- The whole-cell cultures are used for the monitoring study.
- Round-bottom flasks (25 ml).
- Acidified ethyl acetate: [0.01 % (v/v) acetic acid in ethyl ace-
tate] is utilized for the liquid-liquid extraction of PYO, HHQ,
and PQS from bacterial cultures. Add 10μl of acetic acid
(99.7 %) to 100 ml of ethyl acetate (seeNote 9).
2.2.2 Solid-Phase
Extraction (SPE)
- Solid-phase extraction (SPE): Using a mixed-mode strong
cation-exchange (MCX) SPE cartridge (30 mg) (see Note
10 ). All SPE procedures are performed manually using a 5 ml
plastic syringe (seeNote 11) in a fume hood. - The formate buffer: 0.5 M formate buffer (HCOOH) at
pH 2.0 is used for equilibration and washing buffer for MCX
SPE. Prepare a stock solution of 1 M formate buffer. Add about
25 ml DW to a 100 ml volumetric flask, then slowly add
3.850 ml of concentrated formic acid (98 %) (seeNote 12),
and adjust the final volume to 100 ml with DW. Then, dilute
50 ml of 1 M formate buffer to 100 ml with DW to obtain a
0.5 M HCOOH buffer, pH 2.0 (seeNote 6). - Ammonium formate: 5 % of 4.5 M ammonium formate
(NH 4 HCO 2 ) in methanol (95 % CH 3 OH: 5 % DW, v/v) is
used as an elution solvent for MCX SPE. Prepare a stock solution
of 10 M ammonium formate in a 10 ml volumetric flask. Weigh
6.306 g of ammonium formate, then add 6 ml of DW, sonicate
until the ammonium formate is dissolved, and adjust the final
volume to 10 ml with DW. Subsequently, 4.5 M ammonium
formate is obtained by the dilution of 0.45 ml of 10 M
ammonium formate to 1 ml with DW. Prepare 95 %
CH 3 OH:5 % DW, v/v. Add 4.75 ml of 100 % CH 3 OH to
0.25 ml DW. Lastly, add 0.25 ml of 4.5 M ammonium
formate to 4.75 ml of 95 % CH 3 OH:5 % DW to obtain 5 % of
4.5 M ammonium formate in methanol (95 % CH 3 OH:5 %
DW, v/v).
2.3 Direct Detection
of PYO, HHQ, and PQS
in Bacterial Cultures
Based on In Situ
Cationic Surfactant-
Assisted Membrane
Disruption
- Phosphate buffer: 50 mM phosphate buffer, pH 7.0 is used to
dilute the CTAB stock solution (step 2). Prepare a stock solu-
tion of 0.1 M of sodium phosphate dibasic (Na 2 HPO 4 ). Weigh
1.419 g sodium phosphate dibasic and transfer to a 100 ml
volumetric flask. Add DW to a volume of 90 ml, sonicate until
sodium phosphate dibasic is dissolved, and then adjust the final
volume to 100 ml with DW. Also, prepare a stock solution of
0.1 M of sodium phosphate monobasic (NaH 2 PO 4 ). Weigh
1.560 g sodium phosphate monobasic and transfer to a 100 ml
volumetric flask. Add DW to a volume of 90 ml, sonicate until
sodium phosphate monobasic is dissolved, and then adjust the
final volume to 100 ml with DW. 50 mM phosphate buffer,
Rapid Detection ofP. aeruginosaSignal 111