Methanothermobacter thermautotrophicus (Zeikus and Wolfe) Wasserfallen et al.
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
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 37oC. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC.
6. Growth should be detected in the broth within 48 to 96 hours. No growth should be detected on the aerobic plate or broth.
· 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
It is also recommended that you pressurize the headspace with at least 20 psi to allow for good gas exchange. For good growth, gas the culture every day for 5 to 6 days.
Growth is noted by turbidity in the broth. Growth is observed after approximately 48 hours.
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Nagle DP Jr., et al. 5-Fluorouracil-resistant strain of Methanobacterium thermoautotrophicum. J. Bacteriol. 169: 4119-4123, 1987. PubMed: 3624203