# 1501: METHYLOCALDUM MARINUM MEDIUM


## Main sol. 1501

100	ml	**Solution 1 (10x NMS salts)**	
900	ml	NaCl (2 %)	
0.1	ml	**Solution 2 (Fe EDTA)**	
1	ml	**Solution 3 (Sodium molybdate)**	
1	ml	**Trace elements**	
1.5	%	Agar, for solid medium	
10	ml	**Phosphate buffer**	

1. Prepare the growth medium as follows:
2. Dilute 100 ml solution 1 to 1 litre with 2% NaCl and then add 1 ml of solution 3, 1 ml of
the trace elements, and 0.1 ml of solution 2. If solid media is required add 1.5% agar.
Dispense the medium into the growth vessels. If using sealed vessels it is appropriate to
add 25% methane to the gas phase and autoclave at 121C (15psi) for 15 minutes.
Autoclave the phosphate buffer separately. When the growth medium is cool 10 ml/l of
the phosphate buffer is added - if too warm the phosphate will precipitate. Liquid
cultures should be grown with shaking.


## Trace elements

25	mg	Na2-EDTA	
20	mg	CuSO4 x 5 H2O	
50	mg	FeSO4 x 7 H2O	
40	mg	ZnSO4 x 7 H2O	
1.5	mg	H3BO3	
5	mg	CoCl2 x 6 H2O	
2	mg	MnCl2 x 4 H2O	
1	mg	NiCl2 x 6 H2O	
100	ml	Distilled water	

1. May be stored at 4°C in the dark


## Phosphate buffer

71.6	g	Na2HPO4 x 12 H2O	
26	g	KH2PO4	
1000	ml	Distilled water	

1. Dissolve in about 800 ml of water, adjust the pH to 6.8 and make up to 1 litre.


## Solution 1 (10x NMS salts)

10	g	KNO3	
10	g	MgSO4 x 6 H2O	
2	g	CaCl2 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve the ingredients listed above (in that order) in about 700 ml of 2% NaCl, and then make up to 1 litre with 2% NaCl.


## Solution 2 (Fe EDTA)

3.8	g	Fe(III)-EDTA	
100	ml	Distilled water	


## Solution 3 (Sodium molybdate)

0.26	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water