# 165: ACETIVIBRIO CELLULOLYTICUS MEDIUM


## Main sol. 165

75	ml	**Mineral solution 1**	
75	ml	**Mineral solution 2**	
10	ml	**Modified Wolin's mineral solution**	
20	ml	**FeSO4 x 7 H2O solution (0.1% w/v)**	
0.5	ml	Sodium resazurin (0.1% w/v)	
1.5	g	Na2CO3	
3	g	Cellobiose	
3	g	Cellulose (MN300) (optional)	
1	ml	**Wolin's vitamin solution (10x)**	
0.25	g	L-Cysteine HCl x H2O	
0.25	g	Na2S x 9 H2O	
820	ml	Distilled water	

1. Dissolve ingredients except carbonate, cellobiose, vitamins, cysteine and sulfide, then 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. Add cellobiose, vitamins, 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. Cellobiose and vitamins have to be sterilized by filtration. Prior to use adjust pH of complete medium to 7.2 - 7.4. Cellulose (MN 300, Whatman CF II, Kleenex tissue paper, or HCl treated cotton) can be used as an alternative to cellobiose.


## Mineral solution 1

3.9	g	K2HPO4	
1000	ml	Distilled water	


## Mineral solution 2

2.4	g	KH2PO4	
6	g	(NH4)2SO4	
0.59	g	NaCl	
1.2	g	MgSO4 x 7 H2O	
0.72	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.


## FeSO4 x 7 H2O solution (0.1% w/v)

1	g	FeSO4 x 7 H2O	
1000	ml	H2SO4 (0.1 N)	

1. The ferrous sulfate solution is not stable and should be freshly prepared.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water