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Place the syringe or pipet back into the flask. Perform a Hungate seal (Fig. 4E and F) by pressing a Balch stopper into the mouth of the culture tube as the gassing probe is withdrawn in one smooth motion. Place the gassing probe into an empty tube. Likewise add the anoxic medium to each tube. Crimp an aluminum seal over each stopper (Fig. 4G and H). 8. Now the gas atmosphere in each tube can be adjusted to the needs of the experiment. The medium is designed to have a neutral pH under pressure (173 kPa, 25 psi) of a gas mixture containing 20% CO2.

W. Metcalf. (A) dual chambers made by Coy, (B) intrachamber dry-heated bath for storing melted agar medium prior to pouring of Petri plates, (C) automatic air lock located between chambers, (D) mechanism for holding a #10 stopper in a 3-L flask that contains anoxic medium as it is passed through the negative pressure of the air lock, (E) intrachamber incubator with inoculated Petri plates under an N2:CO2:H2S atmosphere. and the presence of 20% CO2 is essential for handling media with a CO2– carbonate buffering system.

If H2 is to be the substrate, evacuate the N2:CO2 and replace it with H2:CO2. Now inject the appropriate amount of cysteine and sodium sulfide from anoxic solutions of these reducing agents to complete the medium; sterilize the medium and fast exhaust the autoclave. For more details, consult Wolfe and Metcalf (2010). 4. Anoxic, aseptic use of a syringe 1. Prepare a gassing probe (Fig. 3D) for aseptic use by passing the 16G needle through the flame of a gas burner. Heat exchange with the metal is so efficient that only a brief exposure to the flame is necessary.

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