Spironucleus vortens Poynton et al. (ATCC® 50386)

Organism: Spironucleus vortens Poynton et al.  /  Depositor: SL Poynton

Biosafety Level 1
Isolation
lip lesion, angelfish, Pterophyllum scalare, Archer, FL (29 degrees 32' N, 82 degrees 23' W), 1991
Product Format frozen
Type Strain yes
Medium Medium 2154: LYI Entamoeba medium
Growth Conditions
Temperature: 35.0°C
Growth condition: axenic, anaerobic
Protocol: Thaw the ampule according to instructions accompanying the shipment. Add entire suspension to 13 ml of ATCC medium 2154. Screw the cap on tightly and incubate the tube at 25C on a horizontal slant (5-15 degrees). Transfer the culture weekly; invert the tube 5-10 times, aseptically transfer a 0.1 ml aliquot to a fresh tube of medium, and incubate as above.
Cryopreservation

1.  Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.

2.  Adjust the concentration of cells to 2 x 106 - 2 x 107/ml in fresh medium.

3.  While cells are centrifuging prepare a 10% (v/v) solution of sterile DMSO in fresh medium.

a) Add 1.0 ml of DMSO to an ice cold 20 x 150 mm screw-capped test tube;

b) Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 9.0 ml of ice cold medium;

c) Invert several times to dissolve the DMSO;

d) Allow to warm to room temperature.

4.  Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 106 - 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 no less than 15 min and no longer than 30 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 vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at        -1°C/min through the heat of fusion.  At -40°C plunge 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.  The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.

8.   To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of the frozen material. Do not agitate the vial.

9.   Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate a 16 x 125 mm screw-capped test tube containing 13 ml ATCC Medium 2154.

10.      Incubate the culture on a 15° horizontal slant at 35°C with the cap screwed on tightly.

Name of Depositor SL Poynton
Year of Origin 1991
References

Poynton S, et al. Spironucleus vortens n. sp. from the freshwater angelfish Pterophyllum scalare: Morphology and culture. J. Eukaryot. Microbiol. 42: 731-742, 1995.

Dawson SC, et al. Stable transformation of an episomal protein-tagging shuttle vector in the piscine diplomonad Spironucleus vortens. BMC Microbiol. 8:71, 2008. PubMed: 18445284.

Cross References

Nucleotide (GenBank) : U94407 Spironucleus vortens alpha-tubulin gene, partial cds.

Nucleotide (GenBank) : U94406 Spironucleus vortens elongation factor-1 alpha gene, partial cds.

Nucleotide (GenBank) : U93086 Spironucleus vortens small subunit rRNA gene, copy 2, partial sequence.

Nucleotide (GenBank) : U93085 Spironucleus vortens small subunit rRNA gene, copy 1 , partial sequence.

Nucleotide (GenBank) : U94408 Spironucleus vortens translation initiation factor-2 gamma subunit gene, partial cds.