Cercomonas bodo Meyer
Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen. If liquid nitrogen storage facilities are not available, frozen ampules may be stored at or below -70°C for approximately one week. Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C). Storage of frozen material at this temperature will result in the death of the culture.
1. One day before thawing the ampule, prepare bacterized ATCC medium 802 or 1773, i.e. inoculate medium with a bacteriological loop of either Klebsiella pneumoniae subsp. pneumoniae (ATCC 700831) or Enterobacter aerogenes (ATCC 13048) from a nutrient agar slant (ATCC medium 3).
2. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes. Do not agitate the ampule. Do not leave ampule in water bath after it is thawed.
3. Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 12 ml of bacterized ATCC Medium 802 or 1173. Screw the cap on tightly and incubate on a 15° horizontal slant at 25°C.
2. When the culture is at or near peak density, rub the surface of the tube with a sterile cotton swab, and agitate the swab to dislodge the adherent cells. Invert gently 10 times to distribute cells evenly.
3. Transfer approximately 0.25 ml to a 16 x 125 mm screw-capped test tube containing 12 ml of freshly bacterized ATCC medium 802 or 1773.
4. Screw the cap on tightly and incubate on a 15° horizontal slant at 25°C.
DMSO 2.0 ml
Fresh growth medium w/o bacteria 8.0 ml
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 850 x g for 5 min.
3. Adjust the concentration of cells at least 2 x 106/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. 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 16 x 125 mm screw-capped test tube containing 12 ml of ATCC Medium 802 or 1173 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831) or Enterobacter aerogenes (ATCC 13048).
9. Incubate at 25°C with the cap screwed on tightly.
10. Follow the protocol for maintenance of culture.