Mycoplasma phocicerebrale Giebel et al.
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
1. Follow instructions as suggested for the culturing of Mollicutes:
PROCEDURES FOR PROPAGATING MOLLICUTES:
a) Open the vial according to the enclosed instructions.
b) Using a Pasteur or 1.0 ml pipette, withdraw approximately 0.5 to 1.0 ml from a tube containing 5.0 ml. Rehydrate the entire pellet.
c) Aseptically transfer this aliquot back into the tube. Mix well.
d) Make serial dilutions by transferring 0.5 ml from the original tube to a tube containing 4.5 ml. 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.
e) Use an uninoculated tube of broth to serve as a control.
f) 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.
g) 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.
h) Depending on the medium used, growth will be indicated by increased turbidity, a color change, or
2. Tubes and plates are incubated under 5% CO2. The incubation temperature is 37oC.
3. Growth occurs in 24 to 48 hours. Additional incubation is required for colonies to appear on solid medium. The broth will turn alkaline (dark red) with turbidity.
Store vials at freezer temperatures until ready to use.
Additional information on this culture is available on the ATCC web site at www.atcc.org.
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