# 336: OXOBACTER PFENNIGII MEDIUM


## Main sol. 336

972	ml	**Solution A**	
30	ml	**Solution B**	
1	ml	**Solution C**	
10	ml	**Solution D**	
3	ml	**Solution E**	

1. Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, distribute under same gas atmosphere 8.9 ml medium per Hungate-type tube and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solution C is prepared under 100% N2 gas atmosphere and sterilized by filtration. Autoclave solutions D and E under 100% N2 gas. To complete the medium add appropriate amounts of the solutions B to E to the sterile solution A. Adjust pH of complete medium to 7.0 - 7.2, if necessary.


## Solution A

50	ml	**Mineral solution**	
1	ml	**Trace element solution SL-10**	
2	g	Yeast extract	
2	g	Na-vanillate	
300	ml	**Clarified rumen fluid**	
0.5	ml	Sodium resazurin (0.1% w/v)	
620	ml	Distilled water	

1. Adjust pH to 6.9 before autoclaving.


## Solution B

1.5	g	Na2CO3	
30	ml	Distilled water	


## Solution C

1	ml	**Seven vitamins solution**	


## Solution D

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


## Solution E

90	mg	Na2S x 9 H2O	
3	ml	Distilled water	


## Mineral solution

10	g	KH2PO4	
6.6	g	MgCl2 x 6 H2O	
8	g	NaCl	
8	g	NH4Cl	
1	g	CaCl2 x 2 H2O	
1000	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.


## 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.


## 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