pH 7.0 can be obtained by diluting 0.1 M sodium phosphate
dibasic and 0.1 M sodium phosphate monobasic. Add 4 ml of
0.1 M sodium phosphate dibasic to 5 ml of 0.1 M sodium
phosphate monobasic and adjust the final volume to 50 ml with
DW.- CTAB solution: 1 mM CTAB is used for the bacterial mem-
brane disruption. Prepare a stock solution of 25 mM CTAB.
Weight 0.911 g CTAB and transfer to a 200 ml glass bottle.
Add DW to around 80 ml and sonicate for 15–25 min until
CTAB is dissolved. Then, adjust the final volume to 100 ml
with DW (seeNote 13). 1 mM CTAB is obtained by diluting
25 mM CTAB in 50 mM phosphate buffer, pH 7.0. Dilute
40 μl of 25 mM CTAB to 1 ml with 50 mM phosphate buffer at
pH 7.0.
3.P. aeruginosaPA14 bacterial cultures: PA14 cultures are grown
overnight in LB and then diluted into 20 ml of fresh LB as
described in Subheading2.2.1,step 1. Diluted PA14 cultures
are grown at 37C for 7 h with shaking at 200 rpm (total
volume 20 ml) [17].
3 Methods
3.1 Monitoring the
Production of PYO,
HHQ, and PQS in PA14
Cultures
- 1 ml sample aliquot of the bacterial culture before incubating at
37 C is extracted with 1 ml of acidified ethyl acetate (1:1,
whole-cell culture:acidified ethyl acetate, v/v) (seeNote 14).
Then, separate and collect the top layer of the ethyl acetate into
a 25 ml round-bottom flask. - Add another 1 ml of the acidified ethyl acetate to the 1 ml of
the bacterial culture and follow the same procedure as men-
tioned instep 1. - Evaporate the ethyl acetate using a rotary evaporator, reconsti-
tute the residue by dissolution in 1 ml of 0.5 M formate buffer,
pH 2.0, and sonicate for 5 min. - Wash the SPE cartridge with 1 ml of 100 % methanol followed
by conditioning with 1 ml of 0.5 M formate buffer, pH 2.0. - Load 1 ml of the reconstituted sample (step 3) to the precon-
ditioned SPE cartridge, applying gentle pressure (seeNote 11)
to ensure the maximum retention of the analytes on the sta-
tionary phase. - Wash the cartridge with 0.2 ml of a 0.5 M formate buffer,
pH 2.0, to assist the retention of the analytes and then wash
with 0.2 ml of 100 % methanol to remove any neutral
contaminants.
112 Alyah Buzid et al.