- Add 200μl of candidate dispersant solution (a buffer such as
PBS can be used as a negative control). - Place in plate reader (if the plate reader has an agitation capa-
bility), then allow that to happen for a short period (1 min or
so), and then measure OD 600. If the plate reader does not have
agitation capability, then expose the plate to some type of
gentle vibration for a few minutes before measuring OD 600. - Release of biofilm bacteria into the planktonic population will
be indicated by an increased OD reading in the respective wells.
This increase should be evident within 1–5-min exposure.
Increased OD 600 readings at longer times may reflect the
growth of planktonic cultures in the liquid.
4 Notes
- For long-term storage ofC. violaceumstrains, grow an over-
night culture in broth with shaking at 30C, then mix 1.2 ml of
the overnight strain with 0.4 ml sterile 50% (v/v) glycerol,
place into a 2-ml sterile cryogenic storage tube, and store at
80 C. To revive the frozen culture, simply scrape some cells
from the top of the frozen culture onto LB and incubate
overnight at 30C. For short-term (2–3 days) storage, leave
at room temperature (20–25 C), since Chromobacterium
strains do not survive very well at 4C. - We routinely use Si-C18F TLC Plate Reversed Phase Octade-
cyl, JT Baker (Cat no. 7013-04). - Some AHLs can be purchased commercially through compa-
nies such as Sigma-Aldrich (St. Louis, MO). - We routinely use a TLC glass tank Z204161 from Sigma-
Aldrich (17.5 cm16.0 cm6.2 cm). We have found the
C. violaceumCVO26 bioassay to respond to low micromole to
high nanomole concentrations to its cognate AHL (C6-HSL).
This strain is best suited to detecting short-chain AHLs (C4-
and C6-HSLs). In contrast, another AHL bioassay strain,A.
tumefaciens A136 (pCF218)(pCF372) developed by Fuqua
and Winans [29], is much more sensitive that CVO26, in that
it can detect its cognate AHL (3-o-C8 HSL) in the low-
picomole range. A. tumefaciensNTL4 (pCF218)(pCF372)
has a similar sensitivity as strain A136 (pCF218)(pCF372)
[36, 37]. These A. tumefaciensstrains will detect a wider
variety of AHLs with acyl groups ranging in size from 6 carbons
to 12 carbons [35]. Zhu and Winans developed an even more
sensitiveA. tumefaciensstrain, designated KYC55 (PJZ372)
(PJZ384)(PJZ410) [30] capable of detecting long-chain
AHLs (C6- to C14-HSLs). BothA. tumefaciensbiosensors
20 Starla G. Thornhill and Robert J.C. McLean