# 334a: METHANOTHRIX SOEHNGENII MEDIUM


## Main sol. 334a

942	ml	**Solution A**	
25	ml	**Solution B**	
10	ml	**Solution C**	
10	ml	**Solution D**	
1	ml	**Solution E**	
1	ml	**Solution F**	
1	ml	**Solution G**	
10	ml	**Solution H**	

1. Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, dispense under same gas atmosphere into anoxic serum vials or balch tubes (e.g., 20 ml medium in 50 ml bottles) and autoclave. Prior to inoculation complete the medium by adding sterile solutions C - H prepared under 100% N2 gas and solution B prepared under 80% N2 and 20% CO2 gas atmosphere. Solutions E, F and G are sterilized by filtration. Adjust the pH of the complete medium to 7.2.
2. Notes: Medium is only stable for a few weeks and has to be prepared freshly. Use 20% (v/v) inoculum. Sodium sulfide should be as pure as possible (use only clean crystals for the preparation of stock solutions). It has been noted that impurities of sulfide stock solutions can inhibit growth.


## Solution A

0.22	g	KH2PO4	
0.86	g	Na2HPO4 x 2 H2O	
6.8	g	Na-acetate	
1	ml	Na2-EDTA (0.1% w/v)	
10	ml	**Trace elements solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
100	ml	**Cell-free culture supernatant of <i>S. associata</i>**	
830	ml	Distilled water	


## Solution B

1.25	g	Na2CO3	
25	ml	Distilled water	


## Solution C

0.3	g	NaCl	
0.11	g	CaCl2 x 2 H2O	
0.1	g	MgCl2 x 6 H2O	
10	ml	Distilled water	


## Solution D

0.3	g	NH4Cl	
10	ml	Distilled water	


## Solution E

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


## Solution F

5	mg	Butyl vinyl ether (ALDRICH 110299)	
1	ml	Methanol	


## Solution G

5	mg	L-α-Phosphatidylcholine (SIGMA P3556)	
1	ml	Methanol	


## Solution H

0.5	g	Na2S x 9 H2O	
10	ml	Distilled water	


## Trace elements solution

12.8	g	Nitrilotriacetic acid (NTA)	
1.35	g	FeCl3 x 6 H2O	
0.1	g	MnCl2 x 4 H2O	
0.03	g	CoCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnCl2	
0.03	g	CuCl2	
0.01	g	H3BO3	
0.03	g	Na2MoO4 x 2 H2O	
0.12	g	NiCl2 x 6 H2O	
1	g	NaCl	
0.03	g	Na2SeO3 x 5 H2O	
1000	ml	Distilled water	

1. First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 ml.


## Cell-free culture supernatant of <i>S. associata</i>

1. Cultivate <i>Sphaerochaeta associata</i> DSM 26261 in DSM medium 119 supplemented with 1.00 g/l glucose at 28°C until stationary phase is reached (7-10 days). Centrifuge culture at 3500 xg for 30 min, then sterilize supernatant by filtration and add it to the anoxic solution A before autoclaving.
2. Note: It is also possible to add 50.00 ml/l of a grown <i>S. associata</i> culture to solution A before autoclaving, provided bacterial DNA from dead cells is not a problem in the downstream processing of the Methanothrix culture.


## 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