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Main sol. J827

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Internal Solution-ID: S4786

Equipment needed: Filter Autoclave Anaerobic workbench
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 1.000 g 0.946 16.189
MgCl2 x 6 H2O 0.410 g 0.388 1.908
NH4Cl 0.270 g 0.255 4.775
KCl 0.520 g 0.492 6.599
CaCl2 x 2 H2O 0.015 g 0.014 0.097
KH2PO4 0.200 g 0.189 1.39
FeCl2 solution 1.000 ml - -
Trace element solution 1.000 ml - -
Selenite-tungstate solution 1.000 ml - -
Sodium acetate 0.410 g 0.388 4.729
Resazurin 0.250 mg 2.4e-4 0.001
Distilled water 1000.000 ml - -
1 Mix components thoroughly, bring to a boil and cool down under a N2 atmosphere. Add the following solutions from anaerobic stocks, then dispense the medium into culture vessels leaving 40 to 50% volume of a gas phase (e.g., 30 ml of the medium in a 50 ml serum bottle) and autoclave under a N2 atmosphere:
L-Cysteine HCl x H2O
(5%) 		1.000 ml - -
Na2S x 9 H2O
(5%) 		1.000 ml - -
2 After cooling, replace the gas phase with a N2-CO2-H2 (8:1:1, v/v) gas mixture. Add per liter the following filter-sterilized solutions from anaerobic stocks:
NaHCO3
(8%) 		10.000 ml - -
MOPS
(pH 7.4, 1.0 M) 		20.000 ml - -
25 mM Ti(III) NTA solution
(25 mM) 		20.000 ml - -
Vitamin solution 1.000 ml - -
3 To complete the medium, add per liter the following filter-sterilized chlorosolvent:
Trichloroethylene
(10 mg/ml in methanol) 		0.700 ml - -
4 Check final pH to be about 7.2.
J827 DEHALOCOCCOIDES MEDIUM