Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Isolation
Spinal cord homogenate from horse (2-yr-old thoroughbred filly), Panama, circa 1991.
Product Format
frozen
Storage Conditions
Frozen Cultures: -70°C for 1 week; liquid N2 vapor for long term storage
Freeze-dried Cultures: 2-8°C
Live Cultures: See Protocols section for handling information
Genome Sequenced Strain
Yes
Comments
Causative agent of equine protozoal myeloencephalitis (EPM)
Growth Conditions
Temperature: 37 °C Atmosphere: 5% CO2 Cell line: Bovine turbinate cells (ATCC® CRL-1390™)
Cryopreservation
Harvest and Preservation
Harvest the Sarcocystis culture by gently agitating the contents of each flask. Transfer all but approximately 1 mL of the culture medium to 15 mL plastic centrifuge tubes. Detach the remaining infected and uninfected tissue culture cells by scraping the inner surface of the flask with a cell scraper. Pass the resulting cell suspension through a syringe equipped with a 27-gauge ½-inch needle and pool this suspension with the culture fluid.
Spin the cell suspensions at approximately 50 x g for 3 min, to remove the cellular debris.
Transfer the supernatants to new 15 mL plastic centrifuge tubes. Centrifuge at 1300 x g for 10 min.
Pool the parasite pellets and adjust the concentration to ~2.0 x 107 merozoites/mL with fresh Advanced MEM.
*If the concentration is too low, centrifuge at 1300 x g for 10 min and resuspend in the volume of Advanced MEM required to yield the desired parasite concentration.
Mix equal volumes of parasite suspension and fresh medium containing 20% DMSO and 50% HIFBS to yield a final concentration of ~1 x 107 merozoites/mL in 10% DMSO, 25% HIFBS. The time from the mixing of the parasite preparation and the cryoprotective solution before the freezing process begins should be no less than 15 min and no more than 30 min.
Dispense in 0.5 mL aliquots to 1.0-2.0 mL sterile plastic screw-capped cryovials.
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.
Store in either the vapor or liquid phase of a nitrogen refrigerator.
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 thawing.
Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC® CRL-1390™ cells and 10 mL of Advanced MEM with 4% (v/v) HIFBS.
Outgas the flask for 10 seconds with a 95% air, 5% CO2gas mixture.
Incubate in a 35-37°C CO2 incubator with the caps screwed on tightly.
