Mycoplasma arginini Barile et al.

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

1. Open the thawed vial.
2. Make serial dilutions by transferring the entire contents of the vial to a tube containing 4.5 mL of the appropriate broth. Repeat process by transferring 0.5 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.
3. Use an uninoculated tube of broth to serve as a control.
4. 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.
5. 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.
6. Depending on the medium used, growth will be indicated by increased turbidity, a color change, or both.

2. In #243 (HI+ broth), growth is indicated by increased turbidity within 24-48 hours.  In #484 (PPLO broth), this strain will start to show a color change by a darkening of the medium’s red color due to alkaline formation in the first few dilution tubes within 24 to 48 hours. Additional incubation will be required for growth on solid agar.
3. Subsequent, fresh transfers will grow more rapidly than the original culture.

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