Cryoprotective Solution
DMSO, 1.5 ml
Fresh growth medium w/o bacteria, 7.5 ml
MgCl₂ (0.5 mM), 0.5 ml
CaCl₂ (0.5 mM), 0.5 ml

1. Mix the components in the order listed.  Before adding the MgCl₂ and the CaCl₂ allow the solution to return to room temperature.  When the medium is added to the DMSO the solution will warm up due to chemical heat.
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 200 x g for 1 min.
3. Adjust the concentration of cells to 2 x 10⁵/ml in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions.
5. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. 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.
7. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state add 1.0 ml ATCC medium 802 to the frozen ampule and place it 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.
9. Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate onto the surface of an ATCC medium 919 (non-nutrient agar) plate containing an overlay of 15.0 ml of bacterized ATCC medium 802.
10. Incubate at 25°C.
11. Once the culture is established, transfer 0.5 ml to 5.0 ml of bacterized ATCC medium 802.
12. Follow the protocol for maintenance of culture.

. . Genetics 38: 550-560, 1953.