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
Marine invertebrate - carapace of an American lobster, Homarus americanus, Connecticut, United States, August 4, 2002
Product Format
frozen
Storage Conditions
Frozen Cultures: -70°C for 1 week; liquid N2 vapor for long term storage
Freeze-dried Cultures: 2-8°C
Live Cultures: See Protocols section for handling information
Type Strain
no
Medium
ATCC® Medium 1728: Enriched Isonema medium
Growth Conditions
Temperature: 20°C to 25°C
Culture System: Axenic
Cryopreservation
Reagents
Cryoprotective Solution
DMSO, 2.0 mL
Fresh growth medium, 8.0 mL
Harvest and Preservation
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.
Harvest the culture by agitating the contents of each flask (or by scraping the bottom of each flask using a sterile cell scraper). Transfer the cell suspensions to 15 mL plastic centrifuge tubes.
Spin the cell suspensions at approximately 800 x g for 5 min.
Pool the cell pellets and adjust the concentration to 2.0 - 4.0 x 107 cells/mL with a fresh ATCC medium 1728.
NOTE: If the concentration is too low, centrifuge at 500 x g for 5 min and resuspend in the volume of ATCC medium 1728 required to yield the desired concentration.
Mix the cell preparation and 20% (v/v) DMSO in equal portions. The final concentration will be 1.0 - 2.0 x 107 cells/mL and 10% DMSO. The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.
Dispense in 0.5 mL aliquots to 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 ampules into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 2 to 3 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)
Store in either the vapor or liquid phase of a nitrogen refrigerator.
To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes. Do not agitate the ampule. Do not leave ampule in water bath after thawed.
Immediately after thawing, aseptically transfer entire contents to a T-25 tissue culture flask containing 10 mL ATCC medium 1728. Incubate with the cap tightly sealed at 20-25°C.
