Methanosarcina vacuolata Zhilina and Zavarzin
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2. Exchange gas in the Hungate test tube for 80% H2 - 20% CO2.
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 30 minutes before inoculating.
4. When the Hungate test tube is ready to be inoculated, place the frozen LN2 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.
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.
d. Syringes can be made anaerobic by one of two methods. 1. Displace the dead space in the syringe with a sterile
Cells occur in packets of two, four, or eight large cocci. No motility has been detected.
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Zhilina TN, Zavarzin GA. Methanosarcina vacuolata sp.nov., a vacuolated methanosarcina. Int. J. Syst. Bacteriol. 37: 281-283, 1987.
Zavarzin GA, Zhilina TN. Comparative cytology of Methanosarcinae and description of Methanosarcina vacuolata nov. sp.. Microbiology 48: 279-285, 1979.
Validation list no. 66. Int. J. Syst. Bacteriol. 48: 631-632, 1998.