Parauronema acutum Buddenbrock

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 sterile ATCC medium 1651.  Incubate the parent and daughter cultures upright with the caps on loosely at 25°C.

1. Screw the cap on tightly and vigorously agitate the culture.
2. Aseptically transfer a 0.1-0.25 mL aliquot to 5 mL of fresh medium in a 16 x 125 mm screw-capped test tube or T-25 tissue culture flask.
3. Incubate at 25°C (test tubes are incubated upright with the cap loosened one-half turn).
4. Subculture every 3-5 days.

1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.
2. Adjust the concentration of cells to 2 x 10⁶ /mL in fresh medium.
3. While cells are centrifuging prepare a 22% (v/v) solution of sterile DMSO in fresh medium.
4. Mix the cell preparation and the 22% DMSO in equal portions. Thus, the final concentration will be 10⁶ cells/mL and 11% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the beginning of the freezing process should be no less than 15 min and no greater than 60 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.  At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.  Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.  (The cooling rate in this apparatus is approximately -1°C/min.)  
7. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.
8. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.  Do not leave ampule in water bath after thawed.
9. Immediately after thawing, do not leave in the water bath, gently remove the contents of the ampule with a Pasteur pipette and expel slowly into a 16 x 125 mm screw-capped test tube or T-25 tissue culture flask.  Incubate at room temperature (approx. 25°C) for 15 min.
10. At 15 min intervals add 0.25 mL of ATCC medium 1651 dropwise. Continue until the final volume is 2.0 mL.
11. Allow the culture to remain undisturbed for 15 min.
12. Add 0.5 mL of medium dropwise at 15 min intervals until the volume is 4.0 mL.
13. Allow the culture to remain undisturbed overnight.
14. On the morning of day 2 slowly add 4.0 mL of ATCC medium 1651. Allow the culture to remain undisturbed overnight.
15. When the culture is established, follow the protocol for maintenance of culture.

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.

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