Main sol. 915

Equipment needed: Hungate tubes Filter Autoclave Gassing station Centrifuge

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
K₂HPO₄	0.30	g	0.299	1.719
KH₂PO₄	0.30	g	0.299	2.2
(NH₄)₂SO₄	0.30	g	0.299	2.266
NH₄Cl	1.00	g	0.998	18.658
NaCl	0.61	g	0.609	10.417
MgSO₄ x 7 H₂O	0.12	g	0.12	0.486
CaCl₂ x 2 H₂O	0.08	g	0.08	0.543
Modified Wolin's mineral solution	10.00	ml	-	-
Clarified rumen fluid	100.00	ml	-	-
Yeast extract	2.00	g	1.996	-
Sodium resazurin (0.1% w/v)	0.50	ml	5.0e-4	0.002
NaHCO₃	7.50	g	7.485	89.1
D-Glucose	5.00	g	4.99	27.698
Wolin's vitamin solution (10x)	2.00	ml	-	-
L-Cysteine HCl x H₂O	0.45	g	0.449	2.557
Na₂S x 9 H₂O	0.45	g	0.449	1.87
Distilled water	890.00	ml	-	-
1 Dissolve ingredients (except bicarbonate, glucose, vitamins and reducing agents), then sparge medium with 100% CO₂ gas for 30 - 45 min to make it anoxic. Add solid bicarbonate, adjust pH to 6.8 - 7.0 and dispense medium under 100% CO₂ gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add glucose, vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas. The final pH of the medium should be 7.0.

915	BLAUTIA MEDIUM