# J1159: MOLLICUTES H-RF3 MEDIUM


## Main sol. J1159

1. Mix components and adjust pH to 7.5. Bring to a boil and cool down under a N2-CO2 (4:1, v/v) gas stream. Distribute the medium into culture vessels (e.g., 20 ml medium in 60 ml serum bottles) under the same gas mixture, seal with butyl rubber stoppers and autoclave. After cooling, aseptically and anaerobically add per liter the following solutions (autoclaved or *filter-sterilized): 

0.14	g	KH2PO4	
0.2	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
0.54	g	NH4Cl	
1	g	Casamino acids (BD-Difco)	
1	g	Yeast extract	
1	ml	**Trace minerals**	
0.5	mg	Resazurin	
1000	ml	Distilled water	
30	ml	NaHCO3 (*, 8%)	
10	ml	**Trace vitamins** (*)	
6	ml	L-Cysteine HCl x H2O (5%)	
6	ml	Na2S x 9 H2O (5%)	

2. Readjust pH to 7.0, if necessary.


## Trace minerals

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0.


## Trace vitamins

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 pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water