318. TEPIDIBACILLUS MEDIUM

Sea Salts (SIGMA) 30.0 g

NaNO₃ 1.0 g

Wolfe's mineral elixier (see below) 2.0 ml

VOSO₄ × 5 H₂O solution (0.01% w/v) 0.5 ml

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

FeSO₄ × 7 H₂O 0.1 g

NaHCO₃ 1.0 g

Yeast extract 0.5 g

D-glucose 2.5 g

Vitamins solution (see below) 20.0 ml

Distilled water 1000.0 ml

Wolfe's mineral elixier:

MgSO₄ × 7 H₂O 30.0 g

MnSO₄ × H₂O 5.0 g

NaCl 10.0 g

FeSO₄ × 7 H₂O 1.0 g

CoCl₂ × 6 H₂O 1.8 g

CaCl₂ × 2 H₂O 1.0 g

ZnSO₄ × 7 H₂O 1.8 g

CuSO₄ × 5 H₂O 0.1 g

KAl(SO₄)₂ × 12 H₂O 0.18 g

H₃BO₃ 0.1 g

Na₂MoO₄ × 2 H₂O 0.1 g

(NH4)₂Ni(SO₄)₂ × 6 H₂O 2.8 g

Na₂WO₄ × 2 H₂O 0.1 g

Na₂SeO₄ 0.1 g

Distilled water 1000.0 ml. First adjust pH to 1.0 with diluted H₂SO₄, then add and dissolve the salts.

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 ferrous sulfate, bicarbonate, yeast extract, D-glucose and vitamins), adjust pH to 7.0 and sparge medium with 100% N₂ gas for 30 – 45 min to make it anoxic. Dispense the medium under same gas atmosphere into Hungate-type tubes or serum vials and autoclave. Add ferrous sulfate, bicarbonate, yeast extract, glucose and vitamins from sterile anoxic stock solutions prepared under 100% N₂ gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas atmosphere. Prepare the stock solution of ferrous sulfate by dissolving 1% (w/v) FeSO₄ × 7 H₂O in 0.1 N H₂SO₄. The stock solutions of glucose and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 6.8-7.0. Note: It may be necessary to add 10-20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under 100% N₂ and filter-sterilized), if the medium is not completely reduced after inoculation.

 

Updated 01.02.2024