# 274: METHANOLACINIA MEDIUM


## Main sol. 274

0.34	g	KCl	
2.75	g	MgCl2 x 6 H2O	
3.4	g	MgSO4 x 7 H2O	
1.5	g	NH4Cl	
0.14	g	CaCl2 x 2 H2O	
0.14	g	K2HPO4	
6.31	g	NaCl	
10	ml	**Modified Wolin's mineral solution**	
2	ml	Fe(NH4)2(SO4)2 x 6 H2O (0.1% w/v)	
5	g	NaHCO3	
4.15	g	Na-acetate x 3 H2O	
0.5	ml	Sodium resazurin (0.1% w/v)	
20.93	g	MOPS (3-(N-morpholino) propane-sulfonic acid)	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except MOPS, vitamins, cysteine and sulfide in a total volume of 890 ml. The MOPS buffer is dissolved separately in 90 ml of distilled water, the pH is adjusted to 7.0 with 2 M KOH, then add the buffer to the salt solution. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense medium in anoxic Hungate-type tubes under the same gas atmosphere to 30% of their volume and autoclave. Add vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 7.0, if necessary.
2. After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## 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