# 329: METHANOHALOPHILUS HALOPHILUS MEDIUM


## Main sol. 329

0.33	g	NH4Cl	
0.33	g	KH2PO4	
0.33	g	KCl	
0.01	g	K2SO4	
70	g	NaCl	
1	ml	**Trace element solution SL-10**	
0.05	g	Yeast extract (OXOID)	
0.5	ml	Sodium resazurin (0.1% w/v)	
5	g	NaHCO3	
0.33	g	MgCl2 x 6 H2O	
0.33	g	CaCl2 x 2 H2O	
5	g	Trimethylamine-HCl	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	L-Cysteine HCl x H2O	
0.15	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except bicarbonate, magnesium chloride, calcium chloride, vitamins, trimethylamine, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add bicarbonate and equilibrate pH to 6.9 - 7.0, then distribute under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add magnesium chloride, calcium chloride, vitamins (sterilized by filtration), trimethylamine, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use adjust pH of complete medium to 7.2, if necessary.


## Trace element solution SL-10

10	ml	HCl (25%)	
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	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally 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