# 704: BUTYRIVIBRIO SP. MEDIUM


## Main sol. 704

75	ml	**Mineral solution**	
150	ml	**Clarified rumen fluid**	
0.3	g	K2HPO4	
2	g	Trypticase peptone (BD BBL)	
2	g	Yeast extract (OXOID)	
3.1	ml	**Volatile fatty acid mixture**	
2	ml	**Haemin solution** (0.05% w/v)	
0.5	g	Glycerol	
0.5	ml	Sodium resazurin (0.1% w/v)	
4	g	Na2CO3	
1	g	D-Glucose	
1	g	Maltose	
1	g	Cellobiose	
1	g	Starch (soluble)	
0.25	g	L-Cysteine HCl x H2O	
0.25	g	Na2S x 9 H2O	
770	ml	Distilled water	

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


## Mineral solution

6	g	KH2PO4	
12	g	NaCl	
6	g	(NH4)2SO4	
1.6	g	CaCl2 x 2 H2O	
2.5	g	MgSO4 x 7 H2O	
1000	ml	Distilled water	


## Clarified rumen fluid

1. Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C.


## 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.