Mycoplasma pneumoniae Somerson et al.

1. Follow instructions as suggested for the culturing of Mollicutes:

                PROCEDURES FOR PROPAGATING MOLLICUTES:

1. Open the vial according to the enclosed instructions.
2. Using a Pasteur or 1.0 mL pipette, withdraw approximately 0.5 to 1.0 mL from a tube 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 to a tube containing 2.25 mL. Repeat process by transferring 0.25 mL from the second to a third tube, 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 of broth to serve as a control.
6. Plates may be inoculated to check colony morphology. You can also spot each dilution on the surface of plates (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 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 are incubated aerobically. Plates are incubated under 5% CO₂ or in aerobically in a candle jar. 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. It usually takes at least seven days for the first tubes 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 the medium changes to yellow, cells have started to die.
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 #2611 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.

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