Perkinsus marinus (Mackin et al.) Levine
Additional information on this culture is available on the ATCC web site at www.atcc.org.
While every effort is made to insure authenticity and reliability of strains on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of cultures.
ATCC recommends that individuals contemplating commercial use of any culture first contact the originating investigator to negotiate an agreement. Third party distribution of this culture is discouraged, since this practice has resulted in the unintentional spreading of contaminated cultures.
1. Vigorously agitate a culture at or near peak density and aseptically transfer a 0.1 ml aliquot to a T-25 tissue culture flask containing 10 ml of fresh complete medium.
2. Screw cap on tightly and incubate at 25°C.
3. Subculture every 10-14d.
2. Aseptically transfer the cell suspension to 15 ml plastic centrifuge tubes.
3. Centrifuge at 200 x g for 5 min.
4. While cells are centrifuging, prepare a 20% solution of DMSO in ATCC Medium 1886.
5. Remove the supernatant and pool the cell pellets into a final volume of 4.5 ml.
6. Combine the cell suspension with an equal volume of 20% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 10% DMSO.
7. Dispense in 0.5 ml aliquots to 1.0-2.0 ml Nunc vials (special plastic vials for cryopreservation).
8. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. At -40°C, plunge ampules into liquid nitrogen.
9. Store ampules in a liquid nitrogen refrigerator until needed.
10. To establish a culture from the frozen state, place a frozen ampule in a 35°C water bath just enough to cover the frozen material. Allow the ampule to thaw completely (2-3 min).
11. Immediately after thawing, aseptically remove the contents and transfer to a T-25 tissue culture flask containing 10 ml of fresh ATCC medium 1886.
12.Screw the cap on tightly and incubate at 25°C. Observe the culture daily. Transfer the culture when many trophozoites are observed.