Hi, I am trying to prepare DSMZ 120a for methanosarcina according to the mentioned protocol on DSMZ site. But everytime I autoclave, I see the precipitation of medium. The pH of media was 6.8. Can anyone suggest me what to do?
DSMZ120a composition:
K2HPO4 0.35 g
KH2PO4 0.23 g
NH4Cl 0.50 g
MgSO4 x 7 H2O 0.50 g
CaCl2 x 2 H2O 0.25 g
NaCl 2.25 g
FeSO4 x 7 H2O solution (0.1% w/v in 0.1 N H2SO4) 2.00 ml
Trace element solution SL-10 (see medium 320) 1.00 ml
Yeast extract (OXOID) 2.00 g
Casitone (BD BBL) 2.00 g
Na-resazurin solution (0.1% w/v) 0.50 ml
NaHCO3 0.85 g
Vitamin solution (see medium 141) 10.00 ml
Methanol 10.00 ml
L-Cysteine-HCl x H2O 0.30 g
Na2S x 9 H2O 0.30 g
Distilled water 1000.00 ml
Dissolve ingredients (except bicarbonate, vitamins, methanol, cysteine and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic. Then add and dissolve bicarbonate, adjust pH to 6.8 and dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Methanol (50% v/v stock solution) and the reducing agents are each autoclaved separately under 100% N2 gas atmosphere as concentrated solutions in tightly closed tubes. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Appropriate volumes of the solutions are injected into the sterile medium with hypodermic syringes. Adjust pH of the complete medium to 6.5 - 6.8, if necessary.
Possibly magnesium phosphate or iron phosphate have formed, which are quite insoluble.
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