Trachipleistophora hominis Hollister et al.

1. 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 it is thawed.
2. Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC^® CRL-1634™ cells and 10 mL ATCC^® 30-2003 with 10% (v/v) HIFBS.
3. Outgas the flask for 10 seconds with a 95% air, 5% CO₂ gas mixture.
4. Incubate in a 35-37°C CO₂ incubator with the cap screwed on tightly.
5. Observe the culture every 2-3 days under an inverted microscope for the presence of Trachipleistophora growing in the cytoplasm of infected cells. Evidence of large numbers of intracellular Trachipleistophora spores may take 1-3 weeks.

Cell Line Maintenance

1. To establish a cell culture from the frozen state, place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial just sufficiently to cover the frozen material. Do not agitate the vial.
2. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 10.0 mL of fresh ATCC^® 30-2003 with 10% (v/v) Heat-Inactivated Fetal Bovine Serum (HIFBS)* in a T-25 tissue culture flask.
3. Outgas the flask for 10 seconds with a 95% air, 5% CO₂ gas mixture.
4. Incubate in a 35-37°C CO₂ incubator with the cap screwed on tightly.
5. Change the medium 1-2 times per week.               
   *Fetal bovine serum is available from ATCC (catalog number 30-2020). Serum is heat-inactivated by exposure to 56°C for 30 minutes. This treatment will inactivate proteins of the complement pathway. Remove the serum from the refrigerator and aseptically distribute in 100 mL aliquots to sterile 125 mL screw-capped bottles. Immerse bottles in a 35°C water bath for 5 minutes. Do not directly transfer bottles from the refrigerator to 56°C. Transfer the bottles to a 56°C water bath and begin timing for 30 minutes. To avoid contamination, do not allow the level of the water in the bath to come in contact with the lip of the screw cap. It is best to leave one inch between the serum level in the bottle and the lip of the cap and to fill the water bath to a level just slightly above the level of the serum. To assure even heating of the serum, swirl the bottle(s) every ten minutes. Note: Some suppliers provide serum already heat-inactivated.

1. When the cell line forms a confluent layer, remove all the medium and replace it with 3 mL of Phosphate Buffered Saline (PBS) (ATCC^® cat. 30-2200). Incubate T-25 flask at 35-37°C for 25-30 min.
2. Remove all the PBS and replace it with 2 mL of 0.25% (w/v) trypsin dissolved in Hank's Balanced Salt Solution (ATCC^® cat. 30-2101).
3. Gently distribute the trypsin over the monolayer, remove the trypsin, and place the flask at 35-37°C for 10 min.
4. Add 2 mL of ATCC^® 30-2003 with 10% (v/v) HIFBS and detach any cells still adherent by alternately aspirating the medium into a pipette and discharging the contents over the monolayer.
5. Distribute the cell suspension in 0.5 mL aliquots to 4 T-25 flasks containing 10 mL fresh ATCC^® 30-2003 with 10% (v/v) HIFBS.
6. Outgas the flask for 10 seconds with a 95% air, 5% CO₂ gas mixture.
7. Incubate in a 35-37°C CO₂ incubator with the cap screwed on tightly.

1. Remove the medium from a fresh confluent monolayer of ATCC^® CRL-1634™ cells in a T-25 tissue culture flask and replace it with 10 mL of ATCC^® 30-2003 with 10% (v/v) HIFBS.
2. To transfer the Trachipleistophora culture, remove the old medium containing spores of the organism and centrifuge at 1300 x g for 10 min.
3. Remove all but 0.5 mL of the supernatant and resuspend the cell pellet.  Transfer the resuspended pellet to the fresh flask of ATCC^® CRL-1634™ cells.
4. Outgas the flask for 10 seconds with a 95% air, 5% CO₂ gas mixture.
5. Incubate in a 35-37°C CO₂ incubator with the cap screwed on tightly.

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