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seawater, Atlantic Ocean, Southampton, England, 1992

Temperature: 25.0°C

Protocol: ATCCNO: 50519 SPEC: The strain is routinely distributed as a frozen stabilate. See the general instructions for thawing and storage of frozen material before proceeding. Thaw the ampule and aseptically transfer the material to a T-25 tissue culture flask containing 10 ml of ATCC medium 1525. The baterial flora that is present in the shipped culture will support growth. Growth may be improved by using bacterized ATCC medium 1525 with a single species of bacteria. Bacterization is accomplished by inoculating the medium with Enterobacter aerogenes ATCC 13048 approximately 24 hours before use. Other species of bacteria may work equally well but this must be empirically assessed. The culture is routinely passaged every 14 days. This strain does not form a cyst stage and can be lost if it is not transferred regularly. To subculture, agitate and aseptically transfer 0.25 ml to 10 ml of freshly bacterized medium in a T-25 flask. Screw the cap on tightly and incubate at 25C.

Protocol: ATCCNO: 50519 SPEC: The strain is routinely distributed as a frozen stabilate. See the general instructions for thawing and storage of frozen material before proceeding. Thaw the ampule and aseptically transfer the material to a T-25 tissue culture flask containing 10 ml of ATCC medium 1525. The baterial flora that is present in the shipped culture will support growth. Growth may be improved by using bacterized ATCC medium 1525 with a single species of bacteria. Bacterization is accomplished by inoculating the medium with Enterobacter aerogenes ATCC 13048 approximately 24 hours before use. Other species of bacteria may work equally well but this must be empirically assessed. The culture is routinely passaged every 14 days. This strain does not form a cyst stage and can be lost if it is not transferred regularly. To subculture, agitate and aseptically transfer 0.25 ml to 10 ml of freshly bacterized medium in a T-25 flask. Screw the cap on tightly and incubate at 25C.

Cryoprotective Solution

DMSO ?????????????????????????????????????????????????????????????????????????????????? 2.0 ml

Fresh growth medium w/o bacteria???????????????????????????????? 8.0 ml

1.???? 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. ?? Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 800 x g for 5 min.

3.? Adjust the concentration of cells at least 2 x 10⁶/ml in freshmedium.

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. 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.) ?

7.? ?? Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.

8.? ?? To establish a culture from the frozen state place the vial 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.?? Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 ml ATCC medium 1525 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^? ?700831).

9.     Incubate at 25°C with the cap screwed on tightly.

10.   Once the culture is established, vigorously agitate the flask and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 1525.

11.   Follow the protocol for maintenance of culture.