*2.5. Scanning Electron Microscopy (SEM) Observations*

SEM was used to investigate the structural differences between biofilms before and after treatment with selected EOs and their components.

Polymicrobial biofilms were prepared in six-well microtiter plates, where sterile 2 × 2 cm cover slips served as the surfaces to which the cells attached. After 4 h of incubation, the cover slips were rinsed with sterile water, then, fresh medium containing the EOs or their components was added, and the plates were further incubated for 24 h at 37 ◦C. The concentrations of EOs and components used in this experiment were based on the results on dual-species biofilms.

Preparation of the cover slip-biofilm samples for SEM was performed as described previously [31]. Briefly, samples were immersed in 2.5% glutaraldehyde for 2 h at room temperature and then dehydrated using increasing ethanol concentrations. Final dehydration was performed with *t*-butanol–100% ethanol solution at different ratios, followed by absolute *t*-butanol. After replacing the *t*-butanol with a fresh volume of it and storing the samples at 4 ◦C for 1 h, they were freeze-dried overnight. Before SEM analysis, a gold membrane was applied, the whole field was examined, and photographs were taken from relevant areas with a Hitachi S4700 scanning electron microscope (Hitachi, Tokyo, Japan). Changes in the three-dimensional structure were mainly visualized with small scale magnification, while higher magnification was used for a more detailed view of the cell wall degrading effect of EOs.
