# 1057: ETHANOLIGENES MEDIUM ## Main sol. 1057 4 g Tryptone (BD BBL) 2 g Beef extract (BD BBL) 1 g Yeast extract (OXOID) 4 g NaCl 1.5 g K2HPO4 0.1 g FeSO4 x 7 H2O 0.2 g MgCl2 x 6 H2O 1 ml **Trace element solution** 0.5 ml Sodium resazurin (0.1% w/v) 0.5 g L-Cysteine HCl x H2O 10 g D-Glucose 1 ml **Wolin's vitamin solution (10x)** 1000 ml Distilled water 1. Dissolve ingredients (except cysteine, glucose and vitamins), sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Add cysteine and adjust pH to 6.0, then dispense medium under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Prior to inoculation add glucose and vitamins from sterile anoxic stock solutions prepared under 100% N2 gas. The vitamin solution should be sterilized by filtration. Adjust pH of complete medium to 6.0, if necessary. ## Trace element solution 12.8 g Nitrilotriacetic acid (NTA) 1 g FeCl2 x 4 H2O 0.1 g MnCl2 x 4 H2O 0.03 g CoCl2 x 6 H2O 0.1 g CaCl2 x 2 H2O 0.1 g ZnCl2 0.02 g CuCl2 0.01 g H3BO3 0.03 g Na2MoO4 x 2 H2O 0.1 g NiCl2 x 6 H2O 1 g NaCl 0.03 g Na2SeO3 x 5 H2O 0.04 g Na2WO4 x 2 H2O 1000 ml Distilled water 1. First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 ml. ## 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