Cryptodifflugia operculata (ATCC® PRA-365)

Organism: Cryptodifflugia operculata  /  Depositor: DJG Lahr

Deposited As Cryptodifflugia operculata
Strain Designations Smith College
Biosafety Level 1
Isolation Mixed protozoa culture, Carolina Biological
Product Format frozen
Storage Conditions Frozen: -70°C or colder
Freeze-Dried: 2°C to 8°C
Live Culture: See Protocols Section
Medium Medium 802: Sonneborn's Paramecium medium
Medium 2348: Freshwater Diplophrys medium
Growth Conditions Temperature: 20°C to 25°C
Atmosphere: Aerobic
Culture system: Xenic, with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831™) or Enterobacter aerogenes (ATCC® 13048™) as a food source
Cryopreservation Reagents
Cryoprotective Solution
DMSO, 1.5 mL
Fresh growth medium w/o bacteria, 8.5 mL

Harvest and Preservation
  1. Mix the components of the cryoprotective solution in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.
  2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 400 x g for 5 min.
  3. Adjust the concentration of cells to at least 2 x 105/mL in fresh medium.
  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 complete medium bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831™) or Enterobacter aerogenes (ATCC® 13048™).
  9. Incubate at 20-25°C with the cap screwed on tightly.
  10. Follow the protocol for maintenance of culture.
Name of Depositor DJG Lahr
Chain of Custody ATCC <-- DJG Lahr
Year of Origin 2007
References

Lahr DJ, et al. Comprehensive phylogenetic reconstruction of amoebozoa based on concatenated analyses of SSU-rDNA and actin genes. PLoS One 6(7): e22780, 2011. PubMed: 21829512

Cross References

Nucleotide (GenBank) : JF694297 Actin

Nucleotide (GenBank) : JF694280 18S rRNA gene