CRYOPRESERVATION: Alternative Thawing Procedure

?1.? Aseptically add 0.5 ml of sterile modified PYNFH medium (ATCC Medium 1034) containing 8% (w/v) sucrose to the ampule.? Immediately, place in a 35°C water bath, until thawed. Immerse the ampule just sufficient to cover the frozen material. Do not agitate the ampule.

2.?? Immediately after thawing, aseptically remove the contents of the ampule and gently add the material to the edge of a 20 x 100 mm petri plate containing ATCC Medium 919 (non-nutrient agar) and position on a 15 degree slant.  The cell suspension will pool at the edge of the plate.

3.?? Continue to double the volume of the cell suspension at 10

minute intervals by adding ATCC medium 1034) containing 4% sucrose (w/v).  When the volume reaches 16.0 ml place the plate in horizontal position and incubate at 25°C. 

4.?? On the following day, gently remove the cell suspension for the plate and transfer to a T-25 tissue culture flask.  Note the volume of the suspension and add a volume of fresh medium containing 4% sucrose equal to the volume of the cell suspension.  Incubate the culture at 25°C.

5.?? After culture has been established subculture into fresh

????? normal medium without sucrose.