- Use minimum two medium flasks. Hook, for example, chan-
nels 1–8 up to the medium flask which contains the test QSI
compound and hook the growth medium flask up to channels
9–16 as the controls (seeNote 4). The medium flasks (not the
connecting tubing’s) can be kept on ice in order to avoid turn
over (chemical instability) of the test compound, and flasks
with freshly prepared contents can be prepared each day. - Grow an overnight culture withP. aeruginosa(PAO1) or a
GFP-tagged PAO1. - Before inoculation with the overnight culture, the flow is
stopped and the tubes are clamped off between the flow chan-
nels and the bubble traps. - Inoculate the flow chambers using 250μl of the overnight
culture diluted to an OD 600 of 0.1 in 0.9% (wt/vol) NaCl.
Inject the diluted culture in the flow channels by using a
syringe needle which is inserted in the tubing next to the flow
channel inlet. Close the injection hole with a thin layer of
silicone. - The flow of media is arrested for 1 h to allow efficient coloni-
zation of the glass surface. Flip the flow cell upside down
placing it on the glass surface. - Flip the flow cells back in “upright” position (glass slides are
facing upwards) and start the flow of media. Remove the
clamps and let the medium perfuse the flow chambers at a
constant rate of approximately 3.3 ml/h using the peristaltic
pump. - We usually allow the biofilm to develop and mature in the flow
chambers for 3 days. - At day 3 the growth medium is changed to fresh growth
medium containing antibiotic and antibiotic with QSI. The
medium is changed by:
(a) Stopping the flow.
(b) Clamp the tube of between the flow cells and bubble
traps.
(c) Empty the bubble traps by pulling the syringe off and
quickly again.
(d) Remove the barrel tip cap and fill the bubble traps with
fresh growth medium containing, for example, antibio-
tics, e.g., at 90 rpm.
(e) When the bubble traps are filled up, stop the system and
remove the clamps.
(f) Start the system again on 1.75 rpm (approximately
3.3 ml/h).
In vitro Determination of Quorum Sensing Inhibition 281