Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

DMSO xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 2.0 ml

Fresh growth medium w/o bacteriaxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 8.0 ml

1.xa0xa0 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. xa0 Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 500 x g for 5 min.

3.xa0 Adjust the concentration of cells at least 2 x 10⁶/ml in fresh medium.

4. Allow the concentrated cells to remain undisturbed for approximately 1 hour.xa0 Make sure that the concentrated suspension has plenty of air, i.e., the surface to volume ratio of the suspension should be high.xa0 This resting period allows the cells to repair membrane damage that occurs during centrifugation. xa0

5.xa0 Mix the cell preparation and the cryoprotective solution in equal portions.

6.xa0 Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

7. 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.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately

-1°C/min.) xa0

8.xa0 Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.

9.xa0 To establish a culture from the frozen state place the vial in a 35°C water bath.xa0 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,xa0xa0xa0xa0xa0xa0xa0xa0 aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 ml ATCC medium 802 bacterized with Enterobacter aerogenes (ATCC^® 13048).

10. Incubate at 25°C with the cap loosened one half turn.

11. Once the culture is established, vigorously agitate the flask

and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 802.

12. Follow the protocol for maintenance of culture.