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Main sol. J378

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Internal Solution-ID: S4103

Equipment needed: Autoclave Anaerobic workbench Filter
Compound Amount Unit Conc. [g/L] Conc. [mM]
Yeast extract
(BD-Difco) 		2.00 g 100 -
Tryptone
(BD-Difco) 		2.00 g 100 1406.87
Crotonic acid 0.86 g 43 499.477
KH2PO4 0.50 g 25 183.707
MgCl2 x 6 H2O 0.33 g 16.5 81.16
NaCl 0.40 g 20 342.231
NH4Cl 0.40 g 20 373.902
CaCl2 x 2 H2O 0.05 g 2.5 17.005
Trace metal solution 10.00 ml - -
Trace vitamins 10.00 ml - -
NaHCO3 3.50 g 175 2083.16
L-Cysteine HCl x H2O 0.50 g 25 -
Na2S x 9 H2O 0.50 g 25 104.088
Resazurin 1.00 mg 0.05 0.218
1 Dissolve ingredients, except crotonic acid, NaHCO3, L-cysteine x HCl x H2O and Na2S x 9H2O, in 900 ml of distilled water and adjust pH to 7.0-7.5. Bring to a boil for 5-10 sec, then, cool down under a stream of N2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense 0.9 volume of the medium into suitable culture vessels under a stream of N2-CO2 (80:20, v/v) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Add 0.1 volume of a filter-sterilized crotonic acid solution (0.86 g/100 ml, pH 7.0) to the medium. Autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the solutions of crotonic acid, L-cysteine x HCl x H2O and Na2S x 9H2O to the medium.
J378 ALKALIPHILUS CROTONATOXIDANS MEDIUM