PVA–HES hydrogel membranes were prepared by freezing–thawing (F–T) cycle according to the reported procedure of (Peppas and Stauffer, 1991). Briefly, aqueous solution containing 10% (w/v) PVA and 1.5% (w/v) HES and 20 mg of ampicillin sodium salt were carefully dissolved in deionized water. Different proportions of PVA and HES contents (0%, 25%, 33%, 50%, 65%, and 75%) solutions were mixed, sonicated, and vortexed for 3 h. Proper amounts of this mixture were poured in standard disposable polypropylene Petri dishes (inside dimension is 84 mm diameter × 12 mm H), followed by freezing at −20 °C for 18 h and thawing for 6 h at 25 °C for three continuous cycles, to provide mechanically acceptable hydrogel properties for further experiments. The obtained PVA–HES hydrogel membranes were frozen in liquid nitrogen for 10 min before being lyophilized fractures for SEM investigations. All samples were left in deionized water for 72 h to extract leachable sol fraction or unconnected HES from polymer matrix for further characterizations.