# 1597a: METHANONATRONARCHAEUM MEDIUM


## Main sol. 1597a

480	ml	**Solution A**	
480	ml	**Solution B**	
1	ml	**Solution C**	
1	ml	**Solution D**	
5	ml	**Solution E**	
2	ml	**Solution F**	
10	ml	**Solution G**	
5	ml	**Solution H**	
10	ml	**Solution I**	
1	ml	**Solution J**	
5	ml	**Solution K**	
10	ml	**Solution L**	

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

92.5	g	Na2CO3	
17.5	g	NaHCO3	
8	g	NaCl	
0.5	g	K2HPO4	
480	ml	Distilled water	


## Solution B

120	g	NaCl	
1.25	g	K2HPO4	
2.5	g	KCl	
480	ml	Distilled water	


## Solution C

1	ml	**Trace elements solution (Pfennig & Lippert,1966)**	


## Solution D

1	ml	**Selenite-tungstate solution**	


## Solution E

0.2	g	NH4Cl	
0.25	g	MgSO4 x 7 H2O	
5	ml	Distilled water	


## Solution F

2	ml	Methanol	


## Solution G

3.4	g	Na-formate	
0.16	g	Na-acetate	
10	ml	Distilled water	


## Solution H

0.02	g	Yeast extract	
5	ml	Distilled water	


## Solution I

0.15	g	2-Mercaptoethanesulfonic acid (coenzyme M)	
10	ml	Distilled water	


## Solution J

1	ml	**Wolin's vitamin solution (10x)**	


## Solution K

5	ml	**Ferrous sulfide sludge**	


## Solution L

0.25	g	Na2S x 9 H2O	
10	ml	Distilled water	


## Trace elements solution (Pfennig & Lippert,1966)

5	g	EDTA	
2.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.03	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.03	g	CuCl2 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. pH 3.0-4.0


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Ferrous sulfide sludge

15.4	g	FeSO4 x 7 H2O	
12.3	g	Na2S x 9 H2O	
100	ml	Distilled water	

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)

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