Methanococcus maripaludis Jones et al.
BAA-1331 ™
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Product category
Archaea
Product type
Extremophile
Strain designation
C7
Type strain
No
Genome sequenced strain
Yes
Isolation source
Salt marsh
Product format
Frozen
General
Preceptrol
No
Characteristics
Extremophile type
Methanogen
Comments
Genome sequenced strain
Handling information
Medium
ATCC Medium 1439: Methanogenium medium
Temperature
37°C
Atmosphere
80% H2, 20% CO2
Handling procedure
1. Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flaming the top.
2. Exchange the gas in the test tube for 80% H2 20% CO2, do not pressurize over 5psi. If the tubes are over pressurized (20 psi.), inoculating the tubes will prove difficult.
3. Prepare tubes for inoculation: If the medium is pink (see discussion about resazurin B) add 2.0 ml of reducing agent (5% Co-enzyme M stock solution) per 100 ml of medium. Let the medium sit at room temperature for at least 1 hour or until the resazurin becomes colorless, before inoculating.
4. Thaw the frozen vial under a gentle stream of anaerobic gas. Using an anaerobic (see E) 1.0 ml syringe tipped with 22-gauge needle, withdraw the cell suspension from the vial and transfer to the tube of ATCC #1439 medium. Transfer 0.5 ml of the inoculated culture to a second tube of ATCC medium #1439. Pressurize the culture tubes to 20 psi with 80% H2 20% CO2. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate the plate aerobically at 37oC. Incubate culture tubes at 37oC.
5. Growth should be detected in the broth within 1 to 2 days. Enhanced growth can be obtained by incubating the culture with shaking. No growth should be detected on the aerobic plate.
ANAEROBIC CONDITIONS:
A. Balch tubes (available from Bellco Glass, Vineland, NJ; are specially designed for anaerobic work and use an aluminum crimp cap to hold a rubber stopper in place. Needles can easily be inserted through the stopper, and the tubes can be pressurized to 2 atm. Alternatively, serum vials may be used, or screw cap tubes with butyl rubber stoppers, in the latter case the stopper may be removed and the tube placed under a cannula system that dispenses sterile, oxygen free gas for addition of reducing agents or inoculation.
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, titanium citrate and Co-enzyme M (see D).
D. 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.
E. Syringes can be made anaerobic by one of two methods.
Handling notes
Cells occur singly and in pairs. The cells exhibited characteristic F420 autofluorescence by epifluorescence microscopy using a low wavelength filter set.
Always use freshly prepared anaerobic medium. If there is any question about the anaerobic condition of the medium, it can be reduced with the addition of 5.0% Co-enzyem M (2.0 ml per 100 ml of medium).
Quality control specifications
Verification method
Whole-genome Sequencing
History
Depositors
WB Whitman
Type of isolate
Environmental
Cross references
GenBank CP000745 Methanococcus maripaludis C7, complete genome.
Legal disclaimers
Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty
The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.
Disclaimers
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