Mycoplasma genitalium Tully et al.

1. We recommend that you use T-flasks in the propagation of Mycoplasma genitalium so that a larger surface area will be available for the cells to adhere to the glass or plastic surface. Approximately 10 mL of Medium #988 is placed in 25 cm² flasks, and 200 mL can be placed in 125 cm² flasks if larger amounts are desired.
2. Add an additional 5% heat-inactivated fetal bovine serum to medium #988. Open the vial according to enclosed instructions.
3. Transfer the contents of the vial to a 25 cm² flask. 
4.  Incubate the flask in a horizontal position at 37°C for 7-14 days. Note: This strain can be slow to establish growth, however subsequent transfers should grow faster once primary growth is established.  
5. After the incubation period,  observe the flask for flocs in the medium and for cells adhering to the bottom of the flask.  Growth is also indicated by an indicator change from red to yellow.  
6. To prepare the culture for subculturing and/or scaling-up to larger volumes, loosen adherent cells with a suitable, sterile scraper.  Centrifuge the culture and remove the supernatant (spent growth media).  Resuspend the pellet with fresh medium and place  into a new 25 cm² flask with the appropriate volume of fresh medium.  
7. Incubate the flask in a horizontal position at 37°C for 7-14 days. 
8. When the medium has turned yellow, loosen adherent cells with a suitable, sterile scraper.  Use a 10% inoculum when subculturing this item and/or scaling-up to larger volumes to ensure healthy growth in subcultures.  
   
    

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