ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.
Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen. If liquid nitrogen storage facilities are not available, frozen ampules may be stored at or below -70°C for approximately one week. Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C). Storage of frozen material at this temperature will result in the death of the culture.
1. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes. Do not agitate the ampule. Do not leave ampule in water bath after it is thawed.
3. Incubate with the cap tightly sealed at 25°C.
1. Vigorously agitate the flask and aseptically transfer 0.5 ml from a growing culture to a T-25 tissue culture flask containing 10.0 ml of ATCC medium 1525 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831) or Enterobacter aerogenes (ATCC 13048).
2. Incubate flask at 25°C with the cap on tightly.
DMSO 2.0 ml
Fresh growth medium w/o bacteria 8.0 ml
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 to 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 1525 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 1525.
Follow the protocol for maintenance of culture.
Thomas A Nerad, personal communication