Ulothrix gigas (Vischer) Mattox and Bold

1. To thaw a frozen ampule, place 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.
2. Immediately after thawing, aseptically transfer contents to a screw-capped borosilicate test tube containing ATCC Medium 847 broth. Incubate the tube on a 15° horizontal slant at 50-100 µEinsteins/m²/s irradiance at 25°C with the cap loosened one half turn. Maintain under a 14/10 h light-dark photoperiod.

1. Inoculate a tube of fresh broth medium with 0.1 mL from a growing culture at or near peak density.
2. Incubate at 50-100 µEinsteins/m²/s irradiance at 25° C with the cap loosened one half turn. Maintain under a 14/10 h light-dark photoperiod.

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 - 2 x 10⁷/mL in fresh broth medium.
3. While cells are centrifuging prepare a 14% (v/v) solution of sterile DMSO in fresh broth medium.
4. Mix the cell preparation and the 14% DMSO in equal portions. Thus, the final concentration will be 10⁶ - 10⁷ cells/mL and 7% (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 5 min and no greater than 15 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 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 5 mL of ATCC medium 847 broth.
10. Incubate the culture on a 15° horizontal slant at 25°C with the cap screwed on loosely (loosened one-half turn) and incubate under a 14 hour light (~50 µEinsteins/m²/s irradiance)/10 hour dark cycle.

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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