The protocols provide a method to examine the antimicrobial activities of nanoparticle and nanostructured surfaces from preliminary stage investigations to more complex surface examinations. Unlike previously described methods that resulted in incomparable data across studies, these methods produce consistent and comparable results across different nanoparticle materials, nanostructured materials, and microbial species. Maintaining a homogenous distribution of nanoparticles in methods A and B can be difficult.
Vortexing samples thoroughly will ensure nanoparticle suspension, and pipetting three to four times between aliquots will maintain suspensions Begin collecting cultured bacteria by aliquoting one milliliter of the overnight grown bacterial culture into 1.5 milliliter micro centrifuge tubes. After creating a sufficient number of aliquots, centrifuge them to reach the desired seating density. Collect the supernatant into a collection receptacle.
And re-suspend the cell pellet in 0.5 milliliters of fresh growth medium. Combine the suspension from two tubes, and repeat the centrifugalization. After centrifugalization, give a second wash with fresh growth medium and repeat the combining of the content from two tubes into one before centrifuging the tubes.
Give the third wash with 0.33 milliliters of tris buffer or hydroxyethyl aminomethane buffer. Combine the three suspensions, each of 0.33 milliliter volume, into one 1.5 milliliter micro centrifuge. After centrifugalization, remove the supernatant from the pelleted cells, and re-suspend the cell pellet in one milliliter of fresh tris buffer which will be known as the cell suspension or a lag phase bacterial seeding culture.
To form the bacteria and nanoparticle cultures, aliquot the two milliliters of the bacterial seeding culture in each well of a 12 well non-tissue treated polystyrene plate. For each five milliliter micro centrifuge tube containing pre-weighed magnesium oxide particles, add three milliliters of the bacterial seeding culture. And vortex the tube briefly to mix the magnesium oxide nanoparticles with the bacteria.
After mixing, aliquot one milliliter of the magnesium oxide nanoparticles suspension to three separate wells to create the triplicate samples of each premeasured weight of magnesium oxide nanoparticles. Incubate the sample overnight at 37 degrees Celsius and 120 rpm. And transfer a three milliliter sample into an individual 15 milliliter conical tube using a pipette.
Then perform one to 10 serial dilution in a 96 well plate by adding 180 microliters of tris buffer to each well in row B to row G for the appropriate number of columns. Briefly vortex a 15 milliliter conical tube containing bacterial samples, and add 50 microliters of bacterial sample to an individual well in row A.Next, from row A, transfer 20 microliters of bacterial sample into the corresponding well of row B with brief mixing. Using a sterile pipette tip, transfer 20 microliters from the well in row B to the corresponding well in row C.Continue this until row G to complete the serial dilution from 10 to the negative one in row B to 10 to the negative six in row G.Once done, pipette 100 microliters of each well into an appropriate growth agar plate, and spread across the plate to disperse the cell culture.
Place the well plates into an incubator shaker overnight at 37 degrees Celsius, and incubate the agar plates at 37 degrees Celsius for 24 hours. Examine the plate, and count the plates having approximately 25 to 300 colonies. If possible, choose plates of the same dilution value.
For each material to be tested, dilute the overnight bacterial samples by adding three 200 microliter aliquots to the growth medium into three wells, and three 200 microliter aliquots of the bacterial culture into additional wells. Place the 96 well plate into the plate reader and scan it. If necessary, continue adding broth or overnight bacterial culture until the optical density at 600 nanometers, or OD 600, is reached approximately 0.01.
To prepare bacterial nanoparticle suspensions and sterile media nanoparticle suspensions, remove one milliliter of bacterial culture or medium from a triplicate set of 15 milliliter conical tubes. And add it into a five milliliter centrifuge tube containing premeasured nanoparticles. Vortex the tubes to mix the solution.
Distribute the nanoparticles homogeneously by transferring one milliliter aliquots from the five milliliter centrifuge tube to the 15 milliliter conical tube. Once done, aliquot 200 microliters of each sample into the individual wells of a 96 well plate. After determining the seating density described earlier, add the samples and control into the individual wells of a 48 well non-tissue treated polystyrene plate.
Next, aliquot the 0.75 milliliters of the cell suspension into each well containing the samples and controls. Place the 48 well plate into an incubator shaker for 24 hours at 37 degrees Celsius, with shaking at 120 RPM. After incubation, collect two samples from each group, and transfer them individually into labeled five milliliter micro centrifuge tubes.
Add two milliliters of revised simulated body fluid, or RSBF, to each tube. Prepare sterilized nitrous cellulose papers by trimming them into one centimeter of 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 nitric cellose paper from the surface of the auger. Carefully flip the nitric cellulose 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 one milliliter of tris buffer to a sample containing well. After overnight incubation, collect the nitrocellulose paper from each sample surface and place them into five milliliters of tris buffer.
Vortex the collected filter papers and nanosurface material samples for five seconds. Collect the tris buffer suspensions from the sample and place them into individual fresh collection tubes. The antimicrobial effects using method A identified minimum and inhibitory concentration, or MIC, of one milligram per milliliter of magnesium oxide nanoparticles, and minimum bacteriocidal concentrations, or MBC 99.9, of 1.0 and 1.6 milligrams per milliliter of magnesium oxide nanoparticles for gram-negative Escherichia coli and pseudomonas aeruginosa respectively.
The gram-positive staphylococcus epidermis, S.Aureus, and methicillin-resistant staphylococcus aureus, or MRSA, demonstrated the MIC's values of 0.5, 0.7, and one milligrams per milliliter magnesium oxide nanoparticles respectively. NBC 99.99 values of 1.6 and 1.2 milligrams per milliliter were identified for S.epidermis and S.aureus respectively. While MRSA was not reduced beyond NBC 90.
MICs of 1.2 and one milligram per milliliter were identified in drug sensitive and drug-resistant candida species, C albicans, and C albicans fluconazole resistant, or FR respectively. In contrast, magnesium oxide nanoparticles demonstrated MIC values of one and 0.7 milligrams per milliliter, or C.glabrata and C glabrata echinocandin resistant, or ER respectively. Each candidate species reached an NBC 90 of 0.7 to 1.2 milligrams per milliliter, but only C.glabrata ER was reduced to NBC 99.9 at 1.2 milligrams per milliliter.
In method B, MRSA exposed to nanoparticles grew exponentially to an OD of 0.85. Exposure to 1.2 milligrams per milliliter of magnesium oxide nanoparticles and 6.25 milligrams per milliliter of trimethoprim resulted in 80.2%and 81.6%of reduced bacterial growth respectively. Similarly, 2.9 milligrams per milliliter of magnesium hydroxide nanoparticles reduced bacterial growth to 70.3%Exposure to one microliter per milliliter of vancomycin resulted in a 99.99%reduction in the growth of MRSA, suggesting the bacteriostatic activity of magnesium oxide and magnesium hydroxide nanoparticles.
Method C showed no inhibition of bacterial growth with indirect contact. The results indicated that ZC21 had the strongest antibacterial activity against the adhesion and growth of MRSA, for all the samples tested. In Method D, no viable staph aureus was identified in the 1.9A, 1.9 AA, and EPD samples, or their paired filter papers.
However, exposure to the EPD samples after a kneeling reduced bacterial growth to a few cells on the nanostructured surface and the pared filter paper. Identifying nanoparticle and nanostructured surface antimicrobial activities has led to additional investigations in bioengineering applications, including those related to chronic wounds and implant-related infections.
