# 1455: ALTERNATIVE RUMEN BACTERIA MEDIUM


## Main sol. 1455

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	
6	g	Brain heart infusion (BD BBL)	
5	g	Yeast extract	
3.1	ml	**Volatile fatty acid mixture**	
2	ml	**Haemin solution** (0.05% w/v)	
0.5	ml	Sodium resazurin (0.1% w/v)	
6.4	g	NaHCO3	
5	g	Disodium succinate	
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, succinate, vitamins, 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 reach a pH between 6.5 and 7.0. Dispense medium under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add succinate, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The vitamin solution should be sterilized by filtration. Prior to use adjust pH of complete medium to 6.6 - 6.8, if necessary.


## Volatile fatty acid mixture

548.5	ml	Acetic acid	
193.5	ml	Propionic acid	
129	ml	Butyric acid	
32.25	ml	n-Valeric acid	
32.25	ml	iso-Butyric acid	
32.25	ml	DL-2-Methylbutyric acid	
32.25	ml	iso-Valeric acid	


## Haemin solution

50	mg	Haemin	
1	ml	NaOH (1 N)	
100	ml	Distilled water	

1. Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated.


## 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