ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.
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
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 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 is indicated by increased turbidity, a color change, or both.
2. This strain starts to show turbidity in the first few dilution tubes within 48 to 72 hours. Additional incubation may be required for growth on solid medium.
3. Subsequent, fresh transfers grow in 24-48 hours. The freeze‑drying process and the cryoprotectant occasionally slows the growth rate of the initial culture.
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Freundt EA, et al. Proposal of Mycoplasma buccale nom. nov. and Mycoplasma faucium nom. nov. for Mycoplasma orale "Types" 2 and 3, respectively. Int. J. Syst. Bacteriol. 24: 252-255, 1974.
Taylor-Robinson D, Chanock RM. Characterization of a newly identified mycoplasma from the human oropharynx. Am. J. Epidemiol. 81: 180-191, 1965. PubMed: 14261024
. The Mycoplasmatales and the L-phase of bacteria. New York: Appleton-Century-Crofts; 1969.
Skerman VB, et al. Approved lists of bacterial names. Int J Syst Bacteriol 30: 225-420, 1980.