Mycoplasma hyorhinis

1. BHK-21 cells (ATCC^® CCL-10™) are grown in Eagles MEM with non-essential amino acids and Earle's BSS (90%), plus 10% Fetal Bovine Serum under 5% CO₂ (9 mL of the medium and 1 mL of FBS in 25 cm² plastic tissue culture T-flasks) for approximately three days.
2. When the cells appear to be at optimal condition, change to fresh medium.
3. For scaling up: once the monolayer of cell line is formed, remove the entire medium; add 5 mL of PBS, then incubate for 5 minutes in atmosphere of 5% CO₂. After 5 minutes, remove PBS and add 1 mL of trypsin, then incubate for 2 minutes at recommended atmosphere. After two minutes, add 5 mL of fresh medium to the flask, the monolayer begins to detach. Take the entire content out of the flask and place in a centrifuge tube and spin for 5 minutes at 2000 rpm. Remove supernatant and resuspend the pellet with 1 mL of the fresh medium. Inoculate the pellet into two 75 cm² plastic tissue culture T-flasks containing 22.5 mL of fresh medium and 10% FBS (2.5 mL) and incubate at recommended atmosphere for approximately three days.
4. Reconstitute a vial of ATCC^® 29052™ with 2.0 mL of the culture medium, and add approximately 1.0 mL to each of two T-flasks of BHK-21 cells.
5. Incubate at 37°C under 5% CO₂ until the monolayers begin to detach. This will normally take three to five days. Additional time may be required when initially growing from the freeze-dried vial.
6. Pool the monolayers and medium from the flasks and centrifuge at 9000 rpm for 35 minutes.
7. Resuspend the pellet in a small amount of complete tissue culture medium. Additional passages may be made or cells prepared for storage. Either freeze the suspension by adding an equal amount of 20% glycerol, or freeze-dry using an appropriate cryoprotectant.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

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