Euglena gracilis Klebs

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 contents to a 16 x 125 mm screw-capped borosilicate test tube containing ATCC Medium 351. Incubate the tube upright for one hour. 
3. Gently remove as much supernatant as possible (note: methanol can inhibit growth) and refill with an equal volume of fresh medium. 
4. 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.

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 which is at or near peak density by centrifuging at 100 x g for 1 minute
   Note: Centrifugation at the lowest speed and 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 reagent grade methanol solution that has been prepared in fresh ATCC medium 351.
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, before the cooling cycle begins, 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 and -70°C for no longer than one week.
9. To establish a culture from the frozen state follow steps 1-4 listed above under the heading ESTABLISHING A CULTURE FROM A FROZEN AMPULE.
   

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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