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 1.0 ml

Fresh growth medium w/o bacteriaxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 9.0 ml

1.xa0xa0xa0xa0 Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.xa0 Allow to cool.

2. xa0xa0xa0 Harvest cells from a culture in stationary phase (1-2 days after reaching peak density).

3.xa0xa0xa0xa0 Gently discard most of the supernatant and vigorously agitate the flasks to detach the cells.xa0

4.xa0xa0xa0xa0 Determine the cell concentration using a hemocytometer.xa0xa0 Adjust the concentration to 2 x 10⁵/ml in fresh medium.xa0 If the concentration is too low, centrifuge at 200 x g for 5 minutes and resuspend the pellet with the supernatant to the desired volume.xa0

5.xa0xa0 Mix the cell preparation and the cryoprotective solution in equal portions.

6.xa0 xa0xa0 Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

7.xa0 xa0xa0 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 xa0xa0 Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.

9. xa0xa0xa0 To establish a culture from the frozen 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.

10.xa0 Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate the entire contents into a T-25 flask containing 10 ml of bacterized ATCC medium 802.

11.xa0 Incubate at 25°C with the cap on loosely.

12.xa0 Once the culture is established, follow the protocol for maintenance of culture.