Aciduliprofundum boonei

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% N₂ - 20% CO₂.
3. If the medium is pink (see discussion about resazurin), add 0.1 mL reducing agent (1.5% Na₂S-9H₂O, stock solution) per each 8-10 mL of medium. Let the medium sit at room temperature for a minimum of 10 to 20 minutes - until the resazurin becomes colorless - before inoculating.
4. When the Balch tube is ready to inoculate, allow the vial to thaw under a stream of oxygen-free gas.
5. For inoculation, use 1.0 mL syringe tipped with 22-gauge needle. Make the syringe anaerobic (see discussion below). Draw the thawed cell suspension up into the syringe and transfer the entire cell suspension into the primary tube of broth and incubate at 70°C. Secondary tube(s) can be inoculated by transferring 0.5 mL of the primary tube using an anaerobic syringe. Alternately, all steps can be performed in an anaerobic chamber. Plate 0.1 mL of the inoculated culture onto a non-selective medium and incubate aerobically at 37°C.
6. Faint turbidity should be detected in the #2811 broth within 1 to 2 days. There should be no growth detected on the aerobic plate. Growth maybe easier to detect using phase microscopy.

1. Balch tube refers to a specialized type of test tube that is designed to be pressurized and is suited for anaerobic work. The Balch test tubes can be purchased from Bellco Glass (www.bellcoglass.com; stock no. 2048-00150).
2. 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.
3. To obtain a fully reduced medium, it is necessary that the medium anoxic and that a reducing agent is added. Common reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate.
4. Syringes can be mad 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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