Perkinsus marinus (Mackin et al.) Levine (ATCC® 50903)

Strain Designations: LA5-3 [LA-5-3]  /  Depositor: D Bushek  /  Biosafety Level: 2

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Strain Designations LA5-3 [LA-5-3]
Application
This strain is useful for studies of population genetics, systematics, and virulence.
Biosafety Level 2
Isolation
clone derived from strain LA5 (ATCC 50768), which came from eastern oyster, Crassotrea virginica, Louisiana Gulf Coast, 1998
Product Format frozen
Type Strain no
Comments
parasite of eastern oyster, Crassotrea virginica, and possibly several other marine bivalves
This strain is useful for studies of population genetics, systematics, and virulence.
Medium 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.   Harvest cells from several cultures which are in logarithmic to late stationary phase of growth.  Vigorously agitate to suspend the cells.

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.         

Name of Depositor D Bushek
Chain of Custody
ATCC <<--D Bushek<<--K. Hudson
Year of Origin 1998
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