Thawing Tetrahymena cells

Materials

 * Tetrahymena stocks in LN2
 * Water bath (set to 42 °C)
 * Microfuge tubes  1.7 mL
 * Petri dish(es)
 * 1×SPP or NEFF medium
 * Penicillin G & Sodium sulfate
 * Fungizome

Method
''Liquid Nitrogen in the tank is extremely cold. Use cold gloves and under no circumstance touch any tank apparatus with bare hands. Wear splash resistant lab coats and goggles.''

Ensure thawing area is sterile by wiping surfaces with 70% ethanol.
 * 1) Pipette out 1.5 mL SPP or NEFF medium into microfuge tubes and place in water bath and allow equilibrating to 42 °C (~5-10 minutes). Pipette out 10 mL SPP in petridishes and add selective antibiotics.  Addition of penicillin, fungizome and any other antibiotics should be just prior to use in thawing Tetrahymena cultures. 
 * 2) Take one cryo-vial out of the liquid nitrogen freezer. Swirl the tube gently in the 42 °C for 15 seconds until the frozen pellet starts to thaw and come loose from the edges of the cryo-vial.
 * 3) Immediately pour the pre-warmed 1.5 mL medium from the microfuge tube and add to the cryo-vial. Recap the cryo-vial and continue swirling for about a minute or until the ice pellet starts to melt. Once thawing is complete, transfer the entire volume of the thawed cultures from the cryo-vial to the petridishes.
 * 4) Incubate at 30 °C in a moisture chamber.
 * 5) Check for swimming cells that should be visible in 1 hour.
 * 6) Keep plate at 30 °C to allow healthy cells to grow for 24 hours. Ensure that there is no contamination.