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
hemolymph of oyster, Crassostrea virginica, Tilghman Wharf, Tilghman, MD, 1992
Product Format
frozen
Storage Conditions
Frozen: -70°C or colder for 1 week, vapor phase of liquid nitrogen for long-term storage
Axenic/Xenic
Axenic
Type Strain
no
Comments
Tetrazolium-based cell proliferation assay
Medium
ATCC® Medium 1886: Perkinsus broth medium
ATCC® Medium 2684: DME/F12-3 Growth Medium (29ppt)
Growth Conditions
Temperature: 25°C
Cryopreservation
To achieve the best results set up cultures with several different inocula (e.g. 0.25 ml, 0.5 ml, 1.0 ml). Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density.
If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107 cells/ml with fresh growthmedium. If the concentration is too low, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
While cells are centrifuging prepare a 20% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath. Allow the DMSO to solidify. Add the required volume of refrigerated medium. Dissolve the DMSO by inverting the tube several times.
*NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 106 and 107 cells/ml and 10.0% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should be no less than 15 min and no longer than 30 min.
Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
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.)
The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 10 ml of fresh ATCC medium 1886 in a T-25 tissue culture flask. Incubate at 25°C.
Name of Depositor
CF Dungan
Year of Origin
1992
References
Dungan CF, Hamilton RM. Use of a tetrazolium-based cell proliferation assay to measure effects of in vitro conditions on Perkinsus marinus (Apicomplexa). J. Eukaryot. Microbiol. 42: 379-388, 1995.
