*2.6. Aqueous Decomposition Studies*

The decomposition profile of silver oxynitrate and Ag7NO11:SiO2 was evaluated in aqueous media over a course of seven days. In brief, approximately 1 g of silver oxynitrate or Ag7NO11:SiO2 was added to a series of sealed glass vials denoted 2 h, 6 h, 24 h, 72 h, 120 h, and 168 h, each containing 10 mL water. Following addition of the higher oxidation state silver compounds, the vials were sealed and vortexed for 30 s to disperse the solids in solution then stored at room temperature away from direct light. At each specified time interval, the entire contents of the vial were quantitatively transferred to a Buchner funnel with Whatman 42 ashless filter paper. The grey-black solids were washed three times with water and was subsequently rinsed three times acetone. The solids were dried under air until a steady mass was obtained. At each specified time point, powder X-ray diffraction and scanning electron microscopy, as described above, was performed on the isolated solids.

### *2.7. Planktonic Log Reduction Assay*

The planktonic antimicrobial activities of the higher oxidation state silver compounds were evaluated for their efficacy against *Staphylococcus aureus* (ATCC 6538) and *Pseudomonas aeruginosa* (ATCC 9027). Briefly, the equivalent of 10 mg silver (Ag) of the higher oxidation state silver compounds were weighed and added into a triplicate set of 15 mL sealed tubes containing 10 mL Mueller Hinton Broth (MHB) challenged with 1 × 10<sup>6</sup> CFU/mL inoculum of either *S. aureus* and *P. aeruginosa.* Testing included negative control tubes without any added silver compounds. Sterility control were also included; not containing any silver compounds and not inoculated. The test and control tubes were incubated at 37 ◦C for four (4) hours for *S. aureus* or one (1) hour for *P. aeruginosa.* After the final inoculation and at each required incubation time, contents of the reaction tubes were neutralized with 0.4 wt/wt% sodium thioglycolate solution (STS), serially diluted with 0.89 wt/wt% NaCl (Saline) and plated onto Mueller Hinton Agar (MHA). Plates were incubated for 18–24 hrs and then enumerated. Log reduction of the higher oxidation state silver compounds as compared to the control reaction tubes were calculated.

### *2.8. Biofilm Log Reduction Assay*

Biofilm of *Staphylococcus aureus* (ATCC 6538) and *Pseudomonas aeruginosa* (ATCC 9027) were grown in a three-dimensional matrix to determine the e fficacy of higher oxidation state silver compounds against bacterial biofilm. In, brief, three to five layers of sterile cotton gauze were placed in simulated wound fluid (SWF) in 6-well tissue culture plates for each strain tested. To each well, a 1 × 10<sup>6</sup> CFU/mL inoculum was added every 24 hrs for up to 72 hrs. During which time the plates were incubated at 37 ◦C with shaking at 200 rpm. After the incubation period, the gauze was removed from the liquid culture medium. The gauze biofilm was rinsed three times with sterile water to eliminate planktonic bacterium and then placed onto the surface of an MHA plate (Oxoid, Nepean, ON, Canada). The gauze biofilm was overlaid with additional MHA cooled to ca. 50 ◦C such that one-half of the biofilm was embedded in the agar and one-half was exposed. An equivalent of 10 mg silver (Ag) of the higher oxidation state silver compounds were weighted over-laid onto the biofilm and exposed to treatment at 37 ◦C for four (4) hours for *S. aureus* or two (2) hour for *P. aeruginosa*. After the exposure time, the dressings and biofilm (gauze pieces) were carefully removed from the plates and were placed into 10 mL of 0.4 wt/wt% STS. They were vortexed (3 × 1 min) to disrupt the biofilm, then serially diluted in saline (0.89 wt/wt% saline) and spot-plated onto MHA for viable cell counts. Negative controls were made as follows: (i) gauze biofilm were grown and were overlaid with agar as described above; and (ii) no higher oxidation state silver compounds were overlaid on the biofilm in the same manner as the treatment conditions. Log reduction of the higher oxidation state silver compounds versus the control biofilm were calculated.
