Trichomonas vaginalis Donne

1. To thaw a frozen ampule, place it in a 35°C water bath, until thawed (2 to 3 min).  Immerse the ampule just sufficient to cover the frozen material.  Do not agitate the ampule.
2. Immediately after thawing, aseptically transfer contents to a screw-capped test tube containing either 9 mL of ATCC medium 361 (completed with serum) or 13 mL ATCC Medium 2154 adjusted to pH 6.0.  Incubate the tube at 35°C (tube should be vertical for medium 361 or on a 15° horizontal slant for medium 2154).

1. When the culture is at or near peak density, place the tubes on ice for 10 minutes. 
2. Gently invert the culture tube 10 times and aseptically transfer a 0.1 mL to 0.4 mL aliquot to a screw-capped test tube containing either 9 mL of ATCC medium 361 (completed with serum) or 13 mL ATCC Medium 2154 adjusted to pH 6.0. 
3. Incubate the culture at 35°C (tube should be vertical for medium 361 or on a 15° horizontal slant for medium 2154). 
4. Transfer the culture every 3 to 4 days as described in steps 1 through 2.  The transfer interval will depend on the quantity of the inoculum and the quality of the medium.  This should be empirically determined by examining the culture on a daily basis until the growth cycle has stabilized. Do not allow the culture to overgrow. The culture crashes soon after reaching peak density.

1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min. The cells grown in a medium containing agar are concentrated by centrifugation, a solid pellet does not form. The soft pellet is resuspended to desired cell concentration with agar-free supernatant.
2. Adjust the concentration of cells to 2 x 10⁶  to  2 x 10⁷/mL in fresh medium.
3. While cells are centrifuging prepare a 10% (v/v) solution of sterile DMSO in fresh medium.
   1. Add 1.0 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 9.0 mL of ice cold medium.
   3. Invert several times to dissolve the DMSO.
   4. Allow to warm to room temperature.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 10⁶ to  10⁷ cells/mL and 5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should no less than 15 min and no longer than 30 min.
5. Dispense in 0.5 mL aliquots into 1.0 mL to 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 establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of the frozen material. Do not agitate the vial.
9. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate a 16 x 125 mm screw-capped test tube containing either 9 mL of ATCC medium 361 (completed with serum) or 13 mL ATCC Medium 2154 adjusted to pH 6.0.
10. Incubate the culture at 35°C with the cap screwed on tightly (tube should be vertical for medium 361 or on a 15° horizontal slant for medium 2154).

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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