# 397: FERULATE MEDIUM


## Main sol. 397

882	ml	**Solution A**	
30	ml	**Solution B**	
1	ml	**Solution D**	
10	ml	**Solution E**	
10	ml	**Solution F**	
10	mM	Sodium ferulate	

1. Add and dissolve ingredients of solution A and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes and autoclave. Solution B is prepared under 80% N2 and 20% CO2 gas atmosphere and autoclaved. Solutions E and F are autoclaved under 100% N2 gas atmosphere.
2. Solution D is prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated.
3. Note: Some cultures are shipped in semi-solid medium which stimulates growth at the beginning. For agar stabs 3.00 g/l agar are added to the complete medium from a sterile anoxic stock solution (2% w/v). Upon receipt add anoxically 1 - 2 ml of the recommended freshly prepared liquid medium to the agar tube and incubate for 3 - 5 days . After incubation transfer 0.5 ml of the resulting cell suspension in the liquid phase to tubes with liquid medium.
4. Add sodium ferulate to 10 mM final concentration from filter-sterilized anaerobic stock solution.


## Solution A

0.5	g	KH2PO4	
0.33	g	MgCl2 x 6 H2O	
0.4	g	NaCl	
0.4	g	NH4Cl	
0.05	g	CaCl2 x 2 H2O	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
50	ml	**Clarified rumen fluid**, clarified	
1	g	Trypticase peptone (BD BBL)	
0.5	ml	Sodium resazurin (0.1% w/v)	
830	ml	Distilled water	


## Solution B

1.5	g	Na2CO3	
30	ml	Distilled water	


## Solution D

1	ml	**Seven vitamins solution**	


## Solution E

0.3	g	L-Cysteine HCl x H2O	
10	ml	Distilled water	


## Solution F

0.3	g	Na2S x 9 H2O	
10	ml	Distilled water	


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 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.