Methanosarcina barkeri Schnellen
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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. Exchange gas in the Hungate test tube for 80% H2 - 20% CO2. Do not pressurize above 5psi.
3. If the medium is oxidized (see discussion about resazurin below) all 0.1 ml of reducing agent (see above) to the medium and let the medium sit for at least 30 minutes before inoculating.
4. When the Hungate test tube is ready to be inoculated, place the frozen vial under a stream of oxygen free gas and thaw at room temperature.
5. Using a syringe in which the dead space has been filled with an anaerobic gas mixture or reducing agent (see below), withdraw the cell suspension from vial and transfer to a single tube (5 to 6 ml) of the recommended broth.
6. Use several drops of the suspension to inoculate a #260 Tryptic Soy Agar (BD 236950) with 5% Defibrinated Sheep Blood agar plate to be incubated aerobically to check for aerobic contamination
7. While wearing safety glasses, increase the pressure of the 80% H2 - 20% CO2 gas to 20 psi. Incubate the tubes and plate at 37oC for 4 to 7 days.
a. Balch tube refers to a special 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).
b. 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.
c. 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.
Using the syringe transfer method, you must make the transfer as quickly as possible. Sometimes during transfer, the medium will oxidize and turn pink (due to resazurin), however it may reduce itself back to the clear broth color during incubation. If the color does not change back, anaerobic conditions were not met and the culture will not grow.
Additional information on this culture is available on the ATCC® web site at www.atcc.org.
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