# 854: HIPPEA MEDIUM ## Main sol. 854 0.33 g NH4Cl 0.33 g CaCl2 x 2 H2O 0.33 g MgCl2 x 6 H2O 0.33 g KCl 0.33 g KH2PO4 25 g NaCl 3 g MOPS buffer (SIGMA) 10 ml **Modified Wolin's mineral solution** 0.1 g Yeast extract 5 g Na-acetate 0.5 ml Sodium resazurin (0.1% w/v) 10 g Sulfur (powdered) 1 ml **Wolin's vitamin solution (10x)** 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except sulfur, vitamins, and sulfide), adjust pH to 5.5 - 6.0, and sparge the medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense the medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials that contain already an appropriate amount of sulfur powder. Autoclave 20 min at 110°C. Prior to use add vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins are sterilized by filtration. Adjust the pH of the complete medium to 6.0 - 6.2, 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