RM-9 Media for cryopreservation of Tetrahymena Proteose Peptone (Difco 0120) ??????????????????????????????????? 5.0 g
Tryptone ???????????????????????????????????????????????????????????????????????????? 5.0 g
K2HPO4??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? 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 CaC12 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 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.
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 frozen 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 a T-25 flask and position the flask on a 15 degree slant. The cell suspension will pool at the bottom of the flask.
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 8.0 ml place the flask in horizontal position with the cap screwed on tightly and incubate at 25°C.
4.?? On the following day, add 8.0 ml of ATCC medium 1034 (this will dilute the sucrose to 2%).? Incubate the culture at 25°C.
5.?? When the culture has reached peak density, gently agitate and aseptically transfer a 0.1 ml aliquot into 5 ml of ATCC medium 357 in a 16 x 125 mm screw capped glass test tube.? Incubate vertically with the cap screwed on loosely.
6.?? Transfer every 7-10 days by repeating step 5.
