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.
Anaerobic conditions for incubation may be obtained by any of the following:
Culture may have to be established in the primary broth before subcultures can be made.
We suggest adding the reducing agent to the medium at least one hour before the medium is to be inoculated.
Co-enzyme M (mercaptoethanesulfonic acid) (100 X solution): Dissolve 5.0 g in 100 mL of deionized water. Distribute into screw cap test tubes, 5–6 mL per tube and seal with rubber stoppers under N2 gas. Autoclave to sterilize. Excess tubes can be stored at room temperature for up to 2 months. Co-enzyme M is a compound produced by many methanogens. Some methanogens are sensitive to stronger reducing agents such as sodium sulfide. Co-enzyme M is the standard reducing agent we use when working with methanogens.
Sodium sulfide (100 X solution): Dissolve 1.5 g in 100 mL of distilled water. Distribute into screw cap test tubes, 5–6 mL per tube, and seal with Hungate stoppers. Autoclave to sterilize. Excess tubes can be stored frozen for up to 6 months. Once thawed a tube of sodium sulfide should not be used for more than a week. CAUTON: if sodium sulfide comes into contact with a strong acid, hydrogen sulfide (H2S), a very toxic gas, is liberated immediately.
Cysteine (100X solution): Dissolve 3.0 g in 100 mL of distilled water. Distribute into screw cap test tubes, 5–6 mL per tube, and seal with Hungate stoppers. Autoclave to sterilize. Excess tubes can be stored frozen for up to 6 months. Once thawed, a tube of cysteine should not be used for more than a week.
Substrates: Useful Information:
Some methanogens are able to utilize substrates other than H2-CO2, such as acetate, propionate, methanol, etc. These substrates can be added directly to the tubed medium, making it possible to use the same medium for more than one organism. We suggest making up anaerobic stock solutions at 100X. Some of these substrates (organic acids) need to be neutralized with sodium hydroxide.
Amount per 100 mL for 1M solution:
WARNING: EXPLOSION HAZARD:
Methanogens that grow on methanol produce methane gas resulting in over pressurization of growth vessels (3). This creates a potential explosion hazard. We recommend growing cultures in pressure-resistant Balch tubes* to reduce this risk. The cultures should be vented regularly to reduce the gas and prevent overpressure. If it is necessary to grow larger batches of methanol-utilizing methanogens in sealed serum vials, extra caution should be taken. Typically, 3 moles of methane are produced from one mole of methanol.
Always wear protective eye wear when working with methanogens growing in tubes or bottles.
Additional information on this culture is available on the ATCC® web site at www.atcc.org.
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