- Because the Las system, which mediates 3-oxo-C 12 -HSL, is
expressed before the Rhl system which produces the C 4 -HSL,
the maximal concentration of 3-oxo-C 12 -HSL in cultures is
achieved prior to the one of C 4 -HSL. Thus timing of sampling
will critically affect the concentrations of AHLs obtained. - Because the lactone ring of AHLs can spontaneously open at
high pH or the open form can close at low pH, care should be
taken to avoid these conditions if the samples are stored for a
certain period of time prior to analysis. - Keep in mind the concentration factor. At the end, the final
concentration of the internal standard in the injection vial
should be between 1 to 20 mg/l; if it is too high, the detector
signal will be saturated, and a too low concentration will result
in large variations. For instance, in the present example, the
0.2 to 0.5 mg/l HHQ-d4 added to the 10 ml culture sample
before the extraction will end up at a final concentration of 4 to
10 mg/l in the injection vial if the residue is dissolved in 500μl
acetonitrile. - If a sample is turbid, transfer to a microcentrifuge tube and
centrifuge at 12,000gfor 5–10 min. Transfer the cleared
supernatant to a new vial.
Acknowledgments
We thank E ́milie Gauthier for the help in the development of the
AHL quantification method. Work on AHLs and HAQs in the
De ́ziel lab is funded by the Canadian Institutes of Health
Research (CIHR). ED holds the Canada Research Chair in
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