- The biofilm is investigated after approximately 24 h.
- For examination of the biofilm with CSLM, a viability staining
kit is used:
(a) If a non-GFP-taggedP. aeruginosastrain is used, LIVE/
DEAD viability staining kit can be applied. Syto 9 (live;
Invitrogen) and PI (dead) are added at a concentration of
0.005 mM and 0.01 mM, respectively, 15 min before
examination of the flow cells by injecting it the same way
as the bacterial culture explained instep 10(seeNote 5).
(b) If a GFP-taggedP. aeruginosastrain is used, only PI is
added to the flow chambers (seeNote 6). - Biofilm formation in flow chambers is examined by CSLM.
Figure3 is an example of 3-day-old biofilms ofP. aeruginosa
(PAO1) grown in flow chambers treated or untreated with
tobramycin and garlic extract, which we have shown to inhibit
QS inP. aeruginosa[13].
Fig. 3Images of 3-day-old biofilms grown in flow chambers. Biofilm shown in picture (a–d) are grown with the
following; (a) untreated, (b) tobramycin for 24 h, (c) garlic extract, (d) tobramycin for 24 h + garlic extract. Live
bacterial cells are appearing aslight graywhereas dead cells aredark gray. Reproduced from [13] with
permission from Journal of Bacteriology
282 Tim Holm Jakobsen et al.