# 287: METHANOCULLEUS CHIKUGOENSIS MEDIUM


## Main sol. 287

0.3	g	K2HPO4	
0.3	g	KH2PO4	
0.3	g	(NH4)2SO4	
0.61	g	NaCl	
0.14	g	CaCl2 x H2O	
0.13	g	MgSO4 x 7 H2O	
10	ml	**Modified Wolin's mineral solution**	
2.7	g	NH4Cl	
2.5	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
5	g	NaHCO3	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, vitamins, cysteine and sulfide) and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH to 6.8 and then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum bottles and autoclave. After sterilization add cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.8 - 7.0. For incubation use sterile 80% H2 and 20% CO2 gas mixture at one atmospheres of pressure.


## 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