2.13. Minimum Inhibitory Concentration (MIC) Determination

The bacterial strains were obtained from CECTA-USACH collection. To obtain the bacterial stock cultures, a single colony from each bacterial strain were isolated and growth at 35 °C ± 2 (18 h, 120 rpm) in an appropriated broth, Brain Heart Infusion broth (BHI, Merck, Germany) to E. coli (ATCC 25922), S. aureus (ATCC 25923) and E. faecalis (ATCC 29212); Buffered Peptone Water (Merck, Darmstadt, Germany) to S. typhimurium (ATCC 14028); Tryptone Soya Yeast Extract Broth (Merck, Darmstadt, Germany) to L. monocytogenes (ATCC 13932); CASO broth (Merck, Darmstadt, Germany) to P. aeruginosa (ATCC 15442). To the MIC determination a subculture of each bacterial strains was obtained by inoculum from stock culture in Muller Hinton broth (Merck, Darmstadt, Germany) pH 5.5 at 35 °C ± 2 (18 h, 120 rpm). To the MIC determination the Chitosan-CGNA and commercial chitosan were dissolved in 0.1 M acetic acid solution.

For MIC determination, the micro-dilution broth method was used [16,17]. Overnight subculture in MHB (pH 5.5) of each bacterium were adjusted to 10⁷ CFU/mL, and two-fold serial dilutions of Chitosan-CGNA and commercial chitosan were prepared in sterile MHB (pH 5.5). In a 96 well microplate, 20 µL of each bacteria subculture was aggregated to 120 µL of different concentration of Chitosan-CGNA or commercial chitosan and incubated for 20 h at 35 °C with 15 min of shaking at 45 min intervals. The MIC was determined following the Optic Density at 600 nm (OD₆₀₀) to each bacterium in a Microplate Reader Synergy^TM HTX (Figures S1 and S2). In all the assays was included a culture in MHB (pH 5.5) of the respective bacteria without chitosan as negative control, and dilutions of chitosan in sterile MHB (pH 5.5) without bacteria as blank. All experiments were done in triplicate. The MIC endpoint was the lowest concentration of chitosan where no OD₆₀₀ increase was detected after 20 h of incubation.