# 1743: GHIORSEA MEDIUM


## Main sol. 1743

27.5	g	NaCl	
5.38	g	MgCl2 x 6 H2O	
6.78	g	MgSO4 x 7 H2O	
0.72	g	KCl	
0.5	g	NH4Cl	
1.4	g	CaCl2 x 2 H2O	
0.05	g	KH2PO4	
10	ml	**Trace element solution**	
1.7	g	NaHCO3	
1	ml	**Wolin's vitamin solution (10x)**	
1000	ml	Distilled water	

1. Dissolve ingredients (except vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture to make it anoxic and until a pH of 6.5 is reached. Dispense medium under same gas atmosphere into anoxic serum vials to 20% of their volume and autoclave. After autoclaving add vitamins from a sterile stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration. Prior to inoculation add 5% (v/v) sterile H2 and 5% (v/v) sterile air to the headspace by using syringes. Check pH of the complete medium and adjust pH to 6.5, if necessary. Incubate without shaking.
2. Note: After growth has started cell density of the culture can be increased by replenishing H2 and air every two days.


## Trace element solution

0.5	g	Na2-EDTA x 2 H2O	
0.15	g	CoCl2 x 6 H2O	
0.1	g	MnCl2 x 4 H2O	
0.1	g	FeSO4 x 7 H2O	
0.1	g	ZnCl2	
40	mg	AlCl3 x 6 H2O	
40	mg	Na2WO4 x 2 H2O	
30	mg	Na2SeO3 x 5 H2O	
20	mg	NiCl2 x 6 H2O	
20	mg	CuCl2 x 2 H2O	
10	mg	H3BO3	
10	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve EDTA in distilled water and adjust pH to 7 using 2 N NaOH, then dissolve remaining compounds.


## 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