Methanospirillum hungatei corrig. Ferry et al. 1974

1. Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flaming the top.  Formate can be added to a final concentration of 100 mM to individual tubes of Medium #2487 or to the medium before it is tubed.  When formate is used as the substrate 100% nitrogen can be used as the gas. 
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 0.1 mL Na₂S^.9H₂O (1.5% sodium sulfide, stock solution) per 10.0 mL of medium.  Let the medium sit at room temperature for 10 to 20 minutes until the resazurin becomes colorless before inoculating.
4. When the Balch tube is ready to inoculate, thaw the frozen vial at room temperature under a gentle stream of oxygen-free gas.
5. For inoculation, use an anaerobic (see “c” below) 1.0 mL syringe tipped with 22-gauge needle, withdraw the cell suspension from the vial and transfer it to the broth.  Plate 0.1 mL of the inoculated culture onto a non-selective medium and incubate aerobically at 37°C.  Use 0.1 mL of the inoculated culture to inoculate a nonselective aerobic broth and an additional tube of #2487 broth.   Incubate the non-selective aerobic broth tubes at 37°C.  Incubate the anaerobic tube at 37°C.
6. Growth should be detected in the #2487 broth within 24 to 72 hours.  There should be no growth detected on the aerobic plate or in the aerobic broth.

1. 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.
2. 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.
3. Syringes can be made anaerobic by one of two methods. 

1. Displace the dead space in the syringe with a sterile oxygen-free gas.  
2. Displace the dead space in the syringe with a reducing agent.  

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