Tetrahymena thermophila Nanney and McCoy
ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.
ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
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.)
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
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
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.
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 culture has been established subculture into fresh
normal medium without sucrose.
The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.
While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.
This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.
Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.
If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.