# 931: ALCALIPHILIC AMPHIBACILLUS STRAINS MEDIUM


## Main sol. 931

0.2	g	KH2PO4	
0.1	g	MgCl2	
0.5	g	NH4Cl	
0.2	g	KCl	
63.6	g	Na2CO3	
50.4	g	NaHCO3	
0.7	g	Na2S x 9 H2O	
0.2	g	Yeast extract	
5	g	Sucrose	
1	ml	**Modified Wolin's mineral solution**	
10	ml	**Modified Wolin's mineral solution**	
1	mg	Resazurin	
1000	ml	Distilled water	

1. Final pH 9.5-10.0
2. Prepare the medium without adding the vitamins, sucrose, yeast extract, NH4Cl, Na2CO3, and NaHCO3 using anaerobic conditions, under nitrogen. If the medium has been boiled to remove oxygen add the NH4Cl, Na2CO3, and NaHCO3 after the medium had cooled. Dispense into tubes stopper with rubber stoppers (serum tubes or bottles, or screw capped tubes). Autoclave, and to the cooled medium add the vitamins (filter sterilised under N2), sucrose, yeast extract, and Na2S x 9 H2O from anaerobic, sterile stock solutions.


## 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.