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Main sol. 52

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Internal Solution-ID: S67

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