- Microtiter plate reader (preferably with temperature control
and ability to agitate samples). - Multichannel pipets are very useful (200μl volume for 96-well
plates).
3 Methods
3.1 Ethyl Acetate
Extraction [35, 38]
- Grow 20 ml broth culture to stationary phase. If biofilm from
pebbles or other surfaces is desired, sonicate pebbles in sterile
H 2 O for 15 min, and remove 20 ml of the sonicated liquid to a
centrifuge tube. - Centrifuge to remove bacterial cells, at 3000gfor 10 min at
4 C. - Transfer supernatant to clean bottle.
- Extract the cell-free supernatant three times with three volumes
of ethyl acetate. - Pool the ethyl acetate fractions (top layers) and evaporate to
dryness using a rotary evaporator (with water bath set at
40 C). - Suspend residue in 1 ml ethyl acetate and transfer to a small
glass vial. Again evaporate the ethyl acetate, this time using a
Pasteur pipet attached to a vacuum source (aspirator, linked to
a water tap works fine for this) that is placed above the liquid
(careful: it’s easy to suck up liquid here). - Resuspend the residue in 100μl ethyl acetate and store at
80 C until needed.
3.2 AHL Reporter
Agar-Based Plate
Bioassays [53, 54]
3.2.1 Plate-Based Assay
(Works well if both bioassayC. violaceumorA. tumefaciensand test
organism grow on same medium)- If usingA. tumefaciens-based bioassay, pipet 50μl X-gal solu-
tion onto agar and spread across surface with alcohol-sterilized
spreading rod, prior to inoculating with bacteria. - Streak test organism and bioassay organism beside each other
on LB agar plate using a “T” shape. The cross of the “T” would
represent strain CVO26 and the vertical line would represent
the test organism (Fig.1). Following 16–48-h incubation at
30 C, you should see a positive (blue coloration (due to X-gal
hydrolysis) in the case of A. tumefaciens A136 (pCF218)
(pCF372) or KYC55 (pJZ372)(pJZ384)(pJZ410) bioassay,
and purple violacein in the case of C. violaceumbioassay)
production in the bioassay in the region closest to the intersec-
tion of the two strains and a lack of pigmentation away from
this intersection. An alternative streaking design is shown in
Fig.1, which is intended to give a concentration gradient of
Bioassays of Quorum and Biofilm Dispersion Signals 15