Mineral solution 1 75.00 ml Mineral solution 2 75.00 ml Modified Wolin's mineral solution 10.00 ml FeSO4 x 7 H2O solution (0.1% w/v) 20.00 ml Sodium resazurin (0.1% w/v) 0.50 ml Na2CO3 1.50 g Cellobiose 3.00 g Cellulose (MN300) (optional) 3.00 g Wolin's vitamin solution (10x) 1.00 ml L-Cysteine HCl x H2O 0.25 g Na2S x 9 H2O 0.25 g Distilled water 820.00 ml Dissolve ingredients except carbonate, cellobiose, vitamins, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes and autoclave. Add cellobiose, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Cellobiose and vitamins have to be sterilized by filtration. Prior to use adjust pH of complete medium to 7.2 - 7.4. Cellulose (MN 300,Whatman CF II, Kleenex tissue paper, or HCl treated cotton) can be used as an alternative to cellobiose. Mineral solution 1 K2HPO4 3.90 g Distilled water 1000.00 ml Mineral solution 2 KH2PO4 2.40 g (NH4)2SO4 6.00 g NaCl 0.59 g MgSO4 x 7 H2O 1.20 g CaCl2 x 2 H2O 0.72 g Distilled water 1000.00 ml Modified Wolin's mineral solution (from medium 141) Nitrilotriacetic acid 1.50 g MgSO4 x 7 H2O 3.00 g MnSO4 x H2O 0.50 g NaCl 1.00 g FeSO4 x 7 H2O 0.10 g CoSO4 x 7 H2O 0.18 g CaCl2 x 2 H2O 0.10 g ZnSO4 x 7 H2O 0.18 g CuSO4 x 5 H2O 0.01 g AlK(SO4)2 x 12 H2O 0.02 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.03 g Na2SeO3 x 5 H2O 0.30 mg Na2WO4 x 2 H2O 0.40 mg Distilled water 1000.00 ml First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. FeSO4 x 7 H2O solution (0.1% w/v) (from medium 119) FeSO4 x 7 H2O 1.00 g H2SO4 (0.1 N) 1000.00 ml The ferrous sulfate solution is not stable and should be freshly prepared. Wolin's vitamin solution (10x) (from medium 120) Biotin 20.00 mg Folic acid 20.00 mg Pyridoxine hydrochloride 100.00 mg Thiamine HCl 50.00 mg Riboflavin 50.00 mg Nicotinic acid 50.00 mg Calcium D-(+)-pantothenate 50.00 mg Vitamin B12 1.00 mg p-Aminobenzoic acid 50.00 mg (DL)-alpha-Lipoic acid 50.00 mg Distilled water 1000.00 ml
