates, the sister group to Metazoa. Proc. Natl. Acad. Sci. USA. 105: 16641-16646, 2008. PubMed: 18922774