43570
43570 ™
43570 ™
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.
2. Exchange gas in the Hungate test tube for 80% H2 - 20% CO2.
3. If the medium is oxidized (see discussion about resazurin below) add 0.1 ml of reducing agent (see above) to the medium and let the medium sit for 30 minutes before inoculating.
4. When the Hungate test tube is ready to be inoculated, place the frozen LN2 vial under a stream of oxygen free gas and thaw at room temperature.
5. Using a syringe, in which the dead space has been filled with an anaerobic gas mixture or reducing agent (see below), withdraw the cell suspension from the vial and transfer to a single tube (5 to 6 ml) of the recommended broth.
6. Place the inoculated Hungate tube at 45oC. Growth should be detected with in 2 to 4 days.
ANAEROBIC CONDITIONS:
a. Balch tube refers to a special type of test tube that is designed to be pressurized and is suited for anaerobic work. The Balch test tubes can be purchased from Bellco Glass (www.bellcoglass.com; stock no. 2048-00150).
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. 1. Displace the dead space in the syringe with a sterile
Using the syringe transfer method, you must make the transfer as quickly as possible. Sometimes during transfer the medium will oxidize and turn pink (due to resazurin), however it may reduce itself back to the clear broth color during incubation. If the color does not change back, anaerobic conditions were not met and the culture will not grow.
Cells occur in packets of two, four, or eight large cocci. No motility has been detected.
Additional information on this culture is available on the ATCC® web site at www.atcc.org.
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Zinder SH, Mah RA. Isolation and characterization of a thermophilic strain of Methanosarcina unable to use H2-CO2 for methanogenesis. Appl. Environ. Microbiol. 38: 996-1008, 1979.
Zinder SH, et al. Methanosarcina thermophila sp. nov., a thermophilic, acetotrophic, methane-producing bacterium. Int. J. Syst. Bacteriol. 35: 522-523, 1985.