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DSMZ

DSMZ 1597a . METHANONATRONARCHAEUM 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 480 ml
Solution B 480 ml
Solution C 1 ml
Solution D 1 ml
Solution E 5 ml
Solution F 2 ml
Solution G 10 ml
Solution H 5 ml
Solution I 10 ml
Solution J 1 ml
Solution K 5 ml
Solution L 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 L 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 L 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 #

480
Compound Amount Unit
Na2CO3 92.5 g
NaHCO3 17.5 g
NaCl 8.0 g
K2HPO4 0.5 g
Distilled water 480.0 ml

Solution B #

480
Compound Amount Unit
NaCl 120.00 g
K2HPO4 1.25 g
KCl 2.50 g
Distilled water 480.00 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 #

2
Compound Amount Unit
Methanol 2 ml

Solution G #

10
Compound Amount Unit
Na-formate 3.40 g
Na-acetate 0.16 g
Distilled water 10.00 ml

Solution H #

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

Solution I #

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

Solution J #

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

Solution K #

5
Compound Amount Unit
Ferrous sulfide sludge 5 ml

Solution L #

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

Ferrous sulfide sludge #

100
Compound Amount Unit
FeSO4 x 7 H2O 15.4 g
Na2S x 9 H2O 12.3 g
Distilled water 100.0 ml
1 Heat distilled water to 50°C in a 250 ml beaker with a stir bar present. While rapidly stirring the water, add the ferrous sulfate followed immediately by the sodium sulfide. The formed black FeS sludge is decanted into a glass bottle that can be stoppered. The FeS is allowed to settle for several hours and then the overlying water is decanted and replaced. This procedure is repeated at least five times to wash the FeS. After washing, the pH of the FeS solution should be close to neutrality. The FeS suspension can be kept in closed bottles or tubes under a nitrogen atmosphere for at least three months.

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