Culture System: Xenic
DMSO, 2.0 mL
Fresh growth medium, 8.0 mL
Harvest and Preservation
- To achieve the best results, set up cultures with several different inocula (i.e., 0.5 mL, 1.0 mL, and 2.0mL). Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density. Use a sterile cotton swab, cell scraper, or a rubber policeman to detach adherent organisms
- Adjust the concentration to approximately 1 x 107 cells/mL by centrifugation at 700-800 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 approximately 5 x 106 cells/mL and 10% (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.
- Dispense in 0.5 mL aliquots into 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 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 the vial 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 into a T-25 tissue culture flask containing 10 mL ATCC medium 1525 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831™) or Enterobacter aerogenes (ATCC® 13048™).
- Incubate with the cap tightly sealed at 20-25°C.
- Once the culture is established, follow the protocol for maintenance of culture.