Sower's M media for Methanosarcina barkeri 227

with 0.4 M NaCl
       
  1 L 2 L 3 L
Na2CO3 3 g 6 g 9 g
Na2HPO4 0.6 g 1.2 g 1.8 g
MgCl2.6 H2O 10.17 g 20.3 g 30.47 g
CaCl2.2 H2O 0.101 g 0.202 g 0.303 g
KCl 0.76 g 1.52 g 2.28 g
NaCl 23.4 g 46.8 g 70.2 g
0.1% resazurin 1 ml 2 ml 3 ml
trace metals 10 ml 20 ml 30 ml
pH to 6.8 with 12 M HCl  
   
1. When most of the media components have dissolved and the pH is 6.8, bubble N2 for 15-30 minutes. Stopper the flask and take it into the anaerobic
           
2. Dispense 50 ml into each bottle, stopper and crimp.
           
3. Autoclave media for 55 minutes.
           
4. As you need media, exchange gas 3x with N2/CO2 or use continuous gas, releasing pressure for about 10 minutes. If the media had any precipitates after autoclaving, exchanging the gas allows it to go back into solution. The re-dissolving can be slow.
           
5. Add supplements to media for each 50 ml of media as needed:
     
  Not fixing N2 Fixing N2
10% NaHCO3 0.1 ml 0.1 ml
20% Na2S 0.1 ml 0.1 ml
100% MeOH - 0.1 ml
     
Optional ingredients:    
10x vitamins 0.1 ml 0.1 ml
500x cysteine 0.1 ml 0.1 ml
1 M TMA 0.3 ml -
10% YE (Y-1000) 0.1 ml -
0.5 mg/ml puromycin 0.1 ml 0.1 ml
(1mg/ml final)
           
6. Inoculate M Media with 12 ml of a stock culture
References
1. Lobo, A.L. and Zinder, S.H. (1988) Applied and Environmental Microbiology 24, 1656-1661.