# 52: CLOSTRIDIUM KLUYVERI MEDIUM


## Main sol. 52

10	g	K-acetate	
0.31	g	K2HPO4	
0.23	g	KH2PO4	
0.25	g	NH4Cl	
0.2	g	MgSO4 x 7 H2O	
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)	
20	ml	Ethanol absolute	
1	g	Na2CO3	
1	ml	**Seven vitamins solution**	
0.25	g	L-Cysteine HCl x H2O	
0.25	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve all ingredients (except ethanol, carbonate, vitamins, cysteine and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add ethanol, then dispense medium under same gas atmosphere in Hungate-type tubes or serum vials and autoclave. After autoclaving, add carbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas atmosphere and vitamins from an anoxic and filter-sterilized stock solution prepared under 100% N2 gas. Before inoculation, add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Adjust pH of medium to 6.8 - 7.0, if necessary.
2. Note: If reduction of medium is not complete after inoculation, add 10 - 20 mg/l sodium dithionite from a 5% w/v solution freshly prepared under 100% N2 gas and filter sterilized.


## 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	


## 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