In vitro antibacterial activity assay

Three bacterial strains used in this study were as follows: Staphylococcus aureus (ATCC 25923), which is a gram-positive bacterium; and Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853), which are gram-negative bacteria. The strains were cultivated at 37°C in Luria Bertani (LB) medium or LB agar medium. An isolated colony was picked and inoculated in normal saline for preparing bacterial suspensions of 0.5 McFarland standard. Then, the bacterial suspensions with the concentration of 10⁸ colony-forming unit (CFU)/mL were obtained.

MIC and MBC were measured using the doubling dilution method in line with the guidelines of the Clinical and Laboratory Standards Institute to compare the antimicrobial activity of AgSD coarse powders with that of AgSD nanocrystals. First, 100 µL of LB medium was put into each well of the sterile 96-well microtiter plates. Both AgSD/bulk and AgSD/NS were diluted to 512 µg/mL using LB medium. Diluted solutions of AgSD/bulk and AgSD/NS were added separately to the setting well, which was followed by serial twofold dilutions. Then, 100 µL of prepared bacterial dispersions with concentration of 10⁵ CFU/mL was pipetted into each well, except for the sterility control wells. Finally, the MIC value was evaluated visually by comparing the culture turbidity.

Next, 5 µL of culture medium from the dilutions that showed no visible bacterial growth was picked up to evaluate the MBC of the tested AgSD formulations. The selected incubation medium and two more concentrated dilutions were painted on sterile LB agar medium and incubated for 24 hours at 37°C. After that, the bacterial colonies were counted. The MBC was determined as the lowest concentration at which fewer than five colonies were detected on LB nutrient agar after incubation.

The inhibition zone of AgSD/bulk, AgSD/NS, and AgSD/NS-loaded hydrogels against S. aureus, E. coli, and P. aeruginosa was determined for comparing their bactericidal activities. Then, 100 µL of the bacterial suspension (10⁸ CFU/mL) was spread on LB nutrient agar to prepare a confluent ground for bacterial growth. The wells with a diameter of 5 mm were acquired in the agar plates, and 50 µL of different samples were added into these pores. The penicillin and streptomycin solutions were treated as the positive control, and blank hydrogels served as the negative control. The plates were incubated overnight at 37°C, and the diameters of the inhibition zone (mm) were surveyed using a vernier caliper after deducting the original diameter of the well (5 mm) from the total inhibition zone diameter.