Nano percolator

Bacterial strains in the logarithmic phase were diluted with 5 mM HEPES–KOH (pH 7.0) to a bacterial culture value of 1 × 10⁶ CFU/mL. Equal volumes of diluted bacterial culture and PMB or NCR169 peptide were mixed and incubated at room temperature for 5 min, 2 h, and 24 h. After incubation, the cells were fixed with 2.5% (v/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) and spotted on a polycarbonate membrane filter (nano-percolator; JEOL, Tokyo, Japan). The filter was washed with 0.1 M cacodylate buffer (pH 7.4) and treated for 2 h with 1% OsO₄ in 0.1 M cacodylate buffer (pH 7.4) for post-fixation. The samples were dehydrated with an ethanol gradient series (50, 70, 80, 90, and 100%) and twice with 100% ethanol for 10 min each. The sample was then incubated twice with 100% t-butanol for 15 min and freeze-dried (ES-2030; Hitachi, Ltd., Tokyo, Japan). After applying an 8 nm platinum coating, the samples were observed using a scanning electron microscope (S-4700; Hitachi).

The SEM-EDX analysis was performed using a TM3000 microscope (Hitachi high-technologies, Tokyo, Japan) operated at 15 keV. Each purified protein or crashed silk after the reaction was centrifuged at 15,000 rpm for 10 min. The supernatant was removed, and the precipitate was washed three times with pure water. The samples dispersed in water were dropped onto carbon tape or a nano-percolator (JEOL, Tokyo, Japan) and dried under atmospheric conditions before obtaining the images.