spread evenly over TLC plate). Immediately pour mixture
over pre-warmed plate and spread evenly to cover. Layer
should be approx. 3 mm thick.
(d) Allow the agar to solidify, and then place in a plastic tub
(avoids dehydration during incubation). Cover and place
in a 30C incubator for 12–18 h. Check for blue spots.
The most hydrophobic (largest) AHLs will be near the
origin (bottom of the TLC plate) with the smaller (less
hydrophobic) AHLs migrating a longer distance. Sample
results are shown in Fig.5.
(e) Plastic tub can be disinfected after use with bleach or 70%
(v/v) ethanol.3.3 Biofilm
Dispersion Screen
The method listed below is based on the use of a 96-well microtiter
polystyrene plate and was adapted from a flow cell assay. Other
microtiter plates can be used and colonization substrata can be
added (to test for adhesion and release from surfaces in addition
to polystyrene). Specific culturing and dispersion conditions will
need to be determined empirically.- Prior to the screen, plan the arrangement of the cultures and
tests on the microtiter plate. (We often use an Excel spread-
sheet to plan the distribution of the various tests and controls).
Given the 96-well format, one can perform replicates, using
different organisms, culture conditions, and candidate-
dispersing agents, as well as including controls. - Prepare culture inoculum (typically we grow fast-growing
organisms such asP. aeruginosa,Staphylococcus aureus,Escher-
ichia colifor 18–24 h in broth culture and use this culture as an
inoculum). - Partially fill wells on microtiter plate with 200μl media (assum-
ing that well capacity is 300μl). - Inoculate some (but not all the wells) with culture inoculum (it
is very important to have uninoculated controls). We typically
use a 1% (v/v) inoculum. - Cover the microtiter plate and seal the edge with parafilm.
- Incubate on an orbital shaker. The swirling will cause biofilms
to form along the edges of the wells and is useful for organisms
such asP. aeruginosathat typically form pellicles at air–liquid
interfaces. Incubation time can be determined empirically (typ-
ically older biofilms>48-h incubation [48] will detach more
readily than younger biofilms). - Using a multichannel pipettor, remove the culture (and place
into a container where it can be autoclaved for safe disposal).
Bioassays of Quorum and Biofilm Dispersion Signals 19