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.

Gatti S, et al. Extraintestinal microsporidiosis in AIDS patients: clinical features and advanced protocols for diagnosis and characterization of the isolates. J. Eukaryot. Microbiol. 44: 79S, 1997. PubMed: 9508459

Croppo GP, et al. Western blot and immunofluorescence analysis of a human isolate of Encephalitozoon cuniculi established in culture from the urine of a patient with AIDS. J. Parasitol. 83: 66-69, 1997. PubMed: 9057698

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

Moura H, et al. Detection by an immunofluorescence test of Encephalitozoon intestinalis spores in routinely formalin-fixed stool samples stored at room temperature. J. Clin. Microbiol. 37: 2317-2322, 1999. PubMed: 10364604

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

del Aguila C, et al. Ultrastructure, immunofluorescence, western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS. J. Clin. Microbiol. 36: 1201-1208, 1998. PubMed: 9574677

Bornay-Llinares FJ, et al. Immunologic, microscopic, and molecular evidence of Encephalitozoon intestinalis (Septata intestinalis) infection in mammals other than humans. J. Infect. Dis. 178: 820-826, 1998. PubMed: 9728552

Scaglia M, et al. Asymptomatic respiratory tract microsporidiosis due to Encephalitozoon hellem in three patients with AIDS. Clin. Infect. Dis. 26: 174-176, 1998. PubMed: 9455527

Leitch GJ, et al. Use of fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia. Antimicrob. Agents Chemother. 41: 337-344, 1997. PubMed: 9021189

He Q, et al. Effects of nifedipine, metronidazole, and nitric oxide donors on spore germination and cell culture infection of the microsporidia Encephalitozoon hellem and Encephalitozoon intestinalis. Antimicrob. Agents Chemother. 40: 179-185, 1996. PubMed: 8787902

Da Silva AJ, et al. Detection of Septata intestinalis (Microsporidia) Cali et al. 1993 using polymerase chain reaction primers targeting the small subunit ribosomal RNA coding region. Mol. Diagn. 2: 47-52, 1997.

del Aguila C, et al. In vitro culture, ultrastructure, antigenic, and molecular characterization of Encephalitozoon cuniculi isolated from urine and sputum samples from a Spanish patient with AIDS. J. Clin. Microbiol. 39: 1105-1108, 2001. PubMed: 11230434

Sobottka I, et al. Disseminated Encephalitozoon (Septata) intestinalis infection in a patient with AIDS: novel diagnostic approaches and autopsy-confirmed parasitological cure following treatment with albendazole. J. Clin. Microbiol. 33: 2948-2952, 1995. PubMed: 8576351

Molestina R, Becnel JJ, Weiss LM. Culture and Propagation of Microsporidia. In Microsporidia: Pathogens of Opportunity, First Edition, Chapter 18: pp. 457-467, 2014. Hoboken, NJ: John Wiley & Sons, Inc.