304. HALONATRONUM SACCHAROPHILUM MEDIUM

KH₂PO₄ 0.2 g

MgCl₂ 0.1 g

NH₄Cl 0.5 g

KCl 0.2 g

NaCl 50.0 g

Na₂CO₃ 68,0 g

NaHCO₃ 38,0 g

Na₂S × 9 H₂O 0.7 g

Yeast extract 0.2 g

Sucrose 5.0 g

Trace elements solution (see below) 1.0 ml

Vitamin solution (see below) 10.0 ml

Resazurin 0.01 g

Distilled water 1000.0 ml

Final pH 9.5-10.0

Trace element solution:

Nitrilotriacetic acid 1.5 g

MgSO₄ × H₂O 3.0 g

MnSO₄ × 7 H₂O 0.5 g

NaCl 1.0 g

FeSO₄ × 7 H₂O 0.1 g

CoSO₄ × 7 H₂O 0.18 g

CaCl₂ × 2 H₂O 0.1 g

ZnSO₄ × 7 H₂O 0.18 g

CuSO₄ × 5 H₂O 0.01 g

KAl(SO₄)₂ × 12 H₂O 0.02 g

H₃BO₃ 0.01 g

Na₂MoO₄ × 2 H₂O 0.01 g

NiCl₂ × 6 H₂O 0.03 g

Na₂SeO₃ × 5 H₂O 0.3 g

Na₂WO₄ × 2 H₂O 0.4 g

Distilled water 1000.0 ml

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH

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

Prepare the medium without adding the vitamins, sucrose, yeast extract, NH₄Cl, Na₂CO₃, and NaHCO₃ using anaerobic conditions, under nitrogen. If the medium has been boiled to remove oxygen add the NH₄Cl, Na₂CO₃, and NaHCO₃ after the medium had cooled. Dispense into tubes stopper with rubber stoppers (serum tubes or bottles, or screw capped tubes). Autoclave, and to the cooled medium add the vitamins (filter sterilized under N₂), sucrose, yeast extract, and from anaerobic, sterile stock solutions.