Mycoplasma pneumoniae Somerson et al.

1. Follow instructions as suggested for the culturing of Mollicutes

PROCEDURES FOR PROPAGATING MOLLICUTES

1. Open the vial.
2. Using a Pasteur or 1.0 mL pipette, withdraw approximately 0.5 to 1.0 mL from a tube or T-flask containing 2.5 mL. Rehydrate the pellet.
3. Aseptically transfer this aliquot back into the tube. Mix well.
4. Make serial dilutions by transferring 0.25 mL from the original tube or T-flask to a tube or T-flask containing 2.25 mL. Repeat process by transferring 0.25 mL from the second to a third tube or T-flask, etc. Dilutions are important, not only for titration purposes, but also to keep culture in varying stages of growth. Many strains will die out rapidly once acid or alkaline conditions are reached. It is recommended to prepare several dilutions from the initial tube as the cryoprotectant used in the freeze-drying process often inhibits growth.
5. Use an uninoculated tube or T-flask of broth to serve as a control.
6. Plates may be inoculated to check colonial morphology. You can also spot each dilution on the surface of plate (4 or more/plate) to determine the number of colony-forming units. However, not all strains do well on solid medium.
7. Incubate all tubes or T-flasks and plates under the recommended conditions and appropriate temperature. The time necessary for growth will vary from strain to strain. Growth on plates generally requires additional incubation.
8. Depending on the medium used, growth will be indicated by increased turbidity, a color change, or both. 

2. Tubes or T-flasks are incubated aerobically; plates are incubated under 5% CO₂. Using higher concentrations of CO₂ will result in lowering the pH of the medium. The incubation temperature is 37°C.
3. Mycoplasma pneumoniae strains are very slow growing and produce a very light turbidity. This strain usually takes at least seven to 14 days for the first tubes or T-flasks to start showing growth. Growth is easily recognized by an indicator change from red to orange to yellow. The cells are best transferred when the medium is orange. After medium changes to yellow, cells have started to die. Subsequent transfers should grow in a shorter span of  time.
4. For long term storage of Mycoplasma pneumoniae, freeze-drying or freezing is recommended. Liquid nitrogen storage is the best method. Optimally grown cells are centrifuged at 9000 rpm for 30 minutes, the supernatant poured off, and the packed cells resuspended in a smaller amount of #988 broth. To this, add an equal amount of sterile 20% glycerol as a cryoprotectant. This suspension is aliquoted into small plastic vials and stored at -70°C or below.

Commercially available SP4 Glucose (Remel catalog # R112585 or #R20376 for broth, and Remel catalog # R20276 for agar) may yield the best growth.

This strain requires an additional 5% heat-­inactivated fetal bovine serum be added to ATCC Medium #988 or #2611 or SP4 Glucose broths to sustain growth of this strain.

The flasks should be checked to make sure media is not evaporating and to add media as needed to maintain the volume.

Purified genomic DNA of this strain is available (ATCC 15531D).

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

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