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Main sol. 540

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Internal Solution-ID: S1194

Equipment needed: Autoclave Hungate tubes Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.20 g 0.197 1.448
Na2SO4 0.10 g 0.099 0.694
NH4Cl 0.30 g 0.296 5.526
KCl 0.50 g 0.493 6.608
NaCl 7.00 g 6.897 118.011
MgCl2 x 6 H2O 1.20 g 1.182 5.815
CaCl2 x 2 H2O 0.15 g 0.148 1.005
Yeast extract 1.50 g 1.478 -
Modified Wolin's mineral solution 10.00 ml - -
NiCl2 x 6 H2O
(0.1% w/v) 		1.00 ml 9.9e-4 0.008
Na2WO4 x 2 H2O
(0.1% w/v) 		0.10 ml 9.9e-5 3.0e-4
Ascorbic acid
(0.01% w/v) 		0.50 ml 4.9e-5 2.8e-4
Myo-inositol
(0.01% w/v) 		0.50 ml 4.9e-5 2.7e-4
Niacinamide
(0.01% w/v) 		0.50 ml 4.9e-5 4.0e-4
Choline chloride
(0.01% w/v) 		0.50 ml 4.9e-5 3.5e-4
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
NaHCO3 4.00 g 3.941 46.911
D-Glucose 5.00 g 4.926 27.343
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.30 g 0.296 1.683
Na2S x 9 H2O 0.30 g 0.296 1.231
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, glucose, vitamins and reducing agents), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add solid bicarbonate and equilibrate pH to 6.8 with 80% N2 and 20% CO2 gas mixture, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add glucose, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Vitamins should be sterilized by filtration. Adjust pH of complete medium to 6.8 - 7.0, if necessary.
540 ACETITOMACULUM MEDIUM