Assessing the Antibacterial Activity of Nanostructured Surfaces Using a Focused-Contact Exposure Method

Seed a bacterial culture onto a filter paper placed on agar.

Take the test nanostructured material in a plate and add a medium to its surface.

Invert the filter paper onto the material, ensuring bacterial contact with the nanostructured surface.

Use an inert surface with a bacteria-seeded filter paper as the control.

Add a buffer to both plates to maintain humidity, and incubate.

Surface nanostructures impose stress and disrupt bacterial membranes, causing leakage of intracellular contents.

Additionally, metal ions released from the nanostructures generate reactive oxygen species and damage cellular components, reducing bacterial viability.

Transfer the filter papers to tubes containing the buffer.

Agitate vigorously and sonicate to release adhered bacteria. Collect the suspensions and perform serial dilutions.

Plate the dilutions onto agar, incubate for colony formation, and count the colonies.

Fewer colonies in the test compared to the control indicate the antibacterial activity of the nanostructured surface.

Prepare sterilized nitrocellulose papers by trimming them into 1 centimeter a diameter. Place the cut nitrocellulose paper onto an agar plate containing the appropriate medium.

Next, add 50 microliters of the diluted bacterial culture onto the filter paper before adding 50 microliters of an appropriate medium to the center of each sample surface. Using sterilized tweezers, pick up the nitrocellulose paper from the surface of the agar. Carefully flip the nitrocellulose paper and place it onto the sample surface so that the bacteria are in contact with the 50 microliters of medium and the nanostructured surface of interest.

If the sample is degradable in culture, put a holder underneath it to lift it to avoid contact with the Tris buffer. Maintain the humidity by adding 1 milliliter of Tris buffer to a sample containing well. After overnight incubation, collect the nitrocellulose paper from each sample surface and place them into 5 milliliters of Tris buffer.

Vortex the collected filter papers and nanosurface material samples for 5 seconds. Collect the Tris buffer suspensions from the sample and place them into individual fresh collection tubes.