Acholeplasma laidlawii (Sabin) Edward and Freundt

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

1. Open the vial according to the enclosed instructions.
2. Using a single tube of broth (5 to 6 mL), withdraw approximately 0.5 to 1.0 mL with a Pasteur or 1.0 mL pipette. 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 2.5 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 freeze­drying 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 colonyforming 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 and plates are incubated in 5% CO₂. The incubation temperature is 37°C.
3. This strain will show turbidity in the first few dilution tubes within 24-48 hours. Additional incubation will be required for colonies to appear on solid medium.
4. Subsequent, fresh transfers will grow more rapidly than the original culture. This strain produces good turbidity.

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