# J1033: ST65 MEDIUM


## Main sol. J1033

1. Mix components, except sulfur, bring to a boil and cool down under a CO2 (100%) gas stream. While gassing, add the following components to the medium: 

18	g	NaCl	
4.33	g	MgCl2 x 6 H2O	
0.33	g	NH4Cl	
0.33	g	KCl	
0.165	g	KH2PO4	
0.033	g	CaCl2 x 2 H2O	
10	ml	**Trace elements solution**	
5	g	Sulfur (powder)	
1000	ml	Distilled water	
10	ml	**Trace vitamins**	
2	g	NaHCO3	

2. Bubble CO2 for 3 - 4 min and check pH to be 6.5 - 6.8. Readjust pH to the range if necessary. Autoclave under a CO2 gas atmosphere. Add the following solution to the medium (separately autoclaved under N2): 

10	ml	KNO3 (1 M)	

3. Steam sulfur for 3 hr on each of 3 successive days. Aseptically and anaerobically distribute the medium into culture vessels containing appropriate amount of sulfur (e.g., 10 ml of the medium and 0.05 g sulfur in Hungate tubes) under a CO2 stream and seal with butyl rubber stoppers.


## Trace elements solution

78.4	mg	Fe(NH4)2(SO4)2 x 6 H2O	
23.8	mg	CoCl2 x 6 H2O	
16.6	mg	NiCl2 x 6 H2O	
2.42	mg	Na2MoO4 x 2 H2O	
5.49	mg	Na2WO4 x 2 H2O	
14.4	mg	ZnSO4 x 7 H2O	
0.17	mg	CuCl2 x 2 H2O	
9.45	mg	Na2SeO4	
0.62	mg	H3BO3	
19.8	mg	MnCl2 x 4 H2O	
0.92	mg	SrSO4	
0.5	mg	CrK(SO4)2 x 12 H2O	
2.41	mg	AlCl3 x 6 H2O	
2.08	mg	BaCl2	
14.2	mg	Na2SiO3 x 9 H2O	
11.9	mg	KBr	
16.6	mg	KI	
71	mg	Na2SO4	
1000	ml	Distilled water	


## Trace vitamins

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water