1. To achieve the best results, set up cultures with several different inocula (i.e., 0.25 mL, 0.5 mL, 1.0 mL).xa0 Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density.
2. If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to approximately 2 x 10⁶ cells/mL with fresh growth medium.xa0 If the concentration is too low, centrifuge at 400-500 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
3. While cells are centrifuging prepare a 20% (v/v) solution of sterile DMSO as follows:xa0 Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.xa0 Allow the DMSO to solidify.xa0 Add the required volume of refrigerated medium.xa0 Dissolve the DMSO by inverting the tube several times.xa0
   Note: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be approximately 10⁶ cells/mL and 10.0% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should be no less than 15 min and no longer than 30 min.
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 the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.xa0 At -40°C plunge 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 approximatelyxa0-1°C/min.) xa0
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
8. To establish a culture from the frozen state, quickly add 0.5 mL spent/exhausted ATCC medium 802* supplemented with 12% (w/v) sucrose to the frozen ampule and place it 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.
   *Spent/exhausted medium is medium which has been previously used for cultivation of food-source bacteria and subsequently filter-sterilized for general use. The bacteria consume many of the nutrients in the medium, reducing its capacity to promote additional bacterial growth when re-used. It is prepared by passing supernatant from an advanced culture (i.e., one which is at or near stationary phase) through a 0.22 μm filter.
9. Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate onto the surface of a 20 x 100 mm petri plate containing ATCC medium 919 (non-nutrient agar) with an overlay of 15.0 mL spent ATCC medium 802 supplemented with 6% (w/v) sucrose.
10. Incubate at 20-25°C with the cap on loosely.
11. Once the culture is established (motile cells observed), aseptically transfer approximately 8 mL to an upright 20x150mm glass test tube and gently overlay with an equal volume of ATCC medium 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831™) or Enterobacter aerogenes (ATCC^® 13048™).xa0 The bacterized medium should form a bilayer with the spent medium below.
12. When cells migrate from the layer of spent medium upward into the layer of bacterized medium, aseptically remove a 0.5-1.0 mL aliquot and transfer to a T-25 tissue culture flask containing 10.0 mL of ATCC medium 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831™) or Enterobacter aerogenes (ATCC^® 13048™).
13. Incubate with the cap tightly sealed at 20-25°C.
14. Follow the protocol for maintenance of culture.

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McCoy JW. New features of the Tetrahymenid cortex revealed by protargal staining. Acta Protozool. 13: 135-159, 1974.

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