Trypanosoma conorrhini (Donovan) Shortt and Swaminath (ATCC® 30028)

Organism: Trypanosoma conorrhini (Donovan) Shortt and Swaminath  /  Depositor: EJ Tobie

Application
Vector borne research
Biosafety Level 1
Isolation
kissing bug, Triatoma rubrofasciata, Oahu Island, HI, 1947
Product Format frozen
Type Strain no
Comments
Variation in kinetoplast minicircle and maxicircle DNA sequences
use of plastic ampoules for freeze preservation
Medium ATCC® Medium 1012: Diphasic blood agar medium
Growth Conditions
Temperature: 25.0°C
Duration: axenic
Protocol: ATCCNO: 11745 SPEC: See general instructions for thawing and storage of frozen material before proceeding. Add thawed contents to a single 16 x 125 mm glass screw-capped test tube of the appropriate medium. Incubate the culture vertically with the cap screwed on tightly. It is essential to establish cultures initially in small volumes. Once established, the culture can be scaled up to larger volumes. Vigorously agitate the culture and aseptically transfer 0.1 ml of culture to a fresh tube of medium weekly.
Subcultivation
Protocol: ATCCNO: 11745 SPEC: See general instructions for thawing and storage of frozen material before proceeding. Add thawed contents to a single 16 x 125 mm glass screw-capped test tube of the appropriate medium. Incubate the culture vertically with the cap screwed on tightly. It is essential to establish cultures initially in small volumes. Once established, the culture can be scaled up to larger volumes. Vigorously agitate the culture and aseptically transfer 0.1 ml of culture to a fresh tube of medium weekly.
Cryopreservation

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 tube of ATCC medium 1011. 

10.          Incubate vertically at 25°C with the cap screwed on tightly.

11.          Maintain as described above. 

Name of Depositor EJ Tobie
Chain of Custody
ATCC <<--EJ Tobie<<--S. Wood <<--- E.J. Tobie
Year of Origin 1947
References

Wood LT. The cultivation of Trypanosoma conorhini. J. Parasitol. 33: 85, 1947.

Wood LT. The occurence of Trypanosoma conorhini Donovan in the reduviid bug, Triatoma rubrofasciata (Degeer) from Oahy, T.H. Proc. Hawaii. Entomol. Soc. 12: 651, 1946.

Simione FP Jr., et al. The use of plastic ampoules for freeze preservation of microorganisms. Cryobiology 14: 500-502, 1977. PubMed: 891238

Gomez-Eichelmann MC, et al. Comparison of several lizard Leishmania species and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids [published erratum appears in Mol. Biochem. Parasitol. 28: 301, 1988]. Mol. Biochem. Parasitol. 27: 143-158, 1988. PubMed: 3344003