Long-Term Storage of Tetrahymena

Materials

 * Tetrahymena cells
 * 1×SPP or NEFF medium
 * Starvation Buffer (10 mM Tris-Cl pH 7.5)
 * 10% Dimethyl sulfoxide (DMSO)
 * 50 mL conical Falcon tube(s)
 * Cryo-vials
 * Nalgene cryo-container (filled with isopropanol)

Growth

 * 1) Grow 100 mL cells in modified NEFF medium at 30 °C in a platform shaker (for 24 hours typically).

Starvation

 * 1) Determine the appropriate volume of growth culture necessary to yield 100 mL of cells at a final concentration of 2×10^5 cells/mL and place this volume in 50 mL conical Falcon tube(s).  It is important that volume in which cells are resuspended has an OD540 = 0.3. Very high values will result in dead cells and poor growth following thawing.  
 * 2) Centrifuge at 1,100 g (2,250 rpm in back-bench or instrument room centrifuge) for 3 minutes, and decant the supernatant immediately.
 * 3) Resuspend cells in 100 mL of sterile Tris-Cl Starvation Buffer. Place the cells at 30 °C in a shaking incubator for 1-2 days.

Freezing

 * 1) Centrifuge 100 mL of the starved cells at 1,100 g for 3 minutes.
 * 2) Decant the supernatant, leaving behind a total volume of 1 mL of cells in the Tris buffer.
 * 3) Resuspend the cell pellet gently by tapping the bottom of the tube, and immediately add 4 ml of 10% DMSO to the resuspended cells. Mix gently but thoroughly.
 * 4) Aliquot 300 µL of cells in DMSO to the desired number of cryovials.   Include one extra vial to test thaw immediately after freezing to ensure that the batch is viable. 
 * 5) Incubate the DMSO-treated cells in the sealed cryo-vials for 30 minutes at room temperature.
 * 6) Place the cryo-vials in a Nalgene cryo-container. Place the entire container in a low temperature freezer (-70 C). Leave the container in the low-temperature freezer overnight to several days. Transfer the cryo-vials from the cryo-container to the liquid nitrogen tank.   Do not allow cryo-vials to be warmed up during transfer. 
 * 7) Test thaw one cryo-vial to ensure cells are viable.  Swimming cells are generally visible after 1 hour. Numerous cells should be observed after 24 hours.