## 2 Materials and Methods
### Methods
#### 2.4.4 Efficacy Test of Ozone-Induced Free Radical Solution in Controlling Typical Pathogens  
_Bacillus subtilis_ served as the experimental subject for investigating the antimicrobial efficacy and mechanisms of the ozone-induced free radical solution via suspension sterilization tests. The concentration of the _Bacillus subtilis_ suspension in the experiment ranged from 1 \(\times\) 10\({}^{5}\) to 3 \(\times\) 10\({}^{5}\) cfu/mL. For enumeration, a gradient group is selected where the colony count in the culture dish is below 300 cfu for accurate counting.  
Inhibitory Effect of ozone-induced free radical solution on _Bacillus subtilis_  
To assess the inhibitory effect of varying concentrations of the ozone-induced free radical solution, 0.5 mL of bacterial suspension was combined with 4.5 mL of the solution at different concentrations in a test tube, and the mixture was vigorously mixed for 1 min. Six serial dilutions were performed, and 0.1 mL of each dilution was inoculated onto nutrient broth agar Petri dishes. Following a 3-day incubation period, colonies were counted. Exploring the impact of different exposure times on the inhibitory effect, 0.5 mL of bacterial suspension was mixed with 4.5 mL of 0.5 mg/L ozone-induced free radical solution in a test tube, and agitated for durations of 1, 3, 5, and 10 min, respectively, with all subsequent steps mirroring those previously described.  
Analysis of the Effect of ozone-induced free radical solution on the Structure of _Bacillus subtilis_  
Structural alterations in _Bacillus subtilis_ pre- and post-treatment with ozone-induced free radical solution were observed via scanning and transmission electron microscopy, elucidating the solution's microbicidal mechanism [26; 27]. The experiment featured a control group (sterile water) and treatment groups with 0.3 mg/L and 0.5 mg/L ozone-induced free radical solution concentrations. Initially, samples were pretreated, with the bacterial suspensions exposed to ozone-induced free radical solution collected via centrifugation. Following a sterile water rinse, samples were fixed in 2.5% glutaraldehyde and refrigerated at 4 \({}^{\circ}\)C for a minimum of 2 h. The fixative was removed, and samples were rinsed three times with 0.1 M, pH 7.0 phosphate buffer, 15 min per rinse; thereafter, samples were fixed in 1% osmotic acid solution for 1-2 h. Subsequently, osmotic acid waste was discarded, and the samples underwent three additional rinses with 0.1 M, pH 7.0 phosphate buffer, each lasting 15 min. Samples underwent dehydration through sequential exposure to ethanol solutions at gradient concentrations (30%, 50%, 70%, 80%, 90%, and 95%) for 15 min, followed by two subsequent treatments with 100% ethanol, each for 20 min.  
Scanning electron microscopy (SEM) observation: The pretreated samples underwent treatment with an ethanol and isoamyl acetate mixture (1:1 volume ratio) for 30 min, followed by pure isoamyl acetate treatment for 1 h. This process was succeeded by critical point drying and coating. Images of the treated samples were captured using a scanning electron microscope at an acceleration voltage of 15 kV.  
Transmission electron microscopy (TEM) observation: The pretreated samples underwent treatment with pure acetone for 20 min. Subsequently, the sample received treatment  
Figure 4: Reaction equation of DPBF with singlet oxygen.  
with an embedding agent and acetone mixture (1:1 volume ratio) for 3 h, followed by embedding and overnight heating at 70 \({}^{\circ}\)C. Embedded samples were sectioned into 70 to 90 nm slices using an ultra-microtome. Sections were stained with lead citrate and a 50% ethanol-saturated solution of uranium acetate for 5 to 10 min, respectively, before being imaged with a transmission electron microscope at an acceleration voltage of 90 kV.