Chlorella pyrenoidosa Chick

Potential strain for oil or fat production

Storage and Culture Initiation

Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen.  If liquid nitrogen storage facilities are not available, frozen ampules may be stored at or below -70°C for approximately one week.  Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C).  Storage of frozen material at this temperature will result in the death of the culture.

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 the entire contents to a single 16 x 125 mm screw-capped test tube containing 5 ml of ATCC medium 1908 broth.  Incubate the tube on a 15^o horizontal slant with the cap screwed on loosely (loosened one half turn) at 25°C in the dark. Alternatively, add the entire thawed contents to the surface of a 20 x 100 mm Petri plate containing 20 ml of ATCC medium 1908 agar.  Wrap the plate culture with parafilm and incubate upright in the dark.

1. For a plate culture, transfer cells with an inoculating loop to a plate of fresh agar medium from a growing culture at or near peak density. For a broth culture, inoculate a tube of fresh broth medium with 0.1 ml from a growing culture at or near peak density.
2. Incubate at 25°C in the dark, with the cap loosened one half turn in the case of a test tube culture.
3. Subculture every 14-21 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 - 2 x 10⁷/ml in fresh medium.
3. While cells are centrifuging prepare a 10% (v/v) solution of sterile methanol in fresh medium.
4. Mix the cell preparation and the 10% methanol in equal portions. Thus, the final concentration will be 10⁶ - 10⁷ cells/ml and 5% (v/v) Methanol. The time from the mixing of the cell preparation and methanol 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 add to a centrifuge tube containing 5 ml of ATCC medium 1908 without agar.  Centrifuge at 300 x g for 5 min.
10. Remove most of the supernatant (=methanol, which can inhibit growth) and then resuspend the pellet.  Transfer the culture to a 16 x 125 mm screw-capped test tube containing 5 ml of ATCC medium 1908 broth or to the surface of an ATCC medium 1908 agar plate (20 x 100 mm Petri plate containing 20 ml of ATCC medium 1908 agar).
11. Incubate the culture at 25°C in the dark.

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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