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Main sol. 912

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Internal Solution-ID: S1874

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 1.00 g 0.997 17.06
MgCl2 x 6 H2O 0.50 g 0.499 2.452
KH2PO4 0.20 g 0.199 1.465
K2HPO4 0.25 g 0.249 1.431
NH4Cl 0.30 g 0.299 5.592
KCl 0.30 g 0.299 4.012
CaCl2 x 2 H2O 15.00 mg 0.015 0.102
Trace element solution SL-10 1.00 ml - -
Selenite-tungstate solution 1.00 ml - -
MOPS
(SIGMA) 		2.10 g 2.094 10.005
D-Glucose 0.90 g 0.897 4.981
Yeast extract 0.19 g 0.189 -
Peptone
(BD Bacto) 		0.19 g 0.189 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
NaHCO3 2.50 g 2.493 29.67
Clarified rumen fluid 50.00 ml - -
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.40 g 0.399 2.271
Na2S x 9 H2O 0.40 g 0.399 1.66
Distilled water 950.00 ml - -
1 Dissolve ingredients (except bicarbonate, rumen fluid, vitamins, cysteine and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add bicarbonate, adjust pH to 7.2, distribute medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add rumen fluid, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Vitamins should be sterilized by filtration. Check pH of complete medium and adjust to 7.2, if necessary.
912 TREPONEMA R8 MEDIUM