# 321: METHANOCORPUSCULUM AGGREGANS MEDIUM


## Main sol. 321

1	g	NH4Cl	
0.4	g	K2HPO4 x 3 H2O	
0.4	g	MgCl2 x 6 H2O	
50	ml	**Minerals solution**	
5	g	Na-formate	
1	g	Na-acetate	
1	g	Trypticase peptone (BD BBL)	
1	g	Yeast extract (OXOID)	
10	ml	**Modified Wolin's mineral solution**	
5	ml	**Sludge fluid**	
5	ml	**Clarified rumen fluid**, alternative (optional)	
0.5	ml	Sodium resazurin (0.1% w/v)	
1.5	g	Na2CO3	
0.2	g	L-Cysteine HCl x H2O	
0.2	g	Na2S x 9 H2O	
935	ml	Distilled water	

1. Dissolve ingredients except carbonate, cysteine and sulfide, then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary.
2. After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 2 bar overpressure.


## Minerals solution

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


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## 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.


## Sludge fluid

4	g	Yeast extract	
1000	ml	Sludge	

1. Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark.