Methanothermobacter thermautotrophicus (Zeikus and Wolfe) Wasserfallen et al.
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2. If needed, exchange the gas in the test tube for 80% H2 -20% CO2.
3. If the medium is pink (see discussion about resazurin) add 2.0 ml of reducing agent (3% cysteine 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. Thaw the frozen vial, and using a gassed 1.0 ml syringe tipped with 22 gauge needle and withdraw the cell suspension from the vial and transfer it to the Balch tube. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate the plate aerobically at 30oC. Incubate broth tube at 65oC.
5. Growth should be detected in the broth within 3 to 4 days. No growth should be detected on the aerobic plate.
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.
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Kern R, et al. Isolation and structural identification of a chromophoric coenzyme F420 fragment from culture fluid of Methanobacterium thermoautotrophicum. Arch. Microbiol. 136: 191-193, 1983.
Escalante-Semerena JC, Wolfe RS. Tetrahydromethanopterin-dependent methanogenesis from non-physiological C1 donors in Methanobacterium thermoautotrophicum. J. Bacteriol. 161: 696-701, 1985. PubMed: 3838170
Skerman VB, et al. Approved lists of bacterial names. Int J Syst Bacteriol 30: 225-420, 1980.
Wasserfallen A, et al. Phylogenetic analysis of 18 thermophilic Methanobacterium isolates supports the proposals to create a new genus, Methanothermobacter gen. nov., and to reclassify several isolates in three species, Methanothermobacter thermautotrophicus comb. nov., Methanothermobacter wolfeii comb. nov., and Methanothermobacter marburgensis sp. nov. Int. J. Syst. Evol. Microbiol. 50: 43-53, 2000. PubMed: 10826786