To begin, prepare the graphene oxide/copper nanocomposites. On day one, inoculate one colony of the bacteria from the agar plate into 10 milliliters of broth using a loop. Incubate the broth at 37 degrees Celsius using a shaking incubator set at 200 RPM for 24 hours.
On day two, dilute the nanocomposite mixture using DPBS to obtain three different concentrations and prepare the control solutions for the experiment. Then dispense 100 microliters of the nanocomposite suspension and control solutions into 96-well plates. Based on the CFUs, dilute the bacterial suspension to one times 10 to the power of six CFU per milliliter in Tryptic Soy Broth.
Inoculate 100 microliters of the diluted bacterial suspension into the sample wells in the 96-well plate and incubate the plate at 37 degrees Celsius in a shaking incubator set at 200 RPM for 24 hours. On day three, vigorously mix the sample and bacterial suspensions with a 200 microliter micropipette before serially diluting the sample bacteria mixture tenfold with sterile distilled water. Then spread 100 microliters of the diluted bacterial suspension on an agar plate with a spreader and incubate the plate at 37 degrees Celsius for 24 hours.
On day four, count the bacterial colonies and determine the CFU values to confirm the antibacterial activity of the nanocomposites using the equation. The nanocomposites demonstrated significant antibacterial efficacy against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, eradicating 99.8%and 84.7%of those bacteria respectively at 500 micrograms per milliliter concentration.
