# 1597b: NATRANAEROFABA MEDIUM


## Main sol. 1597b

500	ml	**Solution A**	
500	ml	**Solution B**	
1	ml	**Solution C**	
1	ml	**Solution D**	
5	ml	**Solution E**	
10	ml	**Solution F**	
5	ml	**Solution G**	
10	ml	**Solution H**	
10	ml	**Solution I**	

1. Sterilize solutions A and B in closed thick-walled screw-top bottles (e.g., SCHOTT) for 20 min at 120°C. The pH of solution A after sterilization should be 10. There is some precipitate forming that settles at the bottom after 3 - 4 days. It is best to remove precipitates by decantation before using solution A for medium preparation.
2. Combine solution A with solution B and sparge medium with 100% N2 gas for at least 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions C to I are sterilized separately under 100% N2 gas. Vitamins and pyruvate should be sterilized by filtration. To complete the medium appropriate amounts of solutions C to I are added to the combined sterile solutions A and B in the sequence as indicated. Final pH of the medium should be 9.5.
3. Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum.


## Solution A

92.5	g	Na2CO3	
17.5	g	NaHCO3	
8	g	NaCl	
0.5	g	K2HPO4	
500	ml	Distilled water	


## Solution B

120	g	NaCl	
1.25	g	K2HPO4	
2.5	g	KCl	
500	ml	Distilled water	


## Solution C

1	ml	**Trace elements solution (Pfennig & Lippert,1966)**	


## Solution D

1	ml	**Selenite-tungstate solution**	


## Solution E

0.2	g	NH4Cl	
0.25	g	MgSO4 x 7 H2O	
5	ml	Distilled water	


## Solution F

2.2	g	Na-pyruvate	
10	ml	Distilled water	


## Solution G

0.5	g	Yeast extract	
5	ml	Distilled water	


## Solution H

10	ml	**Wolin's vitamin solution**	


## Solution I

0.25	g	Na2S x 9 H2O	
10	ml	Distilled water	


## Trace elements solution (Pfennig & Lippert,1966)

5	g	EDTA	
2.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.03	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.03	g	CuCl2 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. pH 3.0-4.0


## Selenite-tungstate solution

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


## Wolin's vitamin solution

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water