# Antimicrobial Air Filter Coating with Plant Extracts Against Airborne Microbes
## 2 Materials and Methods
### Preparation of Plant Extract-Cated Filters and the Antimicrobial Test  
Before usage, the PET was sterilized for 15 min at 121 \({}^{\circ}\)C. The coating solution was prepared by mixing 6 mL of TMOS and plant extract solutions with 0.8 mL of 0.2 M HCl. Before mixing to TMOS, each plant extract solution was prepared by diluting in distilled water. After adding the plant extract solution, the mixture was sonicated for 10 min and aged in a refrigerator for 12 h. One milliliter of plant extract coating solution was dispersed onto the surface of the air filter material using an atomizer (TAMIYA, Inc., Shizuoka, Japan). The coating process was carried out on a clean bench, and after coating, the filter material was dried at room temperature for 12 h. Negative control samples of non-coated PET were prepared by using PET materials as received without surface modification.  
The antimicrobial activities of the air filters were measured following the SPS-KACA009-139 protocol developed by the Korea Air Cleaning Association [38]. One colony of _M. luteus_ was removed from the agar plate and cultured in 5 mL enriched nutrient broth in a shaking incubator (180 rpm) at 37 \({}^{\circ}\)C for 24 h. The cell culture so obtained was injected into three flasks each containing 50 mL enriched nutrient broth and cultured until the exponential growth phase was reached. Then, 40 mL of cell solution was centrifuged at 1610 g for 10 min. After centrifugation, the supernatant was discarded, and the cell pellet was re-suspended in 10 mL of fresh enriched nutrient broth. Three re-suspended cell solutions were collected and injected into a bottle of humidifier containing 270 mL of distilled water. The air filter antimicrobial test was performed in a chamber (Figure 1) sterilized by 30 min of ultraviolet (UV) irradiation at 365 nm. The humidifier containing the cell solution was operated for 2 min to spray the bacterial aerosols. The fan located at the back of the air filter pushed the bacterial aerosols through the air filter. After bacterial aerosols exposure, the air filter was separated from the test apparatus andthen incubated at 37\({}^{\circ}\) for 48 h to investigate long-term survival on the air filter surface. To detach the microorganisms, the air filter was immersed in 30 mL of distilled water and vortexed for 2 min. In this work, we considered that bacteria were detached from the air filters with the same efficiency in each experiment. The cell solution was streaked on nutrient agar plates and incubated at 37 \({}^{\circ}\)C for 48 h. The resulting colonies were counted and used to calculate the inactivation rate, using Equation (1):  
\[\text{Inactivation rate (\%)}=\left((\text{CFU}_{\text{control}}-\text{CFU}_{ \text{experiment}})/(\text{CFU}_{\text{control}})\right)\times 100, \tag{1}\]  
where \(\text{CFU}_{\text{experiment}}\) and \(\text{CFU}_{\text{control}}\) are the number of bacteria retrieved from the antimicrobial and pristine (control) filters, respectively. CFU means colony-forming unit.  
The anti-pathogen activities of the plant extract-coated air filters were investigated following the Korean standard testing method, KS K 0693:2016 [39], using _Staphylococcus aureus_ and _Klebsiella pneumonia_. In this test, the cell exposing manner was different. Cell solution was poured on air filter placed in glass bottle. After incubating for 48 h, distilled water was added to the bottle and used it to count cell number. All experiments were conducted in triplicate.