# 712: PSEUDOBUTYRIVIBRIO MEDIUM


## Main sol. 712

0.23	g	K2HPO4	
0.23	g	KH2PO4	
0.45	g	NaCl	
0.45	g	(NH4)2SO4	
0.06	g	CaCl2 x 2 H2O	
0.09	g	MgSO4 x 7 H2O	
5	g	Yeast extract	
5	mg	Indigocarmine	
6.4	g	NaHCO3	
5	g	D-Glucose	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, vitamins, glucose, cysteine and sulfide), then sparge medium with 100% CO2 gas for 30 - 45 min to make it anoxic. Add the bicarbonate and equilibrate the medium with the CO2 gas to pH 6.8. Distribute medium under CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Thereafter, add vitamins, glucose, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins are sterilized by filtration. Adjust pH of complete medium to 6.7 - 6.8, if necessary.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water