Perkinsus chesapeaki McLaughlin et al. (ATCC® PRA-202)

Organism: Perkinsus chesapeaki McLaughlin et al.  /  Depositor: C Dungan

Strain Designations EBNPMl-4/E3/E10
Biosafety Level 2
Isolation
Maryland, United States
Isolation date: September 10, 2001. Cloned November 5, 2001
Product Format frozen
Type Strain no
Comments
This isolate is a clone derived from Perkinsus chesapeaki strain EBNPMl-4 ATCC PRA-201. That strain was isolated from the clam Mulinia lateralis collected subtidally in Eastern Bay, Maryland.
Medium ATCC® Medium 1886: Perkinsus broth medium
Cryopreservation

1.   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.

2.  If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107cysts/ml with fresh growth medium.  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.

3.  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.

4.  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 before the freezing process is begun should be no less than 15 min and no longer than 30 min.

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 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.)  

7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.

8.   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.

9.   Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 10 ml of fresh ATCC medium 2684 in a T-25 tissue culture flask.  Incubate at 25-28°C.

Name of Depositor C Dungan
Special Collection NSF - Protistology
Geographical Isolation Chesapeake Bay
Year of Origin September 10, 2001. Cloned November 5, 2001
References

Reece KS, et al. Molecular epizootiology of Perkinsus marinus and P. chesapeaki infections among wild oysters and clams in Chesapeake Bay, USA. Dis. Aquat. Org. 82: 237-248, 2008.