- To measure fluorescence and growth of monitor strains, an
automated multi label plate reader is used. Growth is measured
at an optical density at 450 nm. Fluorescence is measured as
excitation and emission wavelength at 485 nm and 535 nm,
respectively. - AB minimal growth medium: 1 mM MgCl 2 , 0.1 mM CaCl 2 ,
and 0.01 mM FeCl 3 in Milli-Q water; sterilize the solution by
autoclaving. Thereafter, add 10% (vol/vol) of A10 (see descrip-
tion below). Store at 4C. - A10: 20 g/l of (NH 4 ) 2 SO 4 , 60 g/l of Na 2 HPO 4 , 30 g/l of
KH 2 PO 4 , and 30 g/l of NaCl in Milli-Q water. Adjust pH to
6.4 and sterilize the solution by autoclaving. Store at 4C for
several months. - Glucose.
- Bacto™Casamino Acid.
2.2 In Vitro
Continuous-Culture
Biofilm Flow Cell
System
- Bacterial strains:P. aeruginosa(PAO1-ATCC) obtained from
thePseudomonasGenetic Stock Center (www.pseudomonas.
med.ecu.edu, strain PAO0001), and P. aeruginosa(PAO1-
ATCC) constitutively expressing a stable GFP [2]. - AB trace minimal growth medium (ABT): 1 mM MgCl 2 ,
0.1 mM CaCl 2 , and 0.01% (vol/vol) trace metals solution
(see description below) in Milli-Q water. Sterilize the solution
by autoclaving. Thereafter add 10% (vol/vol) A10 (seeSub-
heading2.1,item 5). Store at 4C. - Trace metal solution: 200 mg/l CaSO 4 ·2H 2 O, 200 mg/l
FeSO 4 ·7H 2 O, 20 mg/l MnSO 4 ·H 2 O, 20 mg/l CuSO 4 ·5
H 2 O, 20 mg/l ZnSO 4 ·7H 2 O, 10 mg/l CoSO 4 ·7H 2 O,
12 mg/l Na 2 MoO 4 ·H 2 O, and 5 mg/l H 3 BO 3. Can be stored
at room temperature for several months. Remember to mix
before use. - 0.3 mM glucose.
- Bacto™Casamino Acid.
- 16-channel peristaltic pump.
- Equipment required to assemble the system:
(a) Silicon tubing, int. diameter: 2 mm, ext. diameter: 4 mm
(labeled “A” in Fig.2).
(b) Silicon tubing, int. diameter: 1 mm, ext. diameter: 3 mm
(labeled “B” in Fig.2).
(c) Straight connectors 1/8^00 (labeled “1” in Fig.2).
(d) T-connectors 1/8^00 (labeled “2” in Fig.2).
(e) Reducing connectors 1/8^00 1/16^00 (labeled “3” in
Fig.2).
(f) Barrel tip cap orange (labeled “4” in Fig.2).
In vitro Determination of Quorum Sensing Inhibition 277