3.3 Culture
Preparation for DNA
Microarray
- InoculateP. aeruginosa(PAO1) in AB medium supplemented
with 0.5% (wt/vol) casamino acids. - Grow culture at 180 rpm at 37CtoanOD 600 of approx. 0.5.
- Dilute culture to an OD 600 of 0.05 in 200 ml fresh medium.
- Culture in a 1 l conical flask at 180 rpm at 37C.
- At OD 600 of 0.5 the culture is split into two 100 ml cultures in
500 ml flasks (seeNote 7). - From a 100–1000-fold concentrated stock solution (in the
appropriate solvent), add QSI to one culture flask (the concen-
tration must not affect growth rate ofP. aeruginosa). A similar
volume of the solvent used for preparing the stock solution is
added to the other culture flask, which then serves as a refer-
ence (or no treatment) culture. - Grow cultures with shaking (180 rpm) at 37C.
- Retrieve samples at OD 600 of 2.0 and immediately add RNA
later (1:2) (seeNote 8). - Store samples at 20 C if not used the same day.
- Isolate RNA by using the “RNeasy Mini Purification Kit”
(Qiagen) (seeNote 9).
4 Notes
- Flow chambers and bubble traps can be purchased from DTU
BIOENGINEERING, Department of Biotechnology and Bio-
medicine, Technical University of Denmark. - If the added test compound (QSI) is diluted in solvents that
effect growth of the monitor strain, dilute the solution in 0.9%
(wt/vol) NaCl to a concentration that is not affecting growth
of the monitor strain. - It is important that there are no bubbles in the flow chambers.
If bubbles are present, try to remove them by gently knocking
the flow chamber on the inlet side to the table. Remember to
check for bubbles in the flow chamber during the entire exper-
imental period, but after the flow cells are inoculated with
bacteria removal of bubbles should be avoided. If larger bub-
bles are generated, the biofilm development can be affected
resulting in unusable results. Smaller bubbles can be displaced
by the bacteria and will therefore not affect the result. - The amount of growth media used is dependent on the num-
ber of flow channels used and therefore must be calculated
before initiating the experiment to make sure that the system
does not exhaust the media.
In vitro Determination of Quorum Sensing Inhibition 283