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DSMZ

DSMZ 1597 . METHANOSALSUM NATRONOPHILUM MEDIUM

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Equipment needed: Autoclave Filter Gassing station Hungate tubes
Final gas composition: 100 % N2 read more
Final pH: 9.5
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ml
Compound Amount Unit
Solution A 750 ml
Solution B 250 ml
Solution C 1 ml
Solution D 1 ml
Solution E 5 ml
Solution F 3 ml
Solution G 5 ml
Solution H 10 ml
Solution I 1 ml
Solution J 10 ml
1 Sterilize solutions A and B in closed thick-walled screw-top bottles (e.g., SCHOTT) for 20 min at 120°C. The pH of solution A after sterilization should be 10. There is some precipitate forming that settles at the bottom after 3 - 4 days. It is best to remove precipitates by decantation before using solution A for medium preparation.
2 Combine solution A with solution B and sparge medium with 100% N2 gas for at least 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions C to J are sterilized separately under 100% N2 gas. Vitamins and coenzyme M should be sterilized by filtration. To complete the medium appropriate amounts of solutions C to J are added to the combined sterile solutions A and B in the sequence as indicated. Final pH of the medium should be 9.5.
3 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum.

Solution A #

750
Compound Amount Unit
Na2CO3 71.25 g
NaHCO3 11.25 g
NaCl 4.00 g
K2HPO4 0.75 g
Distilled water 750.00 ml

Solution B #

250
Compound Amount Unit
NaCl 30.00 g
K2HPO4 0.25 g
KCl 0.25 g
Distilled water 250.00 ml

Solution C #

1
Compound Amount Unit
Trace elements solution (Pfennig & Lippert,1966) 1 ml

Solution D #

1
Compound Amount Unit
Selenite-tungstate solution 1 ml

Solution E #

5
Compound Amount Unit
NH4Cl 0.20 g
MgSO4 x 7 H2O 0.25 g
Distilled water 5.00 ml

Solution F #

3
Compound Amount Unit
Methanol 3 ml

Solution G #

5
Compound Amount Unit
Yeast extract 0.02 g
Distilled water 5.00 ml

Solution H #

10
Compound Amount Unit
2-Mercaptoethanesulfonic acid
(coenzyme M) 		0.15 g
Distilled water 10.00 ml

Solution I #

1
Compound Amount Unit
Wolin's vitamin solution (10x) 1 ml

Solution J #

10
Compound Amount Unit
Na2S x 9 H2O 0.25 g
Distilled water 10.00 ml

Trace elements solution (Pfennig & Lippert,1966) #

1000
Compound Amount Unit
EDTA 5.00 g
FeSO4 x 7 H2O 2.20 g
ZnSO4 x 7 H2O 0.10 g
MnCl2 x 4 H2O 0.03 g
H3BO3 0.03 g
CoCl2 x 6 H2O 0.20 g
CuCl2 x 2 H2O 0.03 g
NiCl2 x 6 H2O 0.03 g
Na2MoO4 x 2 H2O 0.03 g
Distilled water 1000.00 ml
1 pH 3.0-4.0

Selenite-tungstate solution #

1000
Compound Amount Unit
NaOH 0.5 g
Na2SeO3 x 5 H2O 3.0 mg
Na2WO4 x 2 H2O 4.0 mg
Distilled water 1000.0 ml

Wolin's vitamin solution (10x) #

1000
Compound Amount Unit
Biotin 20 mg
Folic acid 20 mg
Pyridoxine hydrochloride 100 mg
Thiamine HCl 50 mg
Riboflavin 50 mg
Nicotinic acid 50 mg
Calcium D-(+)-pantothenate 50 mg
Vitamin B12 1 mg
p-Aminobenzoic acid 50 mg
(DL)-alpha-Lipoic acid 50 mg
Distilled water 1000 ml