# 1662: ALKALIPHILUS HYDROTHERMALIS MEDIUM ## Main sol. 1662 0.3 g KH2PO4 0.3 g K2HPO4 0.5 g NH4Cl 0.1 g KCl 5 g NaCl 0.5 g MgCl2 x 6 H2O 0.1 g CaCl2 x 2 H2O 5 g Yeast extract (OXOID) 5 g Trypticase peptone (BD BBL) 10 ml **Modified Wolin's mineral solution** 3 g Na2CO3 10 ml **Na-crotonate solution (1 M)** 0.3 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except carbonate, crotonate and sulfide), adjust pH to 8.5 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add crotonate and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. The crotonate solution should be sterilized by filtration. Adjust pH of the complete medium to 9.0, 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. ## Na-crotonate solution (1 M) 86 g Crotonic acid (ALDRICH 113018) 100 ml NaOH (10 N) 900 ml Distilled water 1. Dissolve crotonic acid in 800 ml distilled water, add around 100 ml of 10 N NaOH and adjust pH to around 7. Then add water to reach a volume of 1000 ml. Sterilize by filtration under 100% N2 gas atmosphere.