The authors appreciate the financial support from the U.S. National Science Foundation (NSF CBET award 1512764 and NSF PIRE 1545852), the National Institutes of Health (NIH NIDCR 1R03DE028631), the University of California (UC) Regents Faculty Development Fellowship, the Committee on Research Seed Grant (Huinan Liu), and the UC-Riverside Graduate Research Mentorship Program Grant awarded to Patricia Holt-Torres. The authors appreciate the assistance provided by the Central Facility for Advanced Microscopy and Microanalysis (CFAMM) at UC-Riverside for the use of SEM/EDS and Dr. Perry Cheung for the use of XRD. The authors would also like to thank Morgan Elizabeth Nator and Samhitha Tumkur for their assistance with the experiments and data analyses. Any opinions, findings, conclusions, or recommendations expressed in this article are those of the authors and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health.

To present the direct co-culture and direct exposure methods, we use magnesium oxide nanoparticles (nMgO) as a model material to demonstrate bacterial interactions. To present the direct culture and focused-contact exposure methods, we use an Mg alloy with nanostructured surfaces as examples.
1. Sterilization of nanomaterials
NOTE: All the nanomaterials must be sterilized or disinfected prior to microbial culture. The methods that can be used include ...

<ol>
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The authors have no conflicts of interest.

We have presented four in vitro methods (A-D) to characterize the antibacterial activities of nanoparticles and nanostructured surfaces. While each of these methods quantifies bacterial growth and viability over time in response to nanomaterials, some variation exists in the methods used to measure the initial bacterial seeding density, growth, and viability over time. Three of these methods, the direct co-culture method (A)17, the direct culture method (C)14, and ...

In the US alone, 1.7 million individuals develop a hospital-acquired infection (HAI) annually, with one in every 17 of these infections resulting in death1. In addition, it is estimated that the treatment costs for HAIs range from $28 billion to $45 billion annually1,2. These HAIs are predominated by methicillin-resistant Staphylococcus aureus (MRSA)3,4 and Pseudomonas aeruginosa4, which are commonly isolated from chronic wound infections and usually require extensive treatment and time to produce a favorable patient outcome.
Over the past several decades, multiple antibiotic classes have been developed to treat infections related to these and other pathogenic bacteria. For example, rifamycin analogs have been used to treat MRSA, other gram-positive and gram-negative infections, and Mycobacterium spp. infections5. In the 1990s, to effectively treat an increasing number of M. tuberculosis infections, additional drugs were combined with rifamycin analogs to increase their effectiveness. However, approximately 5% of M. tuberculosis cases remain resistant torifampicin5,6, and there is increasing concern regarding multi-drug resistant bacteria7. Currently, the use of antibiotics alone may not be sufficient in the treatment of HAIs, and this has provoked an ongoing search for alternative antimicrobial therapies1.
Heavy metals, such as silver (Ag)8,9,10 and gold (Au)11, and ceramics, such as titanium dioxide (TiO2)12 and zinc oxide (ZnO)13, in nanoparticle (NP) form (AgNP, AuNP, TiO2NP, and ZnONP, respectively) have been examined for their antimicrobial activities and have been identified as potential antibiotic alternatives. In addition, bioresorbable materials, such as magnesium alloys (Mg alloys)14,15,16, magnesium oxide nanoparticles17,18,19,20,21, and magnesium hydroxide nanoparticles [nMgO and nMg(OH)2, respectively]22,23,24, have also been examined. However, the previous antimicrobial studies of nanoparticles used inconsistent materials and research methods, resulting in data that are difficult or impossible to compare and are sometimes contradictory in nature18,19. For example, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles varied significantly in different studies. Ipe et al.25 evaluated the antibacterial activities of AgNPs with an average particle size of ~26 nm to determine the MICs against gram-positive and gram-negative bacteria. The identified MICs for P. aeruginosa, E. coli, S. aureus,&#160;and MRSA were 2 &#181;g/mL, 5 &#181;g/mL, 10 &#181;g/mL, and 10 &#181;g/mL, respectively. In contrast, Parvekar et al.26 evaluated AgNPs with an average particle size of 5 nm. In this instance, the AgNP MIC and a MBC of 0.625 mg/mL were found to be effective against S. aureus. In addition, Loo et al.27 evaluated AgNPs with a size of 4.06 nm. When E. coli was exposed to these nanoparticles, the MIC and MBC were reported at 7.8 &#181;g/mL. Finally, Ali et al.28 investigated the antibacterial properties of spherical AgNPs with an average size of 18 nm. When P. aeruginosa, E. coli, and MRSA were exposed to these nanoparticles, the MIC was identified at 27 &#181;g/mL, 36 &#181;g/mL, 27 &#181;g/mL, and 36 &#181;g/mL, respectively, and the MBC was identified at 36 &#181;g/mL, 42 &#181;g/mL, and 30 &#181;g/mL, respectively.
Although the antibacterial activity of nanoparticles has been extensively studied and reported during recent decades, there is no standard for the materials and research methods used to allow for direct comparisons across studies. For this reason, we present two methods, the direct co-culture method (method A), and the direct exposure method (method B), to&#160;characterize and compare the antimicrobial activities of nanoparticles while keeping the materials and methods consistent.
In addition to nanoparticles, nanostructured surfaces have also been examined for antibacterial activities. These include carbon-based materials, such as graphene nanosheets, carbon nanotubes, and graphite29, as well as pure Mg and Mg alloys. Each of these materials has exhibited at least one antibacterial mechanism, including physical damage imposed on cell membranes by carbon-based materials and damage to metabolic processes or DNA through the release of reactive oxygen species (ROS) when Mg degrades. In addition, when zinc (Zn) and calcium (Ca) are combined in the formation of Mg alloys, the refinement of the Mg matrix grain size is enhanced, which leads to a reduction in bacterial adhesion to substrate surfaces in comparison to Mg-only samples14. To demonstrate antibacterial activity, we present the direct culture method (method C), which determines bacterial adhesion on and around nanostructured materials over time through the quantification of bacterial colony-forming units (CFUs) with direct and indirect surface contact.
The geometry of nanostructures on surfaces, including the size, shape, and orientation, could influence the bactericidal activities of materials. For example, Lin et al.16 fabricated different nanostructured MgO layers on the surfaces of Mg substrates through anodization and electrophoretic deposition (EPD). After a period of exposure to the nanostructured surface in vitro, the growth of S. aureus was substantially reduced in comparison to non-treated Mg. This indicated a greater potency of the nanostructured surface against bacterial adhesion versus the nontreated metallic Mg surface. To reveal the different mechanisms of the antibacterial properties of various nanostructured surfaces, a focused-contact exposure method (method D) that determines the cell-surface interactions within the area of interest is discussed in this article.
The objective of this article is to present four in vitro methods that are applicable to different nanoparticles, nanostructured surfaces, and microbial species. We discuss key considerations for each method to produce consistent, reproducible data for comparability. Specifically, the direct co-culture method17 and direct exposure method are used for examining the antimicrobial properties of nanoparticles. Through the direct co-culture method, the minimum inhibitory and minimum bactericidal concentrations (MIC and MBC90-99.99, respectively)&#160;can be determined for individual species, and the most potent concentration (MPC) can be determined for multiple species. Through the direct exposure method, the bacteriostatic or bactericidal effects of nanoparticles at minimum inhibitory concentrations can be characterized by real-time optical density readings over time. The direct culture14 method is suitable for examining bacteria directly and indirectly in contact with nanostructured surfaces. Finally, the focused-contact exposure16 method is presented to examine the antibacterial activity of a specific area on a nanostructured surface through the direct application of bacteria and the characterization of bacterial growth at the cell-nanostructure interface. This method is modified from the Japanese Industrial Standard JIS Z 2801:200016, and is intended to focus on microbe-surface interactions and exclude the effects of bulk sample degradation in microbial culture on antimicrobial activities.

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		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Milipore Sigma</td>
			<td>Z336777</td>
			<td></td>
		</tr>
		<tr>
			<td>80 L NTRL Certified Convection Drying Oven&#160;</td>
			<td>MTI Corporation</td>
			<td>BPG-7082</td>
			<td>https://www.mtixtl.com/BPG-7082.aspx</td>
		</tr>
		<tr>
			<td>(hydroxymethyl) aminomethane buffer pH 8.5; Tris buffer&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>42457</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSpec&#160;THIOFLAVIN T ULTRAPURE GRADE</td>
			<td>Fisher Scientific</td>
			<td>50-850-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron-multiplying charge-coupled device digital camera&#160;</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteraldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brightline, Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma - optical emission spectrometry (ICP-OES)</td>
			<td>PerkinElmer</td>
			<td>8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverse microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma Life Science&#160;</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth + agar</td>
			<td>Sigma Life Science&#160;</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>MacroTube 5.0&#160;&#160;</td>
			<td>Benchmark Scientific</td>
			<td>C1005-T5-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium oxide nanoparticles</td>
			<td>US Research Nanomaterials, Inc</td>
			<td>Stock #:&#160;US3310&#160;&#160;&#160;M</td>
			<td>MgO, 99+%, 20 nm</td>
		</tr>
		<tr>
			<td>MS Semi-Micro Balance</td>
			<td>Mettler Toledo</td>
			<td>MS105D</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose paper</td>
			<td>Fisherbrand</td>
			<td>09-801A</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 12-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351143</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 48-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351178</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 96-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351172</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 100 mm</td>
			<td>VWR</td>
			<td>470210-568</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 15 mm</td>
			<td>Fisherbrand</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td>SP70P</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscopy (SEM)</td>
			<td>TESCAN&#160;</td>
			<td>Vega3 SBH</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>VWR</td>
			<td>97043-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Fisher Scientific</td>
			<td>accuSpin Micro 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5430</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>MP</td>
			<td>1010617</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer&#160;</td>
			<td>Tecan</td>
			<td>Infinite 200 PRO</td>
			<td>https://lifesciences.tecan.com/plate_readers/infinite_200_pro</td>
		</tr>
		<tr>
			<td>VWR Benchmark Incu-shaker 10L</td>
			<td>VWR</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray power defraction</td>
			<td>&#160;Panalytical</td>
			<td>N/A</td>
			<td>PANalytical Empyrean Series 2</td>
		</tr>
	</tbody>
</table>

evaluation of antimicrobial activities of nanoparticles and nanostructured surfaces in vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

The identification of the antibacterial activity of magnesium oxide nanoparticles and nanostructured surfaces has been presented using four in vitro methods that are applicable across different material types and microbial species.
Method A and method B examine bacterial activities when exposed to nanoparticles at a lag phase (method A) and log phase (method B) for a duration of 24 h or longer. Method A provides results regarding the MIC and MBC, while method B determines the inhibito...

Watch this Scientific Journal Video about Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro at JoVE.com

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

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.

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2.6K Views.  University of California, Riverside. 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...