Main sol. 52

Equipment needed: Autoclave Hungate tubes Filter Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
K-acetate	10.00	g	10	101.893
K₂HPO₄	0.31	g	0.31	1.78
KH₂PO₄	0.23	g	0.23	1.69
NH₄Cl	0.25	g	0.25	4.674
MgSO₄ x 7 H₂O	0.20	g	0.2	0.811
Trace element solution SL-10	1.00	ml	-	-
Selenite-tungstate solution	1.00	ml	-	-
Yeast extract	1.00	g	1	-
Sodium resazurin (0.1% w/v)	0.50	ml	5.0e-4	-
Ethanol absolute	20.00	ml	-	-
Na₂CO₃	1.00	g	1	9.435
Seven vitamins solution	1.00	ml	-	-
L-Cysteine HCl x H₂O	0.25	g	0.25	1.423
Na₂S x 9 H₂O	0.25	g	0.25	1.041
Distilled water	1000.00	ml	-	-
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.

52	CLOSTRIDIUM KLUYVERI MEDIUM