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

Filtered artificial seawater (or similar) xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 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 cells from a culture which is at or near peak density by adding 5 ml sterile filtered artificial seawater and washing cells into suspension.xa0 Rub the surface of the plate with a spread bar to detach adhering trophozoites.

3. xa0xa0xa0 Adjust the concentration of cells to at least 2 x 10⁶/ml in fresh medium.

4.xa0 xa0xa0 Mix the cell preparation and the cryoprotective solution in equal portions.

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.xa0 Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.xa0xa0 Immediately after thawing, aseptically transfer the contents of the ampule to the center of a fresh plate of ATCC medium 994.xa0 Distribute the material evenly over the plate using a spread bar.

9.xa0xa0xa0xa0 Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.

10.xa0xa0 Follow the protocol for maintenance of culture.