Paramecium multimicronucleatum Powers and Mitchell
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
1. Transfer 0.5 ml from a growing culture to 5.0 ml of ATCC medium 802 bacterized with Enterobacter aerogenes (ATCC® 13048).
2. Add 1.0 ml of ATCC medium 802 bacterized with Enterobacter aerogenes ATCC 13048 twice weekly. When the tube is filled to within one inch of the top, aspirate from the bottom of the tube and reduce the volume to 5.0 ml.
3. Incubate upright at 25°C with the caps on loosely.
15% (v/v) sterile DMSO
2.5% (w/v) Bovine Serum Album Fraction V
Fresh growth medium w/o bacteria
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 Bacterized 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.
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Allen SL, et al. Intraspecies variability in the esterases and acid phosphatases of Paramecium jenningsi and Paramecium multimicronucleatum: Assignment of unidentified Paramecium: Comparison with the P. aurelia complex. J. Protozool. 30: 155-163, 1983.