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
Freshwater pool in a rock crevice, 2 feet above the spash zone, with the remains of a decaying bird in the pool, Appledore Island, Maine, September 3, 1993
Product Format
frozen
Storage Conditions
Frozen: -70°C or colder Freeze-Dried: 2°C to 8°C Live Culture: See Protocols Section
Type Strain
no
Medium
ATCC® Medium 1873: Seawater microaerophile medium
Growth Conditions
Temperature: 25°C
Atmosphere: Anaerobic
Culture System: Grown with Enterobacter aerogenes ATCC® 13048™ and mixed bacteria. Consult product sheet for culture protocol.
Cryopreservation
Harvest and Preservation
Harvest the cells from a culture that is at or near peak density by centrifuging at 850 x g for 5 minutes.
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 medium. If the concentration is too low, centrifuge at 850 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
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.
Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 106 and 107 cells/mL and 10% (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.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through 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 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)
The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
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.
Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 12 mL of fresh ATCC medium 1873 in a 16 x 125 screw-capped test tube. Incubate on a 15° horizontal slant at 25°C.
