# 503b: FWM MEDIUM (PERCHLORATE)


## Main sol. 503b

950	ml	**Solution A**	
30	ml	**Solution B**	
10	ml	**Solution C**	
1	ml	**Solution D**	
10	ml	**Solution E**	

1. Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic, distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions D and E are prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. Adjust pH of complete medium to 7.0 - 7.2, if necessary.


## Solution A

0.2	g	KH2PO4	
0.25	g	NH4Cl	
1	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.5	g	KCl	
0.15	g	CaCl2 x 2 H2O	
0.15	g	Na2SO4	
10	ml	**Modified Wolin's mineral solution**	
940	ml	Distilled water	


## Solution B

1.5	g	Na2CO3	
30	ml	Distilled water	


## Solution C

1.36	g	Na-acetate	
10	ml	Distilled water	


## Solution D

1	ml	**Wolin's vitamin solution (10x)**	


## Solution E

1.22	g	Sodium perchlorate	
10	ml	Distilled water	


## 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