KH2PO4 0.200 g MgCl2 x 6 H2O 0.100 g NH4Cl 1.000 g KCl 0.200 g NaCl 80.000 g Na2CO3 6.830 g NaHCO3 3.830 g Na2S x 9 H2O 0.200 g Yeast extract 0.200 g Histidine 5.000 g Trace elements (see DSM medium 141) 1.000 ml Vitamin solution (see DSM medium 141) 2.000 ml Resazurin 0.001 g Distilled water 1000.000 ml Final pH 8.9 Prepare the medium without adding the vitamins, histidine, yeast extract, NH4Cl, Na2CO3, and NaHCO3 using anaerobic conditions, under nitrogen. If the medium has been boiled to remove oxygen add the NH4Cl, Na2CO3, and NaHCO3 after the medium had cooled. Dispense into tubes stopper with rubber stoppers (serum tubes or bottles, or screw capped tubes). Autoclave, and to the cooled medium add the vitamins (filter sterilised under N2), histidine, yeast extract, and Na2S x 9 H2O from anaerobic, sterile stock solutions.
