For this demonstration, I’ll be making McCas1 media for Methanococcus maripaludis. There will be differences in protocol according to specific medium type, so this is a general guideline.
- Have initial liquid components (e.g. water, salt solution) stirring at a comfortable speed (~350 rpm is fine). A 1 L Erlenmeyer flask is being used.
- Add components according to the recipe, allowing each to dissolve before adding the next.
- A critical part of anaerobic media is the redox indicator resazurin, a blue compound (Fig. 1). Under anaerobic conditions, resazurin is reduced to hydroresorufin (clear), which may be oxidized to resorufin (pink)2. So, a pink culture is indicative of O2 present.
- Heat the media to 55-65°C to ensure everything is in solution. The thermometer can be held in place by an anaerobic lid stopper (Fig. 1).
Figure 1 - Once the media reaches temperature, bring it (along with any other additional components, such as cysteine) into the chamber.
- You can make the media anaerobic using a gassing manifold and gas exchanging under a stream of N2:CO2, but we have found the chamber method works better for us.
- Have glassware and stoppers in the chamber for at least an hour beforehand.
- Gas exchanging in the chamber can be done by stirring while using a pump and aeration stone (Fig. 2).
Figure 2 - The McCas recipe calls for 15 minutes of stirring, the addition of cysteine, and 15 minutes of additional stirring. An easy way to add the cysteine (or any other solid) is by suspending it in some McCas first.
- The media will stay pink until autoclaving (Fig. 3).
Figure 3 - I am making media to grow M. maripaludis on H2:CO2, so I’ll be aliquotting 50 mL McCas into each 160 mL bottle to promote gas exchange. Different glassware and media amounts may work better for you (Fig. 4).
- Put rubber stoppers in the glassware (Fig 4). Stoppering bottles requires a little more force than Balch tubes.
Figure 4 - Use aluminum crimp seals and a crimper to seal the bottle (Fig 5). I usually place seals on the bottles and then go down the line crimping.
- If your seals require a decrimper, like those used here, try not to crimp too tightly or the seal is more difficult to remove and may require pliers.
Figure 5
- Bring media out of the chamber.
- Optional: gas exchange with gas of choice. (I prefer to sterilely exchange the headspace right before inoculation, especially for H2:CO2.)
- Autoclave on liquid setting of choice.
- Swirl bottles while they’re still hot to promote resuspending of solids (Fig.6). You may have to do this several times until clear, including the next day.
Figure 6
- Moore, B. C. & Leigh, J. A. Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease. J. Bacteriol. 187, 972–979 (2005).
- Dietrich, N., Loubière, K., Jimenez, M., Hébrard, G. & Gourdon, C. A new direct technique for visualizing and measuring gas–liquid mass transfer around bubbles moving in a straight millimetric square channel. Chem. Eng. Sci. 100, 172–182 (2013).
*Melissa Chanderban is an undergraduate researcher in the lab . Melissa is an LSAMP Scholar and has been in the lab since Sept 2014. She has applied to many of the top microbiology graduate programs in the country. She plans to continue her research on Archaea during graduate school.
