Evaluation of antibacterial activity
The bacteria used in this study were Escherichia coli ATCC 8739.
A loop full of bacteria stored at 80 8C was directly inoculated
overnight in a 3 ml Nutrient Broth (NB, Fluka) medium at
37 8C. The antibacterial activity of chitosan and PEGylated chitosan
(1 mg/ml media) was evaluated in a 24-well plate containing
overnight broth cultures diluted to 1:100 NB and adjusted to a
concentration of 105 CFU/ml (1 ml). The plates were incubated
at 37 8C under agitation (100 rpm) and after 24 h samples were
serially diluted in NB 1:100 (performed in 96-well plates). 20 ml
drops were incorporated into NB bacto-agar (Becton Dickinson)
plates to determine cell numbers. Plates were incubated at 37 8C
for 18–24 h; CFU were counted and log reduction was calculated
in comparison to control growth of E. coli in the NB 1:100 medium
(108 CFU/ml). All determinations, including the growth controls,
were duplicated. The antibacterial activity of LDPE based films
was evaluated in a petri dish using film disks (1.2 cm diameter)
consisting 4% chitosan, 3% PEGylated chitosan or pure LDPE in
broth cultures diluted to 1:300 NB contacting 105 CFU. The cell
number analysis was done in the same manner as explained
above.