321. BROCKIA LITHOTROPHICA MEDIUM

NH₄Cl 0.33 g

KH₂PO₄ 0.33 g

KCl 0.33 g

CaCl₂ × 2 H₂O 0.44 g

MgCl₂ × 6 H₂O 0.7 g

NaCl 0.5 g

Trace element solution SL-10 (see below) 1.0 ml

Na-resazurin solution (0.1% w/v) 0.5 ml

Sulfur, powdered 10.0 g

NaHCO₃ 0.8 g

Vitamin solution (see below) 10.0 ml

Na₂S × 9 H₂O 0.5 g

Distilled water 1000.0 ml

Trace element solution SL-10:

HCl (25%; 7.7 M) 10.0 ml

FeCl₂ × 4 H₂O 1.5 g

ZnCl₂ 70.0 mg

MnCl₂ × 4 H₂O 100.0 mg

H₃BO₃ 6.0 mg

CoCl₂ × 6 H₂O 190.0 mg

CuCl₂ × 2 H₂O 2.0 mg

NiCl₂ × 6 H₂O 24.0 mg

Na₂MoO₄ × 2 H₂O 36.0 mg

Distilled water 990.0 ml

Selenite-tungstate solution:

NaOH 0.5 g

Na₂SeO₃ × 5 H₂O 3 mg

Na₂WO₄ × 2 H₂O 4 mg

Distilled water 1000.0 ml

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine-HCl 10.0 mg

Thiamine-HCl × 2 H₂O 5.0 mg

Riboflavin 5.0 mg

Nicotinic acid 5.0 mg

D-Ca-pantothenate 5.0 mg

Vitamin B₁₂ 0.1 mg

p-Aminobenzoic acid 5.0 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

Dissolve ingredients (except sulfur, bicarbonate, vitamins and sulfide), then sparge medium with 80% H₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 6.2-6.4, and dispense medium under 80% H₂ and 20% CO₂ gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur. Sterilize medium by heating cultivation vessels in a boiling water bath for 2-3 hours on each of 3 successive days. After sterilization add bicarbonate from a sterile stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Vitamins (sterilized by filtration) and sulfide are added to the medium from sterile anoxic stock solutions prepared under 100% N₂ gas. Adjust pH of complete medium to 6.5, if necessary.

Updated 06.07.2021