Team:Groningen/protocols/biofilm media

The strains were grown overnight at 37°C on LB broth.
Next, 1 ml of liquid culture was used to inoculate every 10 ml MSgg medium. this in turn was incubated at 27°C for 5 days.

Media for macrocolony formation

2x SG medium (100 mL)

compound	amount	treatment
Nutrient broth (Difco)	1.6 g
KCl	0.2 g
MgSO4.7H2O	0.05 g
agar	1.5 g
		add everything together and autoclave
Ca(NO3)2.4H2O 1 mM	0.1 mL
MnCl2.4H2O 0.1 mM	0.1 mL
FeSO4 1 µM	0.1 mL
0.1% glucose	0.5 mL

LB/Mn/glucose medium (100 mL)

compound	amount	treatment
LB/ 1.5% agar	100 ml	autoclave
MnCl2.4H2O 0.1 mM	0.1mL
0.1% glucose	0.5 mL

MSgg medium (100 mL)

compound	amount	treatment
KH2PO4	0.026 g
K2HPO4	0.061 g
MOPS 100 mM	2.09 g
MgCl2.6H2O 2 mM	0.04 g
agar	1.5 g
		add everything together and autoclave
CaCl2 700 mM	0.1 mL
MnCl2 50 µM	50 µL
FeCl3 50 µM	0.1 mL
ZnCl2 1 µM	0.1 mL
thiamine 2 µM	0.1 mL
0.5% glycerol	0.57 mL
0.5% glutamate	10 mL
tryptophan	1 mL

Grow cells overnight in LB / glass tubes at 37°C.
Inoculate 2 µL spot on plate dry 15 minutes at 30°C
Inoculate 30°C 2-4 days