2.2. Colloidal chitin and chitosan preparation

Colloidal chitin was prepared according to the method of Murthy and Bleakley (2012) with some modifications. Briefly, 1 g chitin powder from shrimp shells (Sigma–Aldrich, St. Louis, MO) was dissolved in 15 ml concentrated hydrochloric acid (HCl) and vigorously stirred for 1 hr in a chemical fume hood at room temperature. Chitin was precipitated as a colloidal suspension by adding 250 ml distilled water. Aliquots of the chitin suspension were dispensed in 50 ml tubes and centrifuged at 4000 rpm for 10 min. Precipitates were washed 3 ×  with distilled water. The pH was adjusted to 5.5 with NaOH. The concentration of colloidal chitin was adjusted to 30 mg/ml. The preparation was then autoclaved at 121°C for 15 min and stored at 4°C until use.

Chitosan (CS) was purchased from Sigma–Aldrich (St. Louis, MO). Chitosan stock solution was prepared by dissolving in 0.2 N HCl at a concentration of 1% (w/v) with stirring overnight at 30°C. The pH of the solution was then adjusted to 5.5 with NaOH. Stock solution of 0.2N HCl (chitosan solvent) was prepared and pH was adjusted to 5.5 to determine the effect of HCl. Stock solutions were sterilized at 121°C for 15 min in an autoclave and stored at 4°C after cooling.