Planktonic and biofilm growth and extraction
Bacterial cells (106) were added to 2 ml of LB broth
and cultured in 9x 15 ml tube (planktonic) or a 12-well plate (biofilm)
for 1-3 days. The 12-well plate cultures were grown statically in a 37°C
incubator (biofilm growth) and the 15 ml tube cultures were grown at
37°C with agitation at 250 rpm to prevent adherence (planktonic growth).
At each time point, samples were taken out and a pipette used to suction
spent media and the planktonic growth was centrifuged at max speed to
pellet cells. One ml of sterile 1x PBS was added to the well and a cell
scraper was used to gently detach biofilm cells. Detached biofilm cells
and planktonic cells were softly pelleted at 3700 rpm for 10 mins.
Biofilm components were extracted as described in Keitheley et
al. (2018) [46] with no modifications. Briefly, the pellet was
resuspended in 1.5 ml of TE buffer (10 mM Tris, 10 mM EDTA, 2.5% NaCl,
pH 8.0) and incubated at 200 rpm for 4 hours at 35 °C. The cells were
pelleted at max speed, 4°C and the resulting extractant filtered and
used for composition analysis. 10 µl of the extractant is plated to
ensure the absence of bacterial cells. A plate count was performed
before and after incubation with TE buffer to determine if cell lysis
occurred.