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

Abstract

Source: Holt-Torres, P. S. et al. Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro. J. Vis. Exp. (2023)

This video demonstrates the use of the focused-contact exposure method to evaluate the antibacterial activity of nanostructured surfaces. It outlines the steps involved in establishing direct contact between bacteria and the nanostructured surface, incubation, bacterial collection, and colony counting to assess viability.

Protocol

1. Focused-contact exposure method (method D)
NOTE: In method D, bacteria on a nitrocellulose filter paper are put in direct contact with an area of interest on the nanostructured surfaces. This method minimizes the interference of bulk sample degradation in bacterial cultures with the bacterial activities.

1. Create a bacterial seeding culture and confirm the actual seeding density.
2. Prepare the samples to a size of 1 × 1 cm² in a square shape. Prepare all the sample types in triplicate. If necessary, place the samples on a three-dimensional (3D) holder that is 15 mm in diameter by 10 mm in height. Place the sample and holder into a well of a non-tissue culture-treated polystyrene well plate.
3. Prepare sterilized nitrocellulose papers by trimming each to a diameter of 1 cm. Place the prepared nitrocellulose papers onto an agar plate containing the appropriate medium.
4. Pipette 50 µL of the diluted bacterial culture onto the filter paper.
5. Pipette 50 µL of an appropriate medium onto the center of each sample surface.
6. 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 µL of medium and the nanostructured surface of interest.
7. Add 1 mL of Tris buffer to each well containing a sample to maintain the humidity. Incubate the polystyrene well plate containing all the samples at 37 °C for 24 h.
8. Collect the nitrocellulose paper from each sample surface. Place each into 5 mL of Tris buffer. Vortex the collected filter papers and nanosurfaced material samples for 5 s.
9. Sonicate each sample for 10 min. Vortex for 5 s after 5 min and again after 10 min.
10. Collect the Tris buffer suspensions from all the samples and place each volume into individual fresh collection tubes.
11. Serially dilute and plate the samples.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tris(hydroxymethyl) aminomethane buffer pH 8.5; Tris buffer	Sigma-Aldrich	42457
Falcon 15 mL conical tubes	Fisher Scientific	14-959-49B
Luria Bertani Broth	Sigma Life Science	L3022
Luria Bertani Broth + agar	Sigma Life Science	L2897
MacroTube 5.0	Benchmark Scientific	C1005-T5-ST
Nitrocellulose paper	Fisherbrand	09-801A
Petri dish 100 mm	VWR	470210-568
Petri dish, 15 mm	Fisherbrand	FB0875713A
Sonicator	VWR	97043-936
VWR Benchmark Incu-shaker 10L	VWR	N/A