# 784a: METHANOCALCULUS NATRONOPHILUS MEDIUM


## Main sol. 784a

0.2	g	KH2PO4	
0.1	g	MgCl2 x 6 H2O	
1	g	NH4Cl	
0.2	g	KCl	
60	g	NaCl	
1	ml	**Trace element solution SL-11**	
0.2	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
68	g	Na2CO3	
38	g	NaHCO3	
1	ml	**Wolin's vitamin solution (10x)**	
1	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, bicarbonate, vitamins and sulfide, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve carbonates and sulfide while gassing the head space only. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Before use add vitamins from an anoxic stock solution sterilized by filtration. Adjust pH of the complete medium to 9.0 - 9.5.


## Trace element solution SL-11

5.2	g	Na2-EDTA x 2 H2O	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 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