Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Isolation
Renal organ from a garden snail (Helix aspersa), heliciculture farm, Valga, Spain
Product Format
test tube
Storage Conditions
Frozen Cultures: -70°C for 1 week; liquid N2 vapor for long term storage
Freeze-dried Cultures: 2-8°C
Live Cultures: See Protocols section for handling information
Type Strain
no
Comments
Morphological and molecular characterization
Medium
ATCC® Medium 1034: Modified PYNFH medium (Available from ATCC as ATCC cat. no. 327-X)
ATCC® Medium 357: Tetrahymena medium
ATCC® Medium 383: Haskins agar for Tetrahymena
Growth Conditions
Temperature: 18°C to 25°C
Culture System: Axenic
Cryopreservation
Reagents
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 CaCl2 solution and mix thoroughly. (Adding the solutions in the order indicated will avoid the precipitation of Ca salts.)
Harvest and Preservation
Transfer Tetrahymena from usual growth medium to RM-9 medium and allow to grow to near peak density.
Harvest cells from a culture by centrifugation at 300 x g for 2 min.
Adjust concentration of cells to 2 x 106/mL in fresh medium.
While cells are centrifuging, prepare a 22% (v/v) sterile solution of sterile DMSO in fresh medium.
Add 2.2 mL of DMSO to an ice cold 20 x 150 mm screw-capped test tube;
Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 7.8 mL of ice cold medium;
Invert several times to dissolve the DMSO;
Allow to warm to room temperature.
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.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
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.
Store in the vapor or liquid phase of a nitrogen refrigerator.
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.
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.
Name of Depositor
R Iglesias
References
Segade P, et al. Morphological and molecular characterization of renal ciliates infecting farmed snails in Spain. Parasitology 136: 771-782, 2009. PubMed: 19402940
