Analysis v 3

The interactions between tested natural substances and planktonic cells were visualized by TEM. The volume 0.75 mL of inoculum (A_625nm 0.08 to 0.1) was added into a 2 mL centrifuge tube and mixed with 0.75 mL of a natural compound of the selected concentration (the effective concentration that resulted in bacterial leakage and the concentration that guaranteed an option to observe the interaction of the tested substance with both Gram-negative and Gram-positive bacteria), or with 0.75 mL of the sterile medium (control). After cultivation (37 °C, 8 h) in a shaking incubator, a drop of a bacterial culture suspension was deposited on a copper carbon-coated electron microscopic grid and incubated at room temperature for about 10 min. After that, the excess liquid was removed by filter paper, and the grid was quickly rinsed with distilled water. Then the grid was deposited onto a solution of 1% sodium silicotungstate pH 7.4 and negatively stained for about 10 s. After the staining, the grid was left to dry and was subsequently inserted into TEM column JEOL JEM-1010 (JEOL Ltd., Tokyo, Japan), operated at 80 kV at various magnifications. Images were recorded by SIS Megaview III CCD camera and analyzed by AnalySIS v. 3.2 software (Olympus Soft Imaging Systems, Münster, Germany).

The interactions between tested metallic NPs and planktonic cells were visualized by TEM. The volume of 0.75 mL of inoculum (10⁷ or 10⁸ CFU/mL) was added into a 2 mL centrifuge tube and mixed with 0.75 mL metallic NPs of selected concentration or 0.75 mL of sterile medium (control). After cultivation (37 °C for 4, 8 and 24 h) in a shaking incubator, a drop of a bacterial culture suspension was deposited on a copper carbon-coated electron microscopic grid and incubated at room temperature for about 10 min. After that, the excess of liquid was removed by filter paper and the grid was quickly rinsed with distilled water. The grid was then deposited into a solution of 1% sodium silicotungstate (pH 7.4) and negatively stained for about 10 sec. After the staining, the grid was left to dry and subsequently inserted into the TEM column JEOL JEM-1010 (JEOL Ltd., Tokyo, Japan) operated at 80 kV at various magnifications. The micrographs were recorded by SIS Megaview III CCD camera and analyzed using AnalySIS v3.2 software (Olympus Soft Imaging Systems, Münster, Germany).