DMSO ?????????????????????????????????????????????????????????????????????????????????? 2.0 ml

Fresh growth medium w/o bacteria???????????????????????????????? 8.0 ml

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.

2.??? Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 800 x g for 5 min.

3.???? Adjust the concentration of cells at least 2 x 10⁶/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. 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.) ?

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 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 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC? 700831) or Enterobacter aerogenes (ATCC? 13048).

9.???? Incubate at 25°C with the cap screwed on tightly.

10.? Once the culture is established, vigorously agitate the flask and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 802.

11.? Follow the protocol for maintenance of culture.