Mesomycoplasma hyopneumoniae (Maré and Switzer) Gupta 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 single tube (5 to 6 mL) of Medium #1699.  Rehydrate the entire 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 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 and plates under the recommended conditions and appropriate temperature.  The time necessary for growth will vary from strain to strain. Initial growth may take 5 to 7 days.  Growth on plates generally requires additional incubation. This strain may take 10 to 14 days.
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 hyopneumoniae 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. The colonies vary in size from tiny to small; rough with irregular margin.  They do not exhibit the usual "fried egg" appearance.
4. For long term storage of Mycoplasma hyopneumoniae, 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 #1699 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.

This Strain requires an additional 5-10% porcine serum (Thermo-Fisher, 26250-084) added to the broth for growth.

Store vials at 4°C until ready to use. 

We have found that using a candle jar for CO₂ conditions works better for those strains whose medium has an indicator present.  CO₂ incubators may lower the pH of the medium enough to cause a color change.  This change may make it difficult to observe growth with those strains that show little turbidity. 

Purified genomic DNA of this strain is available as ATCC^® 25934D™.
Additional information on this culture is available on the ATCC^® web site at www.atcc.org

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