Methanosarcina mazei (Barker) Mah and Kuhn
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2. If needed exchange the gas in the test tube for 80% H2 20% CO2 ; best results are obtained if the tube is pressurized to approximately 1.52 bar.
3. If the medium is pink (see discussion about resazurin) add 2.0 ml of reducing agent (1.5% sodium sulfide, stock solution) per 100 ml of medium. Let the medium sit at room temperature for 10 to 20 minutes, until the resazurin becomes colorless, before inoculating.
4. When the Balch tube is ready to inoculate, thaw the frozen vial at room temperature under a gentle stream of oxygen free gas.
5. For inoculation, use a 1.0 ml syringe tipped with 22 gauge needle, withdraw the cell suspension from the vial and transfer it to the broth. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC. Use 0.1 ml of the inoculated culture to inoculate a nonselective aerobic broth. Incubate the inoculated tubes at 37oC.
6. Growth should be detected in the #1439 broth within 24 to 48 hours. There should be no growth detected on the aerobic plate or broth.
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
This culture will also grow in Thioglycollate, ATCC Medium #177.
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Blotevogel KH, Fischer U. Transfer of Methanococcus frisius to the genus Methanosarcina as Methanosarcina frisia comb. nov.. Int. J. Syst. Bacteriol. 39: 91-92, 1989.
Blotevogel KH, et al. Methanococcus frisius sp.nov., a new methylotrophic marine methanogen. Can. J. Microbiol. 32: 127-131, 1986.
Validation list no. 22. Int. J. Syst. Bacteriol. 36: 573-576, 1986.
Maestrojuan GM, et al. Taxonomy and halotolerance of mesophilic Methanosarcina strains, assignment of strains to species, and synonymy of Methanosarcina mazei and Methanosarcina frisia. Int. J. Syst. Bacteriol. 42: 561-567, 1992.
type strain of Methanococcus frisius