1. Two to three days in advance, prepare fresh tubes containing ATCC medium 1671 and 25% HIHS in an anaerobic jar with tube caps loosened one full turn. Add a BD GasPak (one anaerobic system GasPak per anaerobic culture jar). Close the vessel securely and incubate at 35°C for at least 48 hours. If the GasPak is of the variety that makes use of a palladium catalyst, the catalyst should be replaced biweekly.
2. Using the liquid overlay from the previously-reduced tubes of medium prepared in step 1 above, prepare a 20% (v/v) sterile DMSO solution in Stone's Modification of Locke's Solution in the following manner:
a) Add the required volume of DMSO to a 20 x 150 mm screw-capped test tube;
b) Place the tube on ice and allow the DMSO to solidify (~5 min), then add the required volume of previously-reduced liquid overlay. Chemical heat will be liberated from this combination so allow the solution to cool to room temperature;
c) If time allows, loosen the tube cap one full turn and place in an anaerobic jar with an anaerobic GasPak for at least 48 hours prior to use.
3. When the test tube cultures are at or near peak density remove the tubes from the anaerobic jar and immediately screw the caps on tightly. Opening one tube at a time, gently remove the cells from the bottom of the egg medium slants and pool in a single 16 x 125 mm screw-capped test tube (work quickly to avoid prolonged exposure to air).
4. Adjust the cell concentration to 1.0-2.0 x 107cells/ml using overlay from a reduced tube of medium. If the concentration of cells is too low centrifuge at 500 x g for 5 minutes. Adjust the volume of supernatant to yield the desired final cell concentration.
5. Mix the cell preparation and the cryoprotective agent prepared in step 2 in equal portions. Thus, the final concentration will equal 10% (v/v) DMSO and 5.0 x 106 - 1.0 x 107 cells/ml. The time from the mixing of the cell preparation and glycerol/DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
6. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge 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
8. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.
9. Before thawing an ampule do the following: Place tubes containing ATCC medium 1671 and 25% HIHS in an anaerobic jar with tube caps loosened one full turn. Add a BD GasPak (one anaerobic system GasPak per anaerobic culture jar). Close the vessel securely and incubate at 35°C for at least 48 hours. If the GasPak is of the variety that makes use of a palladium catalyst, the catalyst should be replaced biweekly.
10. Thaw the frozen ampule in a 35°C water bath without agitation until all of the contents are liquid (about 2-3 minutes).
11. Aseptically and gently, lower a sterile Pasteur pipette from which the air has been expelled to the bottom of the liquid in the ampule and slowly aspirate the entire contents into the pipette. Be careful to minimize agitation of the fluid and so not introduce air bubbles from the tip of the pipette.
12. Inoculate a fresh tube of previously-reduced ATCC medium 1671 by inserting the Pasteur pipette tip aseptically through the liquid overlay-air interface (avoid expulsion of air bubbles or culture) and moving the tip of the pipette to the base of the solid-overlay interface. Expel the entire contents of the Pasteur pipette into the culture tube (again avoid expulsion of air bubbles), then tighten the cap immediately unless placing the tube directly into an anaerobic jar.
13. With the cap of the freshly inoculated test tube loosened one full turn, place it in an anaerobic jar containing a BD GasPak and incubate at 35°C.
14. Subculture every 2-3 days.