# 1667: HYPNOCYCLICUS MEDIUM


## Main sol. 1667

0.02	g	Na2SO4	
1	g	KH2PO4	
0.1	g	NH4Cl	
20	g	NaCl	
3	g	MgCl2 x 6 H2O	
0.5	g	KCl	
0.15	g	CaCl2 x 2 H2O	
0.7	g	NaNO3	
1	g	Yeast extract	
1	ml	**Trace element solution SL-10**	
0.5	ml	Sodium resazurin (0.1% w/v)	
1.5	g	Na2CO3	
1	ml	**Seven vitamins solution**	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve all ingredients except carbonate, vitamins and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Vitamins should be sterilized by filtration. Adjust pH of complete medium to 6.8 - 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.


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water