Methanoculleus bourgensis (Ollivier et al.) Maestrojuan et al.

1. Sterilize the top of the Balch tube spraying it with 70% ethanol and then flaming the top.
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 37°C. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37°C.
6. Growth should be detected in the broth within 72 hours. No growth should be detected on the anaerobic or aerobic plates.

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