# 1596: METHANOCALCULUS ALKALIPHILUS MEDIUM


## Main sol. 1596

3	g	NaCl	
1	g	K2HPO4	
20	g	NaHCO3	
15	g	Na2CO3	
0.2	g	NH4Cl	
0.5	g	MgSO4 x 7 H2O	
1	ml	**Trace elements solution (Pfennig & Lippert,1966)**	
1	ml	**Selenite-tungstate solution**	
3.4	g	Na-formate	
0.16	g	Na-acetate	
0.02	g	Yeast extract (OXOID)	
1	ml	**Wolin's vitamin solution (10x)**	
0.25	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve sodium chloride and hydrogenphosphate, then sparge solution with 100% N2 gas for at least 30 - 45 min to make it anoxic. Add and dissolve hydrogencarbonate and carbonate while gassing the head space only, then dispense under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add ammonium chloride, magnesium sulfate, trace elements, formate, acetate, yeast extract, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins should be sterilized by filtration. The pH of the complete medium should be 9.5.
2. 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.


## 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	


## 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