Temperature: 20°C to 25°C
Culture system: Xenic, with Enterobacter aerogenes ATCC 13048 and unidentified microalgae as food sources
DMSO, 2.0 mL
Fresh growth medium w/o bacteria, 8.0 mL
Harvest and Preservation
- Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.
- Harvest cells from a culture that is at or near peak density by centrifugation at 125 x g for 5 min.
- Adjust the concentration of cells at least 1 x 105/mL in fresh medium.
- Mix the cell preparation and the cryoprotective solution in equal portions.
- 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.
- Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
- To establish a culture from the frozen state add 1.0 mL bacterized ATCC medium 802 to the frozen ampule and place it in a 35°C water bath. Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
- Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate onto the surface of an ATCC medium 919 (non-nutrient agar) plate containing an overlay of 15.0 mL of bacterized ATCC medium 802.
- Incubate at 25°C with the cap on loosely.
- Once the culture is established, transfer 0.5 mL to 5.0 mL of bacterized ATCC medium 802.
- Follow the protocol for maintenance of culture.