Pavlova lutheri (Droop) Green

This strain is routinely shipped as a growing culture in a glass 16 x 125 mm screw-capped test tube.  The volume of the cell suspension is approximately 5-6 mL.  When the culture arrives remove it promptly from the shipping container.  Do not store the culture at refrigeration temperatures before handling.  To assure viability, immediately loosen the test tube cap and incubate under light (~50 mEinsteins/m²/s irradiance) at a 15° horizontal slant at 20-25°C for at least one hour before observing the culture.  There should be numerous active trophozoites in suspension.  If the numbers are low the culture may have been exposed to temperature extremes in transit.  Regardless of the state of the culture, seal and agitate the culture, then aseptically transfer a 0.5 mL aliquot to a 16 x 125 mm screw-capped test tube containing 5 mL of sterile ATCC medium 1405.  Incubate the parent and daughter cultures at a 15° horizontal slant at 20-25°C with the caps on loosely for air exchange.  Maintain under a 14 hour light (~50 mEinsteins/m²/s irradiance)/10 hour dark cycle.

Subculture every two weeks to a fresh tube of medium in the following manner:

1. Seal and agitate the parent culture to suspend cells, then aseptically transfer 0.5 mL to a 16 x 125 mm screw-capped test tube containing 5 mL of sterile ATCC medium 1405.
2. Incubate the parent and daughter cultures at a 15° horizontal slant at 20-25°C with the caps on loosely for air exchange.  Maintain under a 14 hour light (~50 mEinsteins/m²/s irradiance)/10 hour dark cycle.

Note:  Addition of penicillin G at 50-75 U/mL and streptomycin at 50-75 µg/mL to the culture may be necessary if the bacterial density in the culture becomes high enough to affect growth.

1. Mix the components of the cryoprotective solution in the order listed.
2. Harvest cells from a culture that is at or near peak density by centrifugation at 800-1000 x g for 5 min.
3. Adjust the concentration of cells to at least 2 x 10⁷/mL in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions.
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 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.)  
7. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place the vial in a 35°C water bath.  Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.   Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate into a 16 x 125 mm screw-capped test tube containing 5 mL of sterile ATCC medium 1405.  Immediately seal and agitate the culture to evenly suspend cells, then aseptically transfer a 0.5 mL aliquot to a second, identical tube of medium.
9. Incubate the parent and daughter cultures at a 15° horizontal slant at 20-25°C with the caps on loosely for air exchange.  Maintain under a 14 hour light (~50 µEinsteins/m²/s irradiance)/10 hour dark cycle.
10. Follow the protocol for maintenance of culture.

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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