1. Transfer tetrahymena from usual growth medium to RM-9 medium and allow to grow to near peak density.
2. Harvest cells from a culture by centrifugation at 300 x g for 2 min.xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0
3. Adjust concentration of cells to 2 x 10⁶/mL in fresh medium.
4. While cells are centrifuging, prepare a 22% (v/v) sterile solution of sterile DMSO in fresh medium.
   1. Add 2.2 mL of DMSO to an ice cold 20 x 150 mm screw-capped test tube;
   2. Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 7.8 mL of ice cold medium;
   3. Invert several times to dissolve the DMSO;
   4. Allow to warm to room temperature.
5. Add a volume of the DMSO solution equal to the cell suspension volume but add in 3 equal aliquots at 2 min intervals. Thus, the final concentration of the preparation will equal 11% (v/v) DMSO and 10⁶ cells /mL.
6. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. Place the ampules in a controlled rate freezing unit. The cooling cycle should be initiated no less than 15 min and no longer than 60 min after the addition of the DMSO to the cell preparation. 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. Atxa0 -50°C ampules are plunged into liquid nitrogen.
8. Store in the vapor or liquid phase of a nitrogenxa0refrigerator.
9. To establish a culture from the frozen state aseptically add 0.5 mL sterile Dryls Salt Solution to an ampule. Immediately place the ampule in a 35°C water bath, until thawed (2-3 min).xa0 Immerse the ampule just sufficient to cover the frozen material. Do not agitate the ampule.
10. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5.0 mL of fresh medium in a 16 x 125 mm screw-capped test tube with a slightly loosened cap. Incubate at 25°C.

Borden D, et al. The inheritance of enzyme variants for tyrosine aminotransferase, NADP dependent malate dehydrogenase, NADP dependent isocitrate dehydrogenase and tetrazolium oxidase in Tetrahymena pyriformis syngen 1. Genetics 74: 595-603, 1974.

Simione FP Jr., et al. The use of plastic ampoules for freeze preservation of microorganisms. Cryobiology 14: 500-502, 1977. PubMed: 891238

Nanney DL, et al. Comparison of sequence differences in a variable 23S rRNA domain among sets of cryptic species of ciliated protozoa. J. Eukaryot. Microbiol. 45: 91-100, 1998. PubMed: 9495037

8310 C: Chemotactic test with freshwater ciliate Tetrahymena thermophila. Washington, DC:American Public Health Association;Standard Methods for the Examination of Water and Wastewater, 2005 APHA8310 - Ciliated Protozoa;.