*2.3. Decontamination Tests*

### 2.3.1. Biological Decontamination Tests

The biological decontamination tests involved the characterization of the antimicrobial activity of the decontamination solutions, followed by the evaluation of the efficacy of the strippable nanocomposites for the removal of biological contaminants from the targeted surfaces. For the characterization of antimicrobial activity of these substances, three of the most used methods appropriate for this type of sample were applied: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and time-kill test. The MIC value is defined as the lowest concentration of the antimicrobial agent that inhibits the visible growth of the micro-organisms tested. This is usually expressed in mg/mL or mg/L. The MBC is defined as the lowest concentration of antibacterial agent needed to kill 99.9% of the final inoculum after incubation for 24 h under a standardized set of conditions. Time-kill assay is the most appropriate method for determining bactericidal effect. It is a good method for obtaining information about the interaction between the antimicrobial agent and the microbial strain. The time-kill test reveals a time-dependent or a concentration-dependent antimicrobial effect [45].

#### Minimal Inhibitory Concentration

The antimicrobial activity of the decontamination solutions was evaluated against *Staphylococcus aureus* (ATCC 6538) as a model for Gram-positive bacteria and *Escherichia coli* (ATCC 8739) and *Pseudomonas aeruginosa* (ATCC 9027) as a model for Gram-negative bacteria. *S. aureus*, *E. coli*, and *Ps. aeruginosa* were chosen, considered standard microorganisms for testing the antimicrobial properties of newly synthesized products [46]. After cultivation overnight in Muller Hinton broth (MHb) (Merck) at 37 ◦C with stirring (200 rpm), the bacterial strains were harvested. Portions of suspension were harvested by centrifugation and resuspended in phosphate buffer saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA). The suspensions were adjusted to approximately 10<sup>6</sup> CFU/mL [47]. Minimum inhibitory concentrations (MIC) were established for each solution by the broth microdilution method [48,49]. Two-fold serial dilutions of each solution were performed in MHb in duplicate. Negative and positive controls were associated [50]. The inhibitory effect of the substances was evaluated starting from 50% concentration (the samples of substances were diluted 1:1 with MHb). A total of 10 μL of the micro-organism suspensions (~104 CFU) was added in each well corresponding to the testing samples and controls. Because the solutions are turbid themselves and the bacterial growth is difficult to discern, at the end of the incubation period, 10 μL of resazurin 0.1% was added to each well. After 2 h of incubation with resazurin, the plates were read.
