1. Harvest cysts from several cultures in stationary phase of growth. Detach cysts using a sterile cell scraper or cotton swab.
2. Aseptically transfer the cyst suspension to 15 ml plastic centrifuge tubes.
3. Centrifuge at ~800 x g for 5 min.
4. While cysts are centrifuging, prepare a 20% solution of DMSO in bacterized ATCC Medium 802. Cool on ice.
5. Remove the supernatant and pool the cell pellets to one-half the final volume desired with fresh growth medium.
6. Combine the cell suspension with an equal volume of 20% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 10% DMSO.
7. Dispense in 0.5 ml aliquots to 1.0-2.0 ml Nunc vials (special plastic vials for cryopreservation).
8. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. 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.).
9. Store ampules in a liquid nitrogen refrigerator until needed.
10. To establish a culture from the frozen state, place a frozen ampule in a 35°C water bath just enough to cover the frozen material. Allow the ampule to thaw completely (2-3 min).
11. Immediately after thawing, aseptically remove the contents and transfer to a T-25 tissue culture flask containing 10 ml of fresh ATCC medium 802 inoculated with Enterobacter aerogenes (ATCC 13048).
12.Screw the cap on tightly and incubate at 25°C. Subculture every 10-15d.