Perkinsus marinus (Mackin et al.) Levine (ATCC® 50509)

Organism: Perkinsus marinus (Mackin et al.) Levine  /  Depositor: D Bushek

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Strain Designations DBNJ-1 [NJ-1]
Application
Host-parasite interactions
Biosafety Level 2
Isolation
eastern oyster, Crassostrea virginica, Delaware Bay, NJ, 1993
Product Format frozen
Type Strain no
Comments
Host-parasite interactions
Races
Medium ATCC® Medium 1886: Perkinsus broth medium
Growth Conditions
Temperature: 25.0°C
Duration: axenic
Protocol: ATCCNO: 50439 SPEC: When the frozen ampule arrives, place it directly into a 35C-water bath and transfer its thawed contents to 14 ml of fresh medium in a T-75 tissue culture flask. Maintain by removing 13 ml of cell suspension weekly and replacing with an equal volume of fresh medium. Alternately, aseptically transfer 1.0 ml of a growing culture to 13 ml of fresh medium in a T-75 tissue culture flask. Please note: This strain has wide tolerances to most environmental variables, i.e., temperature range: 15-35C, salinity range: 10-60 parts per thousand; pH range: 6.0-8.5. The distribution of the species worldwide is not known. In order to avert the introduction of this pathogen into non-endemic areas, all culture wastes must be treated as biohazardous and autoclaved prior to disposal. There are no known mechanisms for eradication of this pathogen from the environment.
Subcultivation
Protocol: ATCCNO: 50439 SPEC: When the frozen ampule arrives, place it directly into a 35C-water bath and transfer its thawed contents to 14 ml of fresh medium in a T-75 tissue culture flask. Maintain by removing 13 ml of cell suspension weekly and replacing with an equal volume of fresh medium. Alternately, aseptically transfer 1.0 ml of a growing culture to 13 ml of fresh medium in a T-75 tissue culture flask. Please note: This strain has wide tolerances to most environmental variables, i.e., temperature range: 15-35C, salinity range: 10-60 parts per thousand; pH range: 6.0-8.5. The distribution of the species worldwide is not known. In order to avert the introduction of this pathogen into non-endemic areas, all culture wastes must be treated as biohazardous and autoclaved prior to disposal. There are no known mechanisms for eradication of this pathogen from the environment.
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 107 cells/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 to the start of the freezing process 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 1886 in a T-25 tissue culture flask.  Incubate at 25°C.

Name of Depositor D Bushek
Year of Origin 1993
References

Bushek D, Allen SK Jr.. Host-parasite interactions among broadly distributed populations of the eastern oyster Crassostrea virginica and the protozoan Perkinsus marinus. Mar. Biol. Prog. Ser. 139: 127-141, 1996.

Bushek D, Allen SK Jr.. Races of Perkinsus marinus. J. Shellfish Res. 15: 103-107, 1996.

Ford SE, et al. Comparison of in vitro-cultured and wild-type Perkinsus marinus. I. Pathogen virulence. Dis. Aquat. Org. 51: 187-201, 2002. PubMed: 12465877

Chintala MM, et al. Comparison of in vitro-cultured and wild-type Perkinsus marinus. II. Dosing methods and host response. Dis. Aquat. Org. 51: 203-216, 2002. PubMed: 12465878

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation