Tetrahymena pyriformis (Ehrenberg) Lwoff

Handling of Test Tube Cultures

This strain is routinely shipped as a growing culture in a glass 16 x 125 mm screw-capped test tube.  The volume of the cell suspension is approximately 5 ml.  When the culture arrives remove it promptly from the shipping container.  Do not store the culture at refrigeration temperatures before handling.  To assure viability, immediately loosen the test tube cap and incubate upright at 25°C for at least one hour before observing the culture.  There should be numerous active trophozoites in suspension.  If the numbers are low the culture may have been exposed to temperature extremes in transit.  Regardless of the state of the culture, aseptically transfer a 0.5 ml aliquot to a 16 x 125 mm screw-capped test tube containing 5 ml of sterilized ATCC medium 357.  Incubate the parent and daughter cultures upright with the caps on loosely at 25°C.

Routine Short-Term Maintenance of Culture:

1. Inoculate 5.0 ml of ATCC medium 357 with 0.1 ml from a Tetrahymena culture at or near peak density.
2. Incubate upright at 25°C with cap loosened one half turn.
3. For routine maintenance subculture every 10-14 d.

Routine Long-Term Maintenance of Culture:

1. Screw the cap on tightly of a Tetrahymena culture at or near peak density and invert several times. Aseptically transfer 0.1 ml aliquot to a fresh vessel of ATCC medium 383.
2. Loosen caps one half turn and incubate vertically at 25°C for 1 d.
3. Place cultures at 15°C for 3-6 wk.
4. For routine maintenance subculture every 3-6 wk; repeat steps 3-5.

1. Transfer Tetrahymena from usual growth medium to ATCC Medium 1034 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.
   1. Add 2.2 ml of DMSO to an ice cold 20 x 150 mm screw-capped test tube;
   2. Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 7.8 ml of ice cold medium;
   3. Invert several times to dissolve the DMSO;
   4. 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 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.
10. 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.
11. 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. 
12. 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.
13. 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.

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