481. DEHALOGENOMONAS MEDIUM (DehM)

Basal salts solution (see below) 10 ml

Resazurin 0.001 g

Carbon source solution (see below) 5 ml

L-cysteine hydrochloride 0.25 g

NaHCO₃ 2.52 g

NaOH 0.065 g

Trace element solution SL-10 (see below) 1ml

Vitamin solution (see above) 1 ml

Selenite-tungstate solution (see below) 1 ml

Titanium citrate solution (see below) 4 ml

1,2-dichloroethane 0.5 mM

Mix basal salts solution, carbon source solution, and resazurin solution, boil and sparge medium with 100% N₂ for 30 – 45 min to make it anoxic. Once cool, add L-cysteine hydrochloride and NaOH solution. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave at 121oC for 15 min. Add other components from sterile anoxic stock solutions prepared under 100% N₂ gas. After inoculation fill 1,2-dichloroethane 0.5 mM in gas phase.

Basal salt solution:

NaCl 100 g

KH₂PO₄ 20 g

NH₄Cl 25 g

KCl 50 g

MgCl₂·x 6 H₂O 40 g

CaCl₂·x 6 H₂O 10 g

Distilled water 1000.00 ml

Carbon source solution:

Sodium acetate 0.41 g

Sodium pyruvate 0.55 g

Sodium lactate 0.57 g

Distilled water 100.00 ml

Trace element solution SL-10:

HCl (25% 7.7 M) 10.0 ml

FeCl₂ x 4 H₂O 1.5 g

ZnCl₂ 70.0 mg

MnCl₂ x 4 H₂O 100.0 mg

H₃BO₃ 6.0 mg

CoCl₂ x 6 H₂O 190.0 mg

CuCl₂ x 2 H₂O 2.0 mg

NiCl₂ x 6 H₂O 24.0 mg

Na₂MoO₄ x 2 H₂O 36.0 mg

Distilled water 990.0 ml

The trace element mixture should be autoclaved in brown bottle tightly closed with rubber-fitted screw caps or stoppers; leave a headspace of approximately 1/3 of the bottle volume. Purge the headspace with H₂/CO₂/N₂ gas mixture (10%/10%/80%).

Selenite-tungstate solution:

NaOH 0.5 g

Na₂SeO₃ x 5 H₂O 3.0 mg

Na₂WO₄ x 2 H₂O 4.0 mg

Distilled water 1000.0 ml

The selenite-tungstate solution can be autoclave in a sealed bottle. Purge the headspace with H₂/CO₂/N₂ gas mixture (10%/10%/80%).

Vitamin solution:

Biotin 2.00 mg

Folic acid 2.00 mg

Pyridoxine-HCl 10.00 mg

Thiamine-HCl 5.00 mg

Riboflavin 5.00 mg

Nicotinic acid 5.00 mg

Ca-D-pantothenate 5.00 mg

Vitamin B₁₂ 0.10 mg

p-Aminobenzoic acid 5.00 mg

(±)-α-Lipoic acid 5.00 mg

Distilled water 1000.00 ml

The vitamin solution should be filter-sterilized (0.2 μm pore size) and kept at 4^oC in the dark. Purge gas headspace with filter-sterilized nitrogen (N₂) or anaerobic gas mix (10% N₂ 10% CO₂ 80% N₂).

Titanium citrate solution:

Titanium(III) chloride 1.5 g

Sodium citrate·2 H₂O 5.95 g

Distilled water 100.00 ml

Add 100 ml distilled water to a beaker, boil in microwave, and then carefully pour the hot water into a 250 ml glass serum bottle that contains a Teflon-coated magnetic stir bar. Purge the water with N₂ gas while cooling in an ice bath. Add 5.95 g sodium citrate·2 H₂O, cap the serum bottle with a butyl rubber stopper, and mix on a stir plate until the sodium citrate is fully dissolved. Slowly add 1.50 g of titanium chloride. Centrifuge the titanium citrate solution at 8,000×g for 20 minutes. Decant the supernatant into a clean beaker, and then filter-sterilize the supernatant and transfer into four 25 ml aliquots, each aliquot placed in a separate, sterile, glass 60 ml serum bottle. Cap the serum bottles with butyl rubber stoppers and aluminum crimp caps.

Updated: 07.01.2024