# 1232: THERMOTOGA TF(D) MEDIUM ## Main sol. 1232 0.5 g NH4Cl 1.9 g MgSO4 x 7 H2O 1.4 g MgCl2 x 6 H2O 1.6 g K2HPO4 1 g Na2HPO4 x 2 H2O 26 g NaCl 10 ml **Modified Wolin's mineral solution** 2 g Yeast extract (OXOID) 2 g Trypticase peptone (BD BBL) 0.5 ml Sodium resazurin (0.1% w/v) 5 g Starch (soluble) 0.56 g CaCl2 x 2 H2O 1 ml **Wolin's vitamin solution (10x)** 0.3 g L-Cysteine HCl x H2O 0.3 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except starch, calcium chloride, vitamins and reducing agents) and adjust pH to 6.5 - 6.8. Sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials (to 30% of volume) and autoclave. Add starch, calcium chloride, vitamins (sterilized by filtration), sulfide and cysteine from sterile anoxic stock solutions prepared under 100 N2 gas. Adjust pH of complete medium to 6.8, if necessary. ## Modified Wolin's mineral solution 1.5 g Nitrilotriacetic acid 3 g MgSO4 x 7 H2O 0.5 g MnSO4 x H2O 1 g NaCl 0.1 g FeSO4 x 7 H2O 0.18 g CoSO4 x 7 H2O 0.1 g CaCl2 x 2 H2O 0.18 g ZnSO4 x 7 H2O 0.01 g CuSO4 x 5 H2O 0.02 g AlK(SO4)2 x 12 H2O 0.01 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.03 g NiCl2 x 6 H2O 0.3 mg Na2SeO3 x 5 H2O 0.4 mg Na2WO4 x 2 H2O 1000 ml Distilled water 1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. ## Wolin's vitamin solution (10x) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water