Antimicrobial Interfaces - Biointerfaces: Where Material Meets Biology - page 414

Protocol to measure cell-cell adhesion within a P.aeruginosa biofilm grown in a CDFF using
Atomic Force Microscopy (AFM)

Gallium, silver or control glass discs (5mm diameter)
were recessed (300 μm) in to the plugs of each of
polytetrafluoroethylene (PTFE) pans in the CDFF

Autoclaving must be avoided to stop
moist heat degrading the glasses

The CDFF was sterilised in a hot air oven (140°C, 3 h)

Upon cooling the CDFF was placed in an incubator (set
at 37°C) the turn table rotated at a speed of 3rpm

P.aeruginosa grown in TSB medium overnight was
added (5%) to inoculation medium (500mL TSB)

The inoculation flask was incubated at 37°C and the
inoculum was magnetically stirred and pumped into the
CDFF ( 0.7 ml min −1 ) for 6h

Please refer to the schematic
diagram of CDFF for details

Upon inoculation, growth medium (1%TSB) was
pumped into the CDFF (0.1 ml min −1 ) via a grow-back
trap

A grow-back trap was used to stop
any potential contamination

After 120h, biofilm grown on different glasses were
harvested (centrifuge 10000 × g for
10min), washed, suspended in 0.5 mL PBS (pH7.4) and
placed in respective containers of gallium, silver or
control glasses

For each condition, place 200 μl of the cell suspension on 1%
polyethyleneimmine (PEI; M r 1200) coated glass substrate
(1×1cm)

For each condition, place 10 μl of the cell suspension on 1%
polyethyleneimmine (PEI; M r 1200) coated silicon (Si) nanoprobe attached to
the cantilever (k sp =0.95Nm -1 )

Store all tips and glass substrates 4°C prior to AFM analyses