# 334: METHANOTHRIX MEDIUM


## Main sol. 334

951	ml	**Solution A**	
25	ml	**Solution B**	
10	ml	**Solution C**	
10	ml	**Solution D**	
1	ml	**Solution E**	
10	ml	**Solution F**	

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 (e.g., 20 ml medium in 50 ml bottles) and autoclave. Prior to inoculation complete the medium by adding appropriate amounts of sterile solutions C, D, E and F prepared under 100% N2 gas and solution B prepared under 80% N2 and 20% CO2 gas atmosphere. Vitamins are sterilized by filtration. Adjust the pH of the complete medium to 7.2.
2. Note: Use 20% (v/v) inoculum.


## 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)	
940	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

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.


## 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