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Main sol. 1237

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Internal Solution-ID: S2459

Equipment needed: Hungate tubes Filter Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.49 g 0.484 3.554
Na2HPO4 1.19 g 1.175 8.275
NaCl 25.00 g 24.679 422.299
NH4Cl 0.30 g 0.296 5.537
MgCl2 x 6 H2O 0.10 g 0.099 0.486
CaCl2 x 2 H2O 0.01 g 0.01 0.067
Modified Wolin's mineral solution 10.00 ml - -
Tryptone
(BD Bacto) 		2.00 g 1.974 -
Proteose peptone
(BD Difco) 		2.00 g 1.974 -
Yeast extract
(OXOID) 		2.00 g 1.974 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
FeCl2
(0.1% w/v in 0.2 N HCl) 		1.50 ml 0.001 0.012
Na2CO3 2.50 g 2.468 23.285
D-Glucose 5.00 g 4.936 27.397
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.50 g 0.494 2.81
Na2S x 9 H2O 0.50 g 0.494 2.055
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except ferrous chloride, carbonate, glucose, vitamins, cysteine and sulfide), adjust pH to 7.5 with NaOH and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add glucose, vitamins, ferrous chloride, cysteine and sulfide form sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. Solutions of ferrous chloride and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 8.5, if necessary.
1237 TEPIDIMICROBIUM MEDIUM