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. Open vial according to enclosed instructions.
2. Under anaerobic conditions, withdraw 0.5 ml of the recommended broth from a single tube (5 to 6 ml) and rehydrate the vial contents.
4. Incubate tubes under an anaerobic atmosphere at 30oC. Incubate one agar plate anaerobically for colony formation, and one aerobically for aerobic contamination check.
5. In 48-72 hours, growth should be evident by turbidity that settles to the bottom of the tube. No growth should occur on agar plate incubated aerobically.
a. Balch tubes (available from Bellco Glass, Vineland, NJ; are specially designed for anaerobic work and use an aluminum crimp cap to hold a rubber stopper in place. Needles can easily be inserted through the stopper, and the tubes can be pressurized to 2 atm. Alternatively, serum vials may be used, or screw cap tubes with butyl rubber stoppers, in the latter case the stopper may be removed and the tube placed under a cannula system that dispenses sterile, oxygen free gas for addition of reducing agents or inoculation.
b. Resazurin is a commonly used redox indicator that is pink when the redox potential is above 50 mv, and colorless when the redox potential is below 110 mv, i.e. highly reducing. Most strict anaerobes require this low redox potential for optimum growth.
c. To obtain a fully reduced medium, it is necessary that the medium be anoxic and that a reducing agent be added. Common reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate.
d. Syringes can be made anaerobic by one of two methods.
This strain is sensitive to Na2S.9H2O. If the medium has been oxidized and needs to be re-reduced, use cysteine HCl (stock concentration of 3%). Add 0.1 ml cysteine per each 5 to 6 ml of #1019 medium. For best results, use freshly prepared medium. The gas mixture used is very important. Avoid the use of H2.
Additional information on this culture is available on the ATCC web site at www.atcc.org.
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black mud sediment, Kysing Fjord
Kai Finster, personal communication