Methanococcus maripaludis Jones et al.

1. Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flaming the top.
2. If needed exchange the gas in the test tube for 80% H₂-20% CO_2.
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 48 to 96 hours.  No growth should be detected on the aerobic plate or broth.

ANAEROBIC CONDITIONS:

• 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
2. Displace the dead space in the syringe with a reducing agent.

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