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Main sol. 683

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Internal Solution-ID: S1431

Equipment needed: Filter Hungate tubes Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 5.80 g 5.783 98.95
MgCl2 x 6 H2O 0.40 g 0.399 1.962
KCl 0.30 g 0.299 4.012
CaCl2 x 2 H2O 0.15 g 0.15 1.017
NH4Cl 0.27 g 0.269 5.033
KH2PO4 0.20 g 0.199 1.465
Trace element solution SL-10 1.00 ml - -
Selenite-tungstate solution 1.00 ml - -
Yeast extract
(OXOID) 		10.00 g 9.97 -
Trypticase peptone
(BD BBL) 		0.50 g 0.499 -
L-Serine 2.00 g 1.994 18.974
L-Threonine 2.00 g 1.994 16.74
N-methylhydantoin
(SIGMA) 		5.64 g 5.623 49.281
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
NaHCO3 4.50 g 4.487 53.407
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.50 g 0.499 2.838
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, cysteine and vitamins) and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Adjust pH to 8.3 with 10 N NaOH, add bicarbonate and cysteine and dissolve. Switch gas phase to 80% N2 and 20% CO2 gas mixture and flush only the head space of the medium vessel. Dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave 15 min at 121°C. Add vitamins to the autoclaved medium from an anoxic stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 8.3, if necessary.
2 Note: If creatinine is used as the substrate replacing N-methylhydantoin, prepare separately an anoxic, filter sterilized stock solution of creatinine under 100% N2 gas atmosphere.
683 TISSIERELLA (AMO.1) MEDIUM