The microorganisms used for antimicrobial activity were Micrococcus luteus
(ATCC 4698), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853),
Klebsiella pneumoniae (ATCC 13883), Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 25923) Salmonella typhi and Enterococcus faecalis (ATCC 29212).
Antibacterial activity assays were performed according to the method described by
Berghe and Vlietinck [24]. Chitosan was dissolved at 50 mg/ml in 0.1% acetic acid (pH
4.68). The inoculum suspension (200 l) of the tested microorganisms, containing
106 colony forming units (CFU/ml) of bacteria cells were spread on Muller–Hinton
agar. The inoculums were allowed to dry for 5 min. Then, bores (3-mm depth, 6-mm
diameter) were made using a sterile borer and were loaded with 50 l of each sample. Well with only acetic acid (without chitosan) was used as a negative control (pH
3.25). Gentamycin was used as positive reference. The Petri dishes were kept, firstly,
for 1 h at 4 ◦C, and then were incubated for 24 h at 37 ◦C. Antibacterial activity was
evaluated by measuring the diameter of the growth inhibition zones in millimeters (including well diameter of 6 mm) for the test organisms and comparing to the
controls. The measurements of inhibition zones were carried out for three sample
replications, and values are the average of three replicates.