# 500: CLOSTRIDIUM (GV) MEDIUM


## Main sol. 500

0.35	g	K2HPO4	
0.23	g	KH2PO4	
0.5	g	NH4Cl	
2.25	g	NaCl	
2	ml	FeSO4 x 7 H2O (0.1% w/v in 0.1 N H2SO4)	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
1	g	Yeast extract	
0.5	ml	Sodium resazurin (0.1% w/v)	
3	g	NaHCO3	
0.5	g	MgSO4 x 7 H2O	
0.25	g	CaCl2 x 2 H2O	
5	g	D-Glucose	
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, magnesium sulfate, calcium chloride, glucose, vitamins, cysteine and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH to 7.0, dispense under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add magnesium sulfate, calcium chloride, glucose, 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 the complete medium to 7.0 - 7.2, if necessary.


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## 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