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Proteose Peptone (Difco 0120) ??????????????????????????????????? 5.0 g

Tryptone ???????????????????????????????????????????????????????????????????????????? 5.0 g

K₂HPO_{4???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????} 0.2 g

Glucose ????????????????????????????????????????????????????????????????????????????? 1.0 g

Liver extract??????????????????????????????????????????????????????????????????????? 0.1 g

Glass distilled water???????????????????????????????????????????????????????? 1.0 L

Dissolve components in glass distilled H₂O and autoclave.

Dryl?s Salt Solution

0.1 M NaH₂PO₄ ^.? 3H₂0_{?????????????????????????????????????????????????????????????????????????????} 10.0 ml

0.1 M Na₂HPO₄ ^. _?7H₂0_{?????????????????????????????????????????????????????????????????????????????} 10.0 ml

0.1 M Sodium citrate ^. 2H₂0 ????????????????????????????????????????? 15.0 ml

0.1 M CaCl₂ ^.? 2H₂0????????????????????????????????????????????????????????? 15.0 ml

Distilled water?????????????????????????????????????????????????????????????? 950.0 ml

Add the first 3 components to the distilled H₂O and mix thoroughly. ?Add the CaCl₂ solution and mix thoroughly. ?(Adding the solutions in the order indicated will avoid the precipitation of Ca salts.)

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.??????????

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.

a)? Add 2.2 ml of DMSO to an ice cold 20 x 150 mm screw-capped test tube;

b)? Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 7.8 ml of ice cold medium;

c)?? Invert several times to dissolve the DMSO;

d)? 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. ?At -50°C ampules are plunged into liquid nitrogen.

8.?? Store in the vapor or liquid phase of a nitrogen refrigerator.

9.?? To establish a culture from the frozen state aseptically add 0.5 ml sterile Dryl's Salt Solution to an ampule. ?Immediately place the ampule in a 35°C water bath until thawed (2-3 min).? Immerse the ampule just sufficiently 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.

CRYOPRESERVATION:

Alternative Thawing Procedure

1.?? Aseptically add 0.5 ml of sterile modified PYNFH medium (ATCC Medium 1034) containing 8% (w/v) sucrose to the ampule.? Immediately, place in a 35°C water bath, until thawed. ?Immerse the ampule just sufficient to cover the frozen material. ?Do not agitate the ampule.

2.?? Immediately after thawing, aseptically remove the contents of the ampule and gently add the material to the edge of a 20 x 100 mm petri plate containing ATCC Medium 919 (non-nutrient agar) and position on a 15 degree slant.  The cell suspension will pool at the edge of the plate.

3.?? Continue to double the volume of the cell suspension at 10 minute intervals by adding ATCC medium 1034 containing 4% sucrose (w/v).  When the volume reaches 16.0 ml place the plate in horizontal position and incubate at 25°C. 

4.?? On the following day, gently remove the cell suspension for the plate and transfer to a T-25 tissue culture flask.  Note the volume of the suspension and add a volume of fresh medium containing 4% sucrose equal to the volume of the cell suspension.  Incubate the culture at 25°C.

5.?? After the culture has been established subculture into fresh medium without sucrose.