Encephalitozoon cuniculi Levaditi et al. (ATCC® PRA-336)

Organism: Encephalitozoon cuniculi Levaditi et al.  /  Depositor: LM Weiss

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Strain Designations type 2
Application
Enteric Research
Food and waterborne pathogen research
Opportunistic pathogen research
Biosafety Level 2
Isolation
Laboratory mouse, 1972
Product Format frozen
Type Strain no
Comments
Grows in various cell lines including MDCK (ATCC CCL-34), RK13 (ATCC CCL-37), and human lung fibroblasts (ATCC CCL-75)
Contaminated with mycoplasma
Medium ATCC® Medium 722: Minimum essential medium (MEM)
Growth Conditions
Temperature: 37.0°C
Growth condition: Grown in RK13 cells (ATCC CCL-37)
Cryopreservation

1.   To harvest the Encephalitozoon culture, detach any remaining tissue culture cells (infected and uninfected) by scraping the surface of the flask with a cell scraper.

2.   Transfer the cell suspension (including parasites) to 15 ml plastic centrifuge tubes. Centrifuge at 1300 x g for 10 min.

3.   Remove all but 0.5 ml of the supernatant from each tube, resuspend the cell pellets, and pool them to a single tube.

4.   Pass the resulting cell suspension through a syringe equipped with a 27 gauge 1/2 in needle to break up any remaining cells. Adjust the parasite concentration to 2.0 - 4.0 x 107 cells/ml with fresh medium or PBS.

      NOTE: If the concentration of parasites is too low, centrifuge at 1300 x g for 10 min and resuspend in the volume of fresh medium or PBS required to yield the desired concentration.

5.   Prepare a cryoprotective solution containing 20% (v/v) DMSO and 20% (v/v) HIFBS in fresh medium or PBS.

6.   Mix the cell preparation and cryoprotective solution in equal portions. The final concentration will be 1.0 - 2.0 x 107 cells/ml, 10% DMSO, and 10% HIFBS. The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.

NOTE: To prevent culture contamination, penicillin-streptomycin solution (ATCC 30-2300) may be added to a final concentration of 50 to 100 I.U./ml penicillin and 50 to 100 µg/ml streptomycin.

7.   Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

8.   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.)

9.   Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.

10.          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.

11.          Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of cells (ATCC CCL-37 or CCL-34) and 10 ml ATCC 30-2003 with 10% (v/v) HIFBS.

12.          Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.

13.          Incubate in a 35-37°C CO2 incubator with the cap screwed on tightly.

Name of Depositor LM Weiss
Year of Origin 1972
References

Didier ES, et al. Identification and characterization of three Encephalitozoon cuniculi strains. Parasitology 111: 411-421, 1995. PubMed: 11023405

Vavra J, et al. Isolation and in vitro cultivation of the mammalian microsporidian Encephalitozoon cuniculi. Folia Parasitol. (Praha). 19: 349-354, 1972. PubMed: 4209547

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