Detection and Inhibition of Bacterial Cell–Cell Communication - Bacterial Pathogenesis: Methods and Protocols - page 62

Control
25 ug/ml
50 ug/ml
100 ug/ml

0.5

0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5 6 7 8 9

Time (Hours)

Fig. 4. Inhibition of the autoinducer 2 (AI-2)-mediated production of hemolysin in
Vibrio vulnificus . Hemolysin production was monitored using sheep red blood cells in
the absence, control, or presence of an AI-2 inhibitor at three concentrations. Samples
were taken at different times during the growth phase. The inhibitor was used at
non-growth inhibitory concentrations.

4. Notes
1. ABt medium can be stored at room temperature, but should not be more than 1
month old when using.
2. It is important to use high-quality solvents or to distill them because many
commercial grade solvents contain plasticizers that are concentrated during the
extraction procedure. These concentrated compounds can be toxic to the indicator
strains or can interfere with the interpretation of results.
3. It is preferable to use a solvent that is miscible in aqueous media, such as ethanol
or DMSO for the bioassay testing. However, if the extract does not dissolve well
in ethanol, it is possible to evaporate off the ethanol and resuspend the sample
in an organic solvent and then dilute into ethanol or DMSO prior to adding to
the bioassay medium. DMSO is not a volatile solvent and hence if the sample
does not dissolve in DMSO, it is not easy to remove the DMSO to try another
solvent.
4. Some materials, such as plants, can lyse in organic solvents. This is only an
issue if the experimental design intends to separate surface active or presented
compounds from intracellular compounds. If the former is the case, then different
organic solvents and extraction times should be tested to identify conditions that
do not lyse the tissues.
5. It is best to grow the organisms in defined minimal medium because some
components of complex media interfere with the AHL assays and can give
false-positive results (24) . Typically, 100 mL of culture supernatant is sufficient
for detection of signals or antagonists.
6. At this point, the maximum concentration has been halved due to the addition
of the cells.