Page 441 - Polymer-based Nanocomposites for Energy and Environmental Applications

398 Polymer-based Nanocomposites for Energy and Environmental Applications

chitosan-g-polyacrylonitrile/silver nanocomposite, prepared by in situ chemical
reduction, showing antimicrobial action against E. coli and S. aureus [8]. However,
there are a few reports indicating the excellent antifouling properties of pressure-
retarded nanocomposite membranes used in the osmosis process [9]. A few papers
have reported that tryptophan-integrated wound-dressing material inhibits biofilm
formation by the chronic wound pathogen, P. aeruginosa [10]. A simple casting
and swelling method was used to prepare polyurethane membranes incorporated with
silver and gold nanoparticles. The Ag nanoparticle-coated implant material proved to
be a better antibiofilm agent than the Au-coated material [11]. An electrospinning
method was used to prepare a wound-dressing material incorporating copper
nanofibers with poly-D,Llactide (PDLLA) and poly(ethylene oxide) (PEO). The cop-
per released from the nanofibers reduced biofilm formation by P. aeruginosa PA01
and S. aureus [12]. In our work, silver nanoparticles were synthesized from a bacterial
source and used in preparing cellulose composite films, which were further employed
in antifouling studies with E. coli.

14.2 Materials and methods

14.2.1 Qualitative determination of biofilm

14.2.1.1 The tube method
Biofilm formation was qualitatively determined by the test tube method, initially.
A sterile tube containing 10 mL of trypticase soy broth (TSB) with 1% glucose was
inoculated with an overnight-grown culture of E. coli. This tube, along with a con-
trol of uninoculated medium, was incubated at 37 °C for 24 h. After the incubation,
the culture was discarded and the tube washed with phosphate buffered saline
(PBS, at pH 7.3) to remove unadhered cells. The test tube was stained with
100 μL of 0.1% crystal violet solution, further washed with deionized water,
and dried [13].

14.2.2 Liquid interface cover slip assay

In this assay, biofilm formation by the bacterial culture was analyzed using a liquid
interface coverslip. A sterile, 24-well microtiter plate was filled with 10 mL of TSB
containing 1% glucose and inoculated with 2000 μL of an overnight-grown culture of
E. coli in the stationary phase. Sterile cover slips were placed at the bottom of the wells
such that these were in contact with the medium throughout an incubation period of
48 h at 37°C. After the incubation, each well was washed with PBS to dislodge
unadhered cells from the cover slip; the dried cover slips were stained with 100 μL
of 0.1% crystal violet and left undisturbed for 15 min, after which they were rinsed
with water repeatedly to wash off any excess dye and then allowed to dry in air. Bio-
film formation on each cover slip was visualized under the microscope [14].