# 778b: CALDIMICROBIUM MEDIUM ## Main sol. 778b 0.33 g KH2PO4 0.33 g NH4Cl 0.33 g KCl 0.33 g MgCl2 x 6 H2O 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 0.2 ml NiCl2 x 6 H2O (0.1% w/v) 1 g Yeast extract 0.5 ml Sodium resazurin (0.1% w/v) 2.5 g NaHCO3 0.33 g CaCl2 x 2 H2O 1.6 g Na2-fumarate 2 g Na2S2O3 x 5 H2O 1 ml **Wolin's vitamin solution (10x)** 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except bicarbonate, calcium chloride, fumarate, thiosulfate, vitamins and sulfide). Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, add solid bicarbonate and adjust pH to 7.2. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add calcium chloride, fumarate, thiosulfate, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Stock solutions of fumarate, thiosulfate and vitamins are sterilized by filtration. Adjust pH of complete medium to 7.0 - 7.2, if necessary. ## Trace element solution SL-10 10 ml HCl (25%) 1.5 g FeCl2 x 4 H2O 70 mg ZnCl2 100 mg MnCl2 x 4 H2O 6 mg H3BO3 190 mg CoCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 24 mg NiCl2 x 6 H2O 36 mg Na2MoO4 x 2 H2O 990 ml Distilled water 1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. ## Selenite-tungstate solution 0.5 g NaOH 3 mg Na2SeO3 x 5 H2O 4 mg Na2WO4 x 2 H2O 1000 ml Distilled water ## 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