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.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.

2. xa0xa0 Harvest Tokophrya cells from a culture that has recently passed peak density by centrifugation at 250-300 x g for 5 min.

3.xa0xa0xa0xa0 Adjust the concentration of cells to at least 2 x 10⁴/ml in fresh medium.

4.xa0 xa0xa0 Mix the cell preparation and the cryoprotective solution in equal portions by adding the cryoprotective solution to the cell suspension in 3 equal aliquots at 2 min. intervals.

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

6. xa0xa0xa0 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

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

8.xa0 xa0xa0 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 transfer to a petri plate or T-25 tissue culture flask containing a bed of non-nutrient agar (ATCC medium 919) and 10 ml ATCC medium 1323.

9.xa0xa0xa0xa0 Aseptically transfer 0.5-2.0 ml of washed Paramecium to the petri plate or T-25 flask (see section on MAINTENANCE OF CULTURE).xa0 Incubate the culture at 20-25°C.

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