# 1277b: CALDIMICROBIUM THIODISMUTANS MEDIUM ## Main sol. 1277b 0.2 g MgCl2 x 6 H2O 0.1 g CaCl2 x 2 H2O 0.1 g (NH4)2SO4 0.1 g KH2PO4 0.1 g KCl 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 45 ml **Amorphous Fe(OH)3** 2.5 g Na2CO3 0.9 g Na2S2O5 1 ml **Seven vitamins solution** 960 ml Distilled water 1. Dissolve ingredients (except carbonate, pyrosulfite and vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into Hungate-type tubes or serum vials and autoclave. Add pyrosulfite from a 10% (w/v) stock solution adjusted to pH 7.5-8.0 with NaOH, made anoxic with 100% N2 gas and sterilized by filtration. Vitamins are added from a stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration and carbonate is added from a sterile stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Adjust pH of complete medium to 7.5 - 8.0. Incubate without shaking. ## 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 ## Amorphous Fe(OH)3 320 ml FeCl3 x 6 H2O (60.00 g/l) 80 ml NaOH (10% w/v), adjust if required 1. Slowly titrate 320 ml of a FeCl3 x 6 H2O stock solution (60.00 g/l) with 10% (w/v) NaOH to pH 8.0-8.5 under agitation (use magnetic stirrer). Total amount of added NaOH approx. 80 - 100 ml. The precipitated Fe(OH)3 should be stored at room temperature overnight with surface covered with water. Thereafter, centrifuge at 2000 rpm for 5 min and discard the supernatant. Wash several times with distilled water. Resuspend the pellet in medium as described above. For storage autoclave under 100% N2 atmosphere. ## Seven vitamins solution 100 mg Vitamin B12 80 mg p-Aminobenzoic acid 20 mg D-(+)-biotin 200 mg Nicotinic acid 100 mg Calcium pantothenate 300 mg Pyridoxine hydrochloride 200 mg Thiamine-HCl x 2 H2O 1000 ml Distilled water