Methanococcus deltae Corder 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. Open the frozen vial and immediately place the vial under a stream of sterile gas, 80% H2-20% CO2 to maintain anaerobicity.
6. Growth should be detected in the broth within 48-72 hours. No growth should be detected on the aerobic plate or broth.
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 up 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.
1. Displace the dead space in the syringe with a sterile
2. Displace the dead space in the syringe with a
Formate maybe substituted for 80% H2-20% CO2 as an energy and carbon source. Growth may take up to one week when the frozen culture is initially inoculated into fresh medium.
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Corder RE, et al. Isolation and characterization of two new methane-producing cocci: Methanogenium olentangyi, sp. nov., and Methanococcus deltae, sp. nov. Arch. Microbiol. 134: 28-32, 1983.
Validation list no. 25. Int. J. Syst. Bacteriol. 38: 220-222, 1988.