Cryoprotective Solution10% (v/v) sterile DMSO
Fresh growth medium w/o bacteria
1.???? Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat. Allow to cool before use.
2.???? Harvest cells from a culture that is at or near peak density by filtration and/or centrifugation at the lowest level and shortest time required to loosely pellet cells (for this strain, typically 300-500 x g for 3-5 min).
3.???? Adjust the concentration of cells at least 2 x 105/ml in fresh medium.
4.???? Mix the cell preparation and the cryoprotective solution in equal portions.
5.???? Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6.???? 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.
7.???? Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8.???? To establish a culture from the frozen state place a 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.
9.???? Immediately after thawing, do not leave in water bath, aseptically transfer the contents of the ampule onto the surface of a 20 x 100 mm agar plate of ATCC medium 919 (non-nutrient agar) containing an overlay of 15.0 ml of ATCC medium 1667 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC? 700831) or Enterobacter aerogenes (ATCC? 13048).
10.? Incubate the plate at 20-25°C.
11.? Once the culture is established, transfer 0.5 - 1.0 ml to a T-25 tissue culture flask or 16 x 125 mm test tube containing 5 ml bacterized ATCC medium 1667.
12.? Incubate at 20-25°C (incubate a test tube upright with the cap on loosely).? Follow the protocol for maintenance of culture.
