Encephalitozoon hellem Didier et al. (ATCC® 50604)

Organism: Encephalitozoon hellem Didier et al.  /  Depositor: GS Visvesvara

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Application
Enteric Research
Food and waterborne pathogen research
Opportunistic pathogen research
Biosafety Level 2
Isolation
urine from human, New York, 1992
Product Format frozen
Type Strain no
Comments
Environmental resistance of spores
flow cytometric analysis
Control strain for enterocytozoan identification
Medium ATCC® Medium 722: Minimum essential medium (MEM)
Growth Conditions
Temperature: 35.0°C
Duration: 5% CO2
Protocol: ATCCNO: 50451 SPEC: Thaw ampule in a 35C water bath and aseptically transfer contents to a T-25 tissue culture flask containing a monolayer of lung fibroblasts (ATCC CCL-75). Twice per week, remove the culture fluid and refeed the culture with fresh medium. Centrifuge the culture fluid at 1300 x g for 10 minutes, discard the supernatant, and transfer the resuspended cell pellet to a fresh monolayer of lung fibroblasts.
Subcultivation
Protocol: ATCCNO: 50451 SPEC: Thaw ampule in a 35C water bath and aseptically transfer contents to a T-25 tissue culture flask containing a monolayer of lung fibroblasts (ATCC CCL-75). Twice per week, remove the culture fluid and refeed the culture with fresh medium. Centrifuge the culture fluid at 1300 x g for 10 minutes, discard the supernatant, and transfer the resuspended cell pellet to a fresh monolayer of lung fibroblasts.
Cryopreservation

1.   Harvest the culture by gently agitating the contents of each flask.  Transfer all but approximately 1 ml of the culture medium to 15 ml plastic centrifuge tubes. Detach the    remaining tissue culture cells (infected and uninfected) by scraping the surface of the flask with a cell scraper. Pass the resulting cell suspension through a syringe equipped with a 27 gauge 1/2 in needle and pool this suspension with the culture fluid.

2.   Spin the cell suspensions at approximately 50 x g for 3 min, to remove the cellular debris.

3.   Transfer the spore suspensions (supernatants) to new 15 ml plastic centrifuge tubes.  Centrifuge at 1300 x g for 10 min.

4.   Pool the spore pellets and adjust the concentration to 2.0 - 4.0 x 107 cells/ml with a fresh solution of Hank's Balanced Salt Solution.

      *If the concentration is too low centrifuge at 1300 x g for 10 min and resuspend in the volume of Hank's Balanced Salt Solution required to yield the desired concentration.

5.   Mix the spore preparation and 20% (v/v) DMSO in equal portions.  The final concentration will be 1.0 - 2.0 x 107 cells/ml and 10% DMSO.  The time from the mixing of the cell preparation and the cryoprotective solution before the freezing process begins should be no less than 15 min. and no more than 30 min.

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

7.   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.)  

8.   Store in either the vapor or liquid phase of a nitrogen refrigerator.

9.   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.

10.          Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC CCL-26 cells and 10 ml ATCC 30-2003 with 3% (v/v) HIFBS.

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

12.          Incubate in a 35°C CO2 incubator with the caps screwed on tightly.

Name of Depositor GS Visvesvara
Special Collection NCRR Contract
Chain of Custody
ATCC <<--GS Visvesvara<<--CDC:V257
Year of Origin 1992
References

Moss DM, et al. Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon. J. Clin. Microbiol. 37: 371-375, 1999. PubMed: 9889221

del Aguila C, et al. Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis. J. Clin. Microbiol. 35: 1862-1866, 1997. PubMed: 9196210

Croppo GP, et al. Identification of the microsporidian Encephalitozoon hellem using immunoglobulin G monoclonal antibodies. Arch. Pathol. Lab. Med. 122: 182-186, 1998. PubMed: 9499364

Kucerova-Pospisilova Z, et al. Environmental resistance of Encephalitozoon spores. J. Eukaryot. Microbiol. 46: 11S-13S, 1999. PubMed: 10519227

Xiao L, et al. Genotyping Encephalitozoon hellem isolates by analysis of the polar tube protein gene. J. Clin. Microbiol. 39: 2191-2196, 2001. PubMed: 11376056

Sobottka I, et al. Acute and long-term humoral immunity following active immunization of rabbits with inactivated spores of various Encephalitozoon species. Parasitol. Res. 87: 1-6, 2001. PubMed: 11199842

Notice: Necessary PermitsPermits

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  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
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