405. MAGNETOSPIRILLUM KUZNETSOVII MEDIUM (MKM)

KH₂PO₄ 0.68 g

NaNO₃ 0.12 g

L(+)-Tartaric acid 0.37 g

Succinic acid 0.37 g

Na-acetate × 3 H₂O 0.05 g

Vitamin solution (see below) 10.0 ml

Trace element solution (see below) 5.0 ml

Fe(III) quinate solution (see below) 2.0 ml

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

Na-thioglycolate 0.05 g

Distilled water 1000.0 ml

Trace element solution:

Nitrilotriacetic acid 1.5 g

MgSO₄ × 7 H₂O 3.0 g

MnSO₄ × 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 mg

Na₂WO₄ × 2 H₂O 0.4 mg

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

Ferric Quinate Solution, 0.01 M:

FeCl₃ × 6 H₂O 4.5 g

Quinic acid 1.9 g

Distilled water 1000.0 ml

Sterilize by filtration under 100% N₂ gas atmosphere.

Dissolve ingredients (except thioglycolate) and adjust pH to 6.75 with NaOH. Sparge medium with 100% N₂ gas for 30-45 min and dispense under the same gas atmosphere into anoxic Hungate – type tubes to 50% of their volume. Before inoculation add thioglycolate from a 0.5% (w/v) stock solution, freshly prepared under 100% N₂ gas and filter-sterilized. Then add sterile air to a concentration of 1.0 % O₂ in the vial.