## 2 Materials and Methods
### In Vitro Antimicrobial Activity Assays  
Bacterial (_Bacillus_ sp., _Streptococcus_ sp.) and fungal (_Penicillium_ sp., _Aspergillus_ sp.) colonies were preliminarily grown as a liquid culture (nutrient or Sabouraud broth), incubating for 16-36 h at 30 \(\pm\) 1 \({}^{\circ}\)C. The antimicrobial activity of the EO and HA solutions was evaluated by agar diffusion disc (ADD) or well plate diffusion (WPD) in vitro methods (Figure 4). Aliquots (10-20 \(\upmu\)L) of bacterial or fungal liquid cultures were uniformly spread by a sterile Drigalsky spatula on nutrient or Sabouraud surface media, and the surface was allowed to dry. The assays were performed, in duplicate, using bacterial or fungal liquid cultures, normalized to the concentration of 1 \(\times\) 10\({}^{6}\) CFU/mL and 1 \(\times\) 10\({}^{4}\) conidia/mL, respectively. In the ADD assay, 10 \(\upmu\)L aliquots of _T. vulgare_ essential oil (50%, 25%,12.5%) solutions were dropped on a sterile paper disc (6 mm in diameter, Dutscher papier, FR) centred on a nutrient or Sabouraud agar medium (9 cm Petri dishes) surface, previously sown with microbial cultures. Incubating at 30 \(\pm\) 1 \({}^{\circ}\)C, confluent microbial growth was clearly observed except in the inhibition halo (i.h.) area, the diameter (mm) of which was related to the antimicrobial activity of the main components. In control assays, paper discs were soaked with 70% ethanol or 3% (vol/vol) benzalkonium chloride solutions. In the WPD assay, a hole 4 mm in diameter was drilled aseptically on the agar, and a 10 \(\upmu\)L aliquot of _T. vulgare_ HA solution was loaded. After incubation at 30 \(\pm\) 1 \({}^{\circ}\)C, the diameters (mm) of the growth inhibition halos were measured. The antimicrobial activity vs. the microbial colony was outlined as: a sensitive strain = i.h. \(>\) 9 mm; a resistant strain = i.h. \(<\) 6 mm. As summarized in Table 1, both EO and HA solutions were able to produce inhibition halos of at least 9 mm, showing good antimicrobial action. Reduced halos in diameter were revealed for benzalkonium-Cl (>6 mm) and for Et-OH (almost no activity). Biocide or biostatic activity was also evaluated, distinguishing minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC). MIC corresponds to any visible microbial growth at the lowest concentration after incubation at 30 + 1 \({}^{\circ}\)C, and MFC was measured by an antimicrobial-free sub-culture, defining the lowest concentration of essential oil able to kill 99.5% of the original inoculum. In 96-well microtiters, 30 microliter aliquots of 12.5%, 25%, 50%, and 100% EO solutions were mixed with an equal volume of microbial cultures and a liquid nutritive medium. Microbial growth after 18-36 h of incubation at 30 + 1 \({}^{\circ}\)C, and the absorbance at 500-600 nm, were measured; as the control, the commercial biocide benzalkonium chloride (3%, \(v/v\)) was utilized [48]. Assays were performed in duplicate. Figure 3: The Sogo Bo sculpture’s surface, whitish spots are recognizable. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Biocide** & **Microbial Taxa** & **ADD Method Inhibition Halo (mm)** \\
**Solution** & _Bacillus_ sp. & _21.5–22.0_ \\ E.O. & _Streptococcus_ sp. & _17.4–18.0_ \\
12.5\% & _Aspergillus_ sp. & _8.5–9.0_ \\  & _Penicillium_ sp. & _12.0–12.5_ \\  & & WPD method Inhibition halo (mm) \\ \end{tabular}
\end{table} 
Table 1: Antimicrobial activity of the _T. vulgaris_ and control solutions vs. microbial taxa isolated in this study. Values of inhibition halos (mm) were derived from duplicate assays. Figure 4: Antimicrobials vs. _Streptomyces_ sp. colonies and related inhibition halo assayed with the ADD method: (**a**) _T. vulgaris_ essential oil (12.5%); (**b**) benzalkonium chloride (3% \(v/v\)); (**c**) Et-OH (70% \(v/v\)). The diameter of \(>\)9 mm in (**a,b**) confirms antibacterial activity in these solutions. A very small diameter (almost not observable) is seen in (**c**), the 70% Et-OH solution.