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1. Harvest cells from a culture which is at or near peak density by centrifuging at 100 x g for 1 minute. Note: Centrifugation at the lowest speed and for the shortest time to allow sedimentation of the cells will maximize recovery.
2. Adjust the concentration of cells to 4 x 10⁶/mL with fresh broth medium.
3. Transfer the concentrated cell suspension to a sterile Petri dish and allow the cells to remain undisturbed for at least one hour.
4. Transfer the cell suspension (note the volume) from the Petri plate to a 15 mL plastic centrifuge tube.
5. Add an equal volume of 6% (v/v) sterile methanol solution that has been prepared in fresh ATCC medium 351 broth. Mix gently but thoroughly.
6. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation). The time from mixing of the cell preparation and the methanol solution to the start of the cooling cycle should be no greater than 15 min.
7. Place vials in a controlled rate freezing unit. 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 -40°C plunge ampules 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.)
8. 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 can be stored between -80°C and -70°C for no longer than one week.
9. To establish a culture from the frozen state, aseptically add 0.5 mL fresh ATCC medium 351 broth to the frozen pellet, then place the ampule in a 35°C water bath until thawed (2-3 min). Immerse the ampule just sufficiently to cover the frozen material. Do not agitate the ampule.
10. Immediately after thawing, aseptically transfer the entire contents to a single 16 x 125 mm screw-capped test tube containing 5 mL of ATCC medium 351 broth. Incubate the tube upright for one hour at 25°C.
11. Gently remove as much supernatant as possible (the methanol cryoprotectant can inhibit growth) and refill with an equal volume of fresh broth medium.
12. Incubate on a 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. Note: Some strains may grow poorly or not at all when recovered from the frozen state directly into 5 mL of broth medium in a test tube. In such cases recovery may be improved by instead using a plate or flask containing a bed of ATCC medium 351 agar and gently increasing the volume of liquid medium incrementally by 1.0 mL every 10 min to a total of 8 mL. The plate or flask should be kept at a slight angle from the horizontal plane to pool the fluid to one side. Once motile cells are observed, they may be aseptically transferred to a single 16 x 125 mm screw-capped test tube containing 5 mL of ATCC medium 351 broth and incubated as indicated above.

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