# 88a: SULFOLOBUS MEDIUM (ANAEROBIC) ## Main sol. 88a 1.3 g (NH4)2SO4 0.28 g KH2PO4 0.25 g MgSO4 x 7 H2O 0.07 g CaCl2 x 2 H2O 0.02 g FeCl3 x 6 H2O 10 ml **Allen's trace element solution** 10 g Sulfur (powder) 0.5 g Yeast extract (OXOID) 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except sulfur, yeast extract and sulfide), adjust pH of the salt solution at room temperature to 4.0 using 1 N H2SO4 and sparge medium with 100% N2 gas to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials that contain already the appropriate amount of sulfur. Sterilize medium by heating cultivation vessels in a boiling water bath for 1 - 2 hours on each of 3 successive days. Add yeast extract and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to inoculation check pH and adjust to 4.0, if necessary. ## Allen's trace element solution 180 mg MnCl2 x 4 H2O 450 mg Na2B4O7 x 10 H2O 22 mg ZnSO4 x 7 H2O 5 mg CuCl2 x 2 H2O 3 mg Na2MoO4 x 2 H2O 3 mg VOSO4 x 2 H2O 1 mg CoSO4 x 7 H2O 1000 ml Distilled water 1. Adjust pH of final solution to 2 with 1 N HCl.