8) Antibacterial Test

The antimicrobial activity of the iPP/ZnOc composites was evaluated using *E. coli* DSM 498T (DSMZ, Braunschweig, Germany) as test microorganisms. The evaluation was performed using the ASTM Standard Test Method E 2149-10 [34]. The preparation of the bacterial inoculum required to grow a fresh 18-h shake culture of *E. coli* DSM 498T in a sterile nutrient broth (LB composition for 1 L: 10 g of triptone, 5 g of yeast extract and 10 g of sodium chloride) The colonies were maintained according to good microbiological practice and examined for purity by creating a streak plate. The bacterial inoculum was diluted using a sterile buffer solution (composition for one litre: 0.150 g of potassium chloride, 2.25 g of sodium chloride, 0.05 g of sodium bicarbonate, 0.12 g of CaCl2¨6H2O and pH = 7) until the solution reached an absorbance of 0.3 ˘ 0.01 at 600 nm, as measured spectrophotometrically. This solution, which had a concentration of 1.5 ˆ 108–3.0 ˆ <sup>10</sup><sup>8</sup> colony forming units/mL (CFUs/mL), was diluted with the buffer solution to obtain a final concentration of 1.5 ˆ 106–3 ˆ 106 CFUs/mL, that was the working bacterial dilution.

The experiments were performed in 50 mL sterilized flasks. One gram of the film was maintained in contact with 10 mL of the working bacterial dilution. After 2 min, 100 mL of the working bacterial dilution was transferred to a test tube, which was followed by serial dilution and plating out on Petri dishes (10 mm ˆ 90 mm) in which the culture media was previously poured. The Petri dishes were incubated at 35 ˝C for 24 h. These dishes represented the *T*<sup>0</sup> contact time. The flasks were then placed on a wrist-action shaker for 1 h, 24 h, 48 h, 5 days and 10 days. The bacterial concentration in the solutions at these time points was evaluated by again performing serial dilutions and standard plate counting techniques. Three experiments were performed for each composition. The number of colonies in the Petri dish after incubation was converted into the number of colonies that form a unit per millilitre (CFUs/m) of buffer solution in the flask. The percentage reduction (*R*%) was calculated using the following formula:

$$R\% \text{ (CFU/mL)} = \left[ (B - A) / B \right] \times 100,\tag{1}$$

where *A* = CFUs/mL for the flask containing the sample after the specific contact time and *B* = CFUs/mL at *T*0.

#### **3. Results and Discussion**
