Scanning electron microscopy was used to observe biofilms, as previously described (Kim et al., 2020 (link)). Briefly, precut pieces of a nylon membrane 0.5 × 0.5 cm were placed in 96-well plates containing C. albicans grown in PDB medium with or without α-methyl, trans-4-methyl, or trans-cinnamaldehydes (50 μg/mL) and incubated for 24 h at 37°C. Cells that adhered to nylon membranes for 24 h were fixed with a glutaraldehyde (2.5%) and formaldehyde (2%) solution, postfixed using osmium tetroxide, and dehydrated using an ethanol series (50, 70, 80, 90, 95, and 100%) and isoamyl acetate. After critical-point drying, cells were sputter-coated with palladium/gold and imaged using an S-4200 scanning electron microscope (Hitachi, Tokyo, Japan) at 15 kV.
To examine C. elegans cuticles, scanning electron microscopy was performed using an S-4800 instrument (Hitachi, Tokyo, Japan), as described previously (Ropiak et al., 2016 (link)). To investigate the effects of the highly potent anthelmintic agents, nematodes were treated with 4-bromo or 4-chloro cinnamaldehydes at 20 μg/mL for 48 h, and then 10 nematodes per treatment were processed for SEM imaging, as previously described (Ropiak et al., 2016 (link)). trans-cinnamaldehyde was used as the control.
To examine C. elegans cuticles, scanning electron microscopy was performed using an S-4800 instrument (Hitachi, Tokyo, Japan), as described previously (Ropiak et al., 2016 (link)). To investigate the effects of the highly potent anthelmintic agents, nematodes were treated with 4-bromo or 4-chloro cinnamaldehydes at 20 μg/mL for 48 h, and then 10 nematodes per treatment were processed for SEM imaging, as previously described (Ropiak et al., 2016 (link)). trans-cinnamaldehyde was used as the control.