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.
PROCEDURES FOR PROPAGATING MOLLICUTES:
a) Open the vial according to the enclosed instructions.
b) Using a single tube of #2611 broth (5 to 6 ml), withdraw approximately 0.5 to 1.0 ml with a Pasteur or 1.0 ml pipette. Rehydrate the entire pellette.
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 both.
2. Tubes may be incubated aerobically, but plates are incubated under 5% CO2. The incubation temperature is 37°C.
3. Turbidity appears in the first few dilution tubes within 24 to 48 hours. Additional incubation is required for colonies to appear on solid medium.
4. Subsequent fresh transfers will grow more rapidly than the original rehydrated culture. This strain produces good turbidity. Colonies on plates are visible to the naked eye on prolonged incubation.
Colonies on #2611 agar are pin-point, transparent and have typical fried-egg morphology.
Additional information on this culture is available on the ATCC® web site at www.atcc.org.
The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.
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