2.5.1. Culture Conditions

Gram-positive (*Staphylococcus aureus* and *Bacillus subtilis*) and Gram-negative (*Escherichia coli* and *Klebsiella Pneumoniae*) microbes were used for antibacterial assays. The standard growth medium Mueller–Hinton (MH, Difco, Sparks, MD, USA) agar was used for the growth of both bacterial types and prepared by sterilization using an autoclave (15 min, 120 ◦C). These two species of microbes were evenly spread on the solid MH agar and incubated aerobically at 37 ◦C for 24 h. The bacterial suspension was measured by using Spectrophotometer BiomerieuxDensicheck Plus at 600 nm to obtain optical density at 0.5.

#### 2.5.2. Qualitative Study

The Gram-positive and Gram-negative bacteria suspensions were evenly spread on the solid MH agar in the sterile Petri plates. Using a sterile cotton swab, both bacteria were swabbed over the surface of the agar plates and dried in a laminar flow air chamber. The hydrogel sample was gently pressed on the agar with bacteria and incubated at 37 ◦C for 24 h in triplicates. The presence of any clear zone around the disc on the MH agar was recorded as an indication of inhibition against the bacteria.
