Cyanidioschyzon merolae P. De Luca, R. Taddei & L. Varano

1. To thaw a frozen ampule, place it in a 35°C water bath until thawed (2-3 min).  Immerse the ampule enough to cover only the frozen material.  Do not agitate the ampule.
2. Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 5.0 mL of ATCC medium 2861. Incubate the tube on a 15° horizontal slant with the cap screwed on loosely (loosened one half turn) at 42°C under constant light.
3. Examine the culture using an inverted microscope for the presence of green microalgae. Growth of the strain may take several weeks to establish.

1. Screw the cap on tightly and vigorously agitate the culture.
2. Aseptically transfer a 0.5 mL aliquot to 5 mL of fresh medium in a 16 x 125 mm screw-capped test tube.
3. Screw caps on loosely (loosened one-half turn) and incubate on a 15° horizontal slant at 42°C under constant light.
4. Subculture every 2-3 weeks.

1. Harvest cells from a culture which is at or near peak density by centrifugation at ~800 x g for 10 min.
2. Adjust concentration of cells to 2 x 10⁷ - 2 x 10⁸ mL in fresh growth medium.  If the concentration is too low, centrifuge at ~800 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired cell concentration.
3. While cells are centrifuging, prepare a 10% (v/v) solution of sterile methanol in fresh growth medium. 
4. Mix the cell suspension and the methanol solution in equal portions. The final concentration will be 10⁷ – 10⁸ cells/mL and 5% (v/v) of methanol. The time from the mixing of the cell preparation and methanol stock solution before the freezing process is begun should be no less than 5 min and no more than 15 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryovials.
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. Store in either the vapor or liquid phase of a nitrogen refrigerator.
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 it is thawed.
9. Remove the vial from the water bath immediately after thawing.  Aseptically transfer the contents of the ampule into 5 mL of fresh ATCC medium 2861. 
10. Incubate the tube on a 15^° horizontal slant with the cap screwed on loosely (loosened one half turn) at 42°C under constant light.
11. Maintain as described above.  

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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