Methanococcus vannielii Stadtman and Barker
35089 ™
35089 ™
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2. If needed, exchange the gas in the test tube for 80% H2-20% CO2, do not pressurize above 5 psi. If the tube is over pressurized, it will be difficult to inoculate.
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 30 minutes (or until the resazurin becomes colorless) before inoculating.
4. Open the frozen vial and immediately place the vial under a stream of sterile anaerobic gas, to maintain anaerobic conditions. Wait for vial contents to thaw.
5. Using an anaerobic syringe (see d below), withdraw the cell suspension from the vial and transfer it to the primary Balch tube. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC. Additional tubes can be inoculated by transferring 0.5 ml of the primary tube to secondary tube(s). Again, use good anaerobic technique. Once inoculated the Balch tubes should be pressurized up to 20 psi with 80% H2-20% CO2.
6. Growth should be detected in the broth within 48-72 hours. No growth should be detected on the aerobic plate or broth.
ANAEROBIC CONDITIONS:
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 20 psi. 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.
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Stadtman TC, Barker HA. Studies on the methane fermentation. X. A new formate-decomposing bacterium, Methanococcus vannielii. J. Bacteriol. 62: 269-280, 1951. PubMed: 14888644
Skerman VB, et al. Approved lists of bacterial names. Int J Syst Bacteriol 30: 225-420, 1980.
type strain