Methanofollis liminatans

1. Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flame the top.
2. If needed, exchange the gas in the test tube for 80% N₂ 20% CO₂.
3. If the medium is pink (see discussion about resazurin) add 0.2 ml Co-enzyme M (5% stock solution) per 10 ml of medium.  Let the medium sit at room temperature for 30 to 40 minutes until the resazurin becomes colorless before inoculating.
4. When the Balch tube is ready to inoculate, thaw the frozen vial at room temperature under a gentle stream of oxygen-free gas.
5. For inoculation, use an anaerobic (see "c" below) 1.0 ml syringe tipped with 22-gauge needle, withdraw the cell suspension from the vial and transfer it to the broth.  Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37^oC.  Use 0.1 ml of the inoculated culture to inoculate a nonselective aerobic broth. Transfer 0.5 ml of the rehydrated culture to an additional tube of modified #2601 broth.   Incubate the broth tubes at 37^oC. 
6. Growth should be detected in the modified #2601 broth within 48 to 96 hours.  There should be no growth detected on the aerobic plate or in the aerobic broth.

ANAEROBIC CONDITIONS:

1. 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.
2. 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.
3. Syringes can be made anaerobic by one of two methods.

1. Displace the dead space in the syringe with a sterile oxygen-free gas.  
2. Displace the dead space in the syringe with a reducing agent. 

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.

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. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.