Mycoplasma dispar Gourlay and Leach

1. Follow instructions as suggested for the culturing of 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 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 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 is indicated by increased turbidity, a color change, or both.
9. Tubes are incubated aerobically, plates are incubated under 5% CO₂ or in a candle jar.  The incubation temperature is 37°C.

2. Mycoplasma dispar strains are very slow growing and produce a flocculent turbidity.  It may be necessary to hold tubes up to a light source to detect growth.  Initial growth may take up to a week to be detected in first tubes.  An indicator change from red to orange to yellow is very slow.  The best indicator of growth will be the flocs in the broth medium.  The cells are best transferred when the medium is orange.  After medium changes to yellow, cells have started to die.  Subsequent transfers usually grow in 48+ hours, but may take longer depending on size of inoculum. 
3. After broth growth is established, freshly inoculated plates will take three or more days to produce colonies. Growth on plates takes three or more days.  
4. For long term storage of Mycoplasma dispar, freeze-drying or freezing is recommended.  Liquid nitrogen storage is the best method. 

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