Reclinomonas americana Flavin and Nerad
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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. Vigorously agitate the flask and aseptically transfer 0.5 ml from a growing culture to a T-25 tissue culture flask containing 10.0 ml of ATCC medium 802 bacterized with Enterobacter aerogenes (ATCC® 13048)
2. Incubate flask at 25°C with the cap loosened one half turn.
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 500 x g for 5 min.
3. Adjust the concentration of cells at least 2 x 106/ml in fresh medium.
4. Allow the concentrated cells to remain undisturbed for approximately 1 hour. Make sure that the concentrated suspension has plenty of air, i.e., the surface to volume ratio of the suspension should be high. This resting period allows the cells to repair membrane damage that occurs during centrifugation.
5. Mix the cell preparation and the cryoprotective solution in equal portions.
6. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. 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
8. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
9. 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 802 bacterized with Enterobacter aerogenes (ATCC® 13048).
10. Incubate at 25°C with the cap loosened one half turn.
11. Once the culture is established, vigorously agitate the flask
and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 802.
12. Follow the protocol for maintenance of culture.
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Flavin M, Nerad TA. Reclinomonas americana N. G., N. Sp., a new freshwater heterotrophic flagellate. J. Eukaryot. Microbiol. 40: 172-179, 1993. PubMed: 8461890
Lang BF, et al. Mitochondrial genomics in protists, an approach to probing eukaryotic evolution. Protist 149: 313-322, 1998.