Methanobacterium alcaliphilum Worakit et al.
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ethanol and then flame the top.
2. If needed exchange the gas in the test tube for 80% H2 20% CO2.
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 an anaerobic (see c below) 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 and an additional tube of #1617 broth. Incubate the non-selective aerobic broth tubes at 37oC. Incubate the anaerobic tube at 37oC.
6. Growth should be detected in the #1617 broth within 48 to 72 hours. There should be no growth detected on the aerobic plate or in the aerobic broth.
a. 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.
b. 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.
c. Syringes can be made anaerobic by one of two methods. 1. Displace the dead space in the syringe with a sterile
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Worakit S, et al. Methanobacterium alcaliphilum sp. nov., an H2-utilizing methanogen that grows at high pH values. Int. J. Syst. Bacteriol. 36: 380-382, 1986.