Trypanosoma cruzi Chagas
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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.
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. To thaw a frozen ampule, place in a 35°C water bath until thawed (2-3 min). Immerse the ampule just sufficiently to cover the frozen material. Do not agitate the ampule.
2. Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing 10 ml ATCC medium 1029. Incubate at 25°C with the cap screwed on tightly.
2. Incubate at 25°C with the cap screwed on tightly.
3. Subculture every 14-21 days as described in step 1. The transfer interval will depend on the quantity of the inoculum and the quality of the medium. This should be empirically determined by examining the culture on a daily basis until the growth cycle has stabilized.
1. Harvest cells from a culture which is at or near peak density by centrifugation at 1,300 g for 5 min.
2. Adjust concentration of cells to 2 x 107/ml in fresh medium.
3. While cells are centrifuging prepare a 10% (v/v) solution of sterile DMSO in fresh medium (broth). The DMSO solution when first prepared will warm up due to chemical heat. The solution should be allowed to return to room temperature prior to use.
4. Mix the cell preparation and the DMSO solution in equal portions. The final concentration will be 107 cells/ml and 5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no more than 15 min.
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 the ampules 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. Store in either the vapor or liquid phase of a nitrogen refrigerator.
8. 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 thawed.
9. Immediately after thawing, do not leave in the water bath, aseptically transfer the contents of the ampule into a fresh flask containing 10 ml ATCC medium 1029.
10. Incubate at 25°C with the cap screwed on tightly.
11. Maintain as described above.
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Dvorak JA, et al. Trypanosoma cruzi: flow cytometric analysis. I. Analysis of total DNA/organism by means of mithramycin-induced fluorescence. J. Protozool. 29: 430-437, 1982. PubMed: 6182288
Engel JC, et al. Trypanosoma cruzi: biological characterization of 19 clones derived from two chronic chagasic patients. I. Growth kinetics in liquid medium. J. Protozool. 29: 555-560, 1982. PubMed: 6816924
Nozaki T, et al. Cellular and molecular biological analyses of nifurtimox resistance in Trypanosoma cruzi. Am. J. Trop. Med. Hyg. 55: 111-117, 1996. PubMed: 8702014
Engel JC, et al. Trypanosoma cruzi: biological characterization of clones derived from chronic chagasic patients. II. Quantitative analysis of the intracellular cycle. J. Protozool. 32: 80-83, 1985. PubMed: 3921699
Nozaki T, Dvorak JA. Intraspecific diversity in the response of Trypanosoma cruzi to environmental stress. J. Parasitol. 79: 451-454, 1993. PubMed: 8501607