Main sol. 769

Equipment needed: Autoclave Hungate tubes Filter Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Sea Salt (SIGMA)	12.0	g	11.988	-
NH₄NO₃	0.1	g	0.1	-
Yeast extract (BD Bacto)	1.0	g	0.999	-
Trypticase peptone (BD BBL)	0.4	g	0.4	-
Sodium resazurin (0.1% w/v)	0.5	ml	5.0e-4	0.002
NaHCO₃	1.0	g	0.999	11.892
Na-phosphate buffer (0.2 M, pH 6.5)	20.0	ml	-	4
Lactose	2.0	g	1.998	5.837
Wolin's vitamin solution (10x)	1.0	ml	-	-
L-Cysteine HCl x H₂O	0.3	g	0.3	1.706
Na₂S x 9 H₂O	0.3	g	0.3	1.248
Distilled water	980.0	ml	-	-
1 Dissolve ingredients (except bicarbonate, phosphate-buffer, lactose, vitamins, cysteine and sulfide), then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add phosphate-buffer, lactose, cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N₂ gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Vitamins are prepared under N₂ gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.5, if necessary.
2 Note: Sea salts can be replaced by 300.0 ml/l of filtered aged sea water.

769	CLOSTRIDIUM VINCENTII MEDIUM