# J981: ANAEROBIC BICARBONATE-BUFFERED MEDIUM WITH PHENOL


## Main sol. J981

0.2	g	KH2PO4	
0.45	g	NH4Cl	
1.7	g	NaCl	
0.5	g	KCl	
0.4	g	MgCl2 x 6 H2O	
0.16	g	CaCl2 x 2 H2O	
4	g	Na2SO4	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
1	ml	**Selenite-tungstate solution**	
1	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly and autoclave under a N2-CO2 (4:1, v/v) gas mixture. After cooling, add the following solutions (filter-sterilized): 

30	ml	NaHCO3 (8.0%)	
1	ml	**Vitamin solution**	
1	ml	**Thiamine solution**	
1	ml	**Vitamin B12 solution 0.05 g/L**	

2. Distribute the medium into sterile culture vessels (e.g., 50 ml per 120 ml bottle) under a N2-CO2 (4:1, v/v) gas mixture, and seal with sterile butyl rubber stoppers. Prior to inoculation, add per liter the following solutions (autoclaved or *filter-sterilized and stored under a N2 atmosphere) in the order listed: 

10	ml	Phenol (freshly prepared, *, 0.1 M)	
10	ml	Na2S x 9 H2O (5%)	


## FeCl2 solution

10	ml	HCl (7.7 M, 25%)	
1.5	g	FeCl2 x 4 H2O	
990	ml	Distilled water	


## Trace element solution

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	
1000	ml	Distilled water	


## Selenite-tungstate solution

0.4	g	NaOH	
6	mg	Na2SeO3 x 5 H2O	
8	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Vitamin solution

4	mg	p-Aminobenzoic acid	
1	mg	Biotin	
10	mg	Nicotinic acid	
5	mg	DL-Calcium pantothenate	
10	mg	Pyridoxine hydrochloride	
100	ml	Sodium phosphate buffer (10 mM, pH 7.1)	


## Thiamine solution

10	mg	Thiamine HCl	
100	ml	Sodium phosphate buffer (25 mM, pH 3.4)	


## Vitamin B12 solution 0.05 g/L

5	mg	Vitamin B12	
100	ml	Distilled water