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.
2.?? Aseptically transfer the cyst suspension to 15 ml plastic centrifuge tubes.
3.?? Centrifuge at ~800 x g for 5 min.
4.?? While cysts are centrifuging, prepare a 20% solution of DMSO in bacterized ATCC Medium 802.? Cool on ice.
5.?? Remove the supernatant and pool the cell pellets to one-half the final volume desired with fresh growth medium.
6.?? Combine the cell suspension with an equal volume of 20% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 10% DMSO.
7.?? Dispense in 0.5 ml aliquots to 1.0-2.0 ml Nunc vials (special plastic vials for cryopreservation).
8.?? Place the vials in a controlled rate freezing unit.? From room temperature cool at -1°C/min to -40°C.? 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.). ?
9.?? Store ampules in a liquid nitrogen refrigerator until needed.
10.????????? To establish a culture from the frozen state, place a frozen ampule in a 35°C water bath just enough to cover the frozen material.? Allow the ampule to thaw completely (2-3 min).
11.????????? Immediately after thawing, aseptically remove the contents and transfer to a T-25 tissue culture flask containing 10 ml of fresh? ATCC medium 802 inoculated with Enterobacter aerogenes (ATCC 13048).
12.Screw the cap on tightly and incubate at 25°C.? Subculture every 10-15d.??????
