Echinamoeba thermarum Baumgartner et al.
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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. For a plate culture, transfer cells with an inoculating loop to a fresh plate of previously-bacterized agar medium from a growing culture at or near peak density. For a broth culture, inoculate a fresh flask of previously-bacterized broth medium with 0.5 ml from a growing culture at or near peak density.
2. Incubate at 50°C. Keep the cap tightly sealed in the case of a flask culture.
DMSO 1.5 ml
Fresh growth medium w/o bacteria 8.5 ml
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 bacterized ATCC medium 2338. Screw the cap on tightly and incubate the culture at 50°C. Alternatively, add the thawed contents of the ampule to the surface of a previously-bacterized Petri plate of ATCC medium 2338 agar. Wrap the plate culture with parafilm and incubate upright at 50°C.
9. Follow the protocol for maintenance of culture.
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Baumgartner M, et al. Cultivation and properties of Echinamoeba thermarum n. sp., an extremely thermophilic amoeba thriving in hot springs. Extremophiles 7: 267-274, 2003. PubMed: 12910386
outflow of Octopus Spring, White Creek, Yellowstone National Park, WY