Methanosarcina sp.
43170 ™
43170 ™
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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.
ANAEROBIC CONDITIONS:
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
Within 48 to 72 hours, growth should be evident by turbidity that settles to the bottom of the test tube. No growth should occur on the blood agar plate incubated aerobically.
The cells are cocci that occur in very large packets that quickly settle to the bottom of the test tube. The packets of cells are so large that they are difficult to observe microscopically. No motility has been detected.
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Touzel JP, et al. Isolation and characterization of a new thermophilic Methanosarcina, the strain CHTI 55. Syst. Appl. Microbiol. 6: 66-71, 1985.