Methanothermococcus thermolithotrophicus (Huber et al.) Whitman
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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. After the Balch tube is ready to be inoculated let the frozen vial thaw at room temperature under a gentle stream of sterile oxygen free gas.
5. Using a 1.0 ml syringe tipped with 22 gauge needle, withdraw the cell suspension from the vial and transfer it to the broth and incubate at 60oC. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC.
6. Within 48 to 72 hours, growth should be evident by turbidity that settles to the bottom of the test tube. No growth should occur on the blood agar plate incubated aerobically.
· 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.
· 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.
· Syringes can be made anaerobic by one of two methods.
1. Displace the dead space in the syringe with a sterile
Cells are Gram negative irregular cocci that occur singly and in pairs and are highly motile. This strain is able to utilize formate or hydrogen and carbon dioxide.
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Validation list no. 14. Int. J. Syst. Bacteriol. 34: 270-271, 1984.
Huber H, et al. Methanococcus thermolithotrophicus, a novel thermophilic lithotrophic methanogen. Arch. Microbiol. 132: 47-50, 1982.
. Validation of publication of new names and new combinations previously effectively published outside the IJSEM.. Int. J. Syst. Evol. Microbiol. 52: 685-690, 2002. PubMed: 12054225
Whitman WBGenus II. Methanothermococcus gen. nov.In: Whitman WBBergey's manual of systematic bacteriology2nd ed.New YorkSpringer-Verlag241-242, 2001