The authors sincerely thank the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Research and Innovation, and the Canada Foundation for Innovation for funding this study.

1. Preparation of Glassware and Cultures
<ol>
	<li>Before proceeding with this experiment, prepare 5% sheep&#8217;s blood agar (SBA) plates for growing MRSA. Incubate SBA plates for 24 hr at 37 &#176;C. After this time, single, well-isolated colonies should be present to be used for subsequent inoculations into liquid media. Store streaked SBA plates with single colonies at 4 &#176;C for 1 - 2 months. 
	NOTE: Sheep&#8217;s blood agar plates come in pre-made packs containing between 20 - 25 plates based on the manu...

<ol>
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The authors have nothing to disclose.

KPFM was employed as a novel technique for obtaining surface electrical data. It has commonly been used as a method for examining charge distribution in chemistry and has only recently begun to be applied for the study of biological systems on the micro- and nano-scales. From the data collected we found that microbes did not appear to readily attach to clean stainless steel and gold surfaces, even after 3 hr of static incubation. Poly-L-lysine functionalized surfaces showed rapid microbial attachment after 30 min. Longer...

Biofilms produced on equipment surfaces and in cutaneous wounds present a problem for the medical industry as biofilms are recalcitrant to removal and can lead to increased rates of disease transmission and antimicrobial resistance. Attachment is the first step in biofilm formation and is the most critical step due to its reversibility1-3. Substrate surface characteristics play a crucial role on microbial attachment. Factors such as surface hardness, porosity, roughness, and hydrophobicity have been shown to effect microbial attachment; however, little research examining the role of substrate surface potential (SP) on microbial adhesion has been done4,5. Negatively charged surfaces prevent the attachment of Bacillus thuringiensis spores to mica, silicon, and gold6. Changes in cellular membrane potential are indicative of changes in cellular attachment and motility5,7. It has been observed that electrically homogeneous surfaces more readily promote microbial adhesion5. Characterizing the surface potential of bacteria at the nanoscale can provide a novel way to understand the adhesion kinetics of bacteria to various surfaces, and thereby can aid in the development of anti-biofouling strategies. Unlike other methods used for characterizing the electro kinetics of bacteria, such as electrophoretic light scattering, zeta potential, and isoelectric point determination, Kelvin probe force microscopy (KPFM) allows for the examination of individual cells instead of entire cultures8-11. This is beneficial when wanting to compare cell-cell or biofilm electrical characteristics with high accuracy and precision.
KPFM is a module of atomic force microscopy (AFM). AFM was developed as a direct result of the scanning tunneling microscope (STM)12. The first published images using STM were done by Gerd Binnig and Heinrich Rorhrer in 198212. Their invention was able to resolve atomic structures by raster scanning a sharp conductive tip over a conductive surface in air. The implications of achieving high-resolution images excited biologists who quickly attempted to use STM to image dried samples of DNA, proteins, and viruses12. STM can also be done in liquids using specialized probe tips13. This was shown by Lindsay et al., who used STM and AFM to image DNA molecules in 10 mM HClO4 and in water, on gold electrodes13. KPFM has been demonstrated in determining the Surface potentials of DNA and protein analysis25 and biomolecules interaction with ligands26.
KPFM operates by measuring the contact potential difference (CPD) between an AFM conductive cantilever tip and an ideally conductive sample (Figure 1, i)14,15. Samples do not necessarily have to be conductive (i.e., biological samples). Imaging can be carried out on mica, glass, and silicon surfaces (non-conductive) as long as the non-conductive surface is thin and there is an underlying conductive material6,7. The CPD is equivalent to the surface potential voltage and can be described as the difference in the work functions between the tip (&#966;tip) and sample (&#966;sample), divided by the negative electron charge (-e). When a conductive AFM tip is brought close to a sample surface (separated by distance d), an electrostatic force (Fes) is generated due to the difference in Fermi energies (Figure 1, ii, a)15. At this point, the vacuum energies of the tip and sample (Ev) are in equilibrium and aligned. Upon bringing the tip closer to the sample surface, the tip and sample surface come into electrical contact and act as parallel plate capacitors (Figure 1, ii, b)14,15. At the point, the Fermi energies of the tip and sample surface become aligned, reaching a steady-state equilibrium (Figure 1, ii, b). The tip and sample surface will be charged and a VCPD will form due to a difference in Ev&#8217;s and work functions. A Fes acts on the electrical contact area due to the formed VCPD. This force is subsequently nullified through the application of an external VDC to the tip that has the same magnitude as the formed VCPD (Figure 1, ii, c). This applied DC voltage eliminates surface charge in the area of electrical contact, and the amount of VDC necessary to eliminate the Fes of VCPD is equal to the difference in the work functions between the sample surface and tip15. It should be noted that the work function of the tip is known and it is provided by manufacturers. In all KPFM methods, an AC voltage (VAC, approximately 100 - 500 mV) is also applied to the tip in order to generate oscillating electrical forces between the tip and sample14. This provides better resolution when measuring changes in VCPD and/or Fes. In this regard, changes in the frequency or amplitude of electrical oscillation can be corrected by VDC, and surface potential maps can be generated. Data from specific areas of these maps can be further analyzed to provide electrical information about specific topographical features.
KPFM can be operated in three modes: (1) lift mode, (2) amplitude modulation (AM) mode, and (3) frequency modulation (FM) mode14,16. Lift mode was the initial incarnation of KPFM. Lift mode relies on a two-pass method in which an oscillated tip is dragged across the surface to obtain a topographical image. For the second pass the tip is raised a preset distance above the sample (10 - 100 nm) and scanned back across the same area. Due to this two-pass method, lift mode, compared to AM- and FM-KPFM, takes the longest time for image acquisition. Raising the tip away from the surface ensures that only long-range Fes is measured. Also, cross talk between topography and surface potential measurements is decoupled at the expense of increased lateral resolution and sensitivity.
AM-KPFM improves lateral resolution and sensitivity by using dual-frequencies to simultaneously measure sample topography and surface potential (one-pass scan)14. In AM mode, the cantilever is mechanically oscillated, generally 5% below its first resonant frequency (f0), and electrically oscillated (through a VAC) at its second resonant frequency (f1). Changes in the amplitude of f0 lead to the generation of topographical data, while changes in the amplitude of f1, due to changes in Fes and VCPD, give surface potential measurement data. f0 and f1 of the cantilever are separated by significant frequencies and energies that signal cross-talk is minimized14. The head electronics box (HEB) of the AFM separates out the two signals to give both topographical and surface potential data simultaneously in one scan. FM-KPFM improves resolution even further than AM-KPFM on biological surfaces14. FM-KPFM works differently than AM-KPFM in that it measures changes in electrostatic force gradients rather than electrostatic force (Fes)15. Like AM mode, FM mode utilizes dual-frequencies and a single-pass scanning mechanism to obtain topographical and surface potential data simultaneously14. In FM mode, the cantilever is mechanically oscillated at f0 and electrically oscillated at a low modified frequency (fmod, typically 1 - 5 kHz). Upon electrostatic interactions, f0 and fmod mix to produce side bands f0 &#177; fmod. These sidebands are very sensitive to changes in electrostatic force, and can be separated from f0 through the AFM&#8217;s HEB. Since FM-KPFM measures changes in electrostatic force gradients, the tips apex shape and its maintenance/integrity play a critical role in overall surface potential resolution14, 15. Surface potential resolution using AM and FM modes are in the range of 1 nm laterally14-16. It should be noted that KPFM imaging can be done in non-polar liquids, and more recently, has been shown to be done in low-ionic (&#60;10 mM) polar liquids (in open-loop KPFM modes which do not require a bias feedback, obviating the application of a DC bias) such as MilliQ water; however, KPFM imaging has yet to be done on live cells in polar solutions17-20. Additional challenges associated with SP imaging in liquid is that solutions commonly used for maintaining cells (i.e., phosphate buffered saline) have high concentrations of mobile ions, which would lead to Faradaic reactions, bias-induced charge dynamics, and ion diffusion/redistribution20. Thus, for this experiment, measurements were taken from dried and dead MRSA cells on poly-L-lysine functionalized stainless steel and gold surfaces under ambient conditions. Imaging can be carried out in air or vacuum conditions on biological samples that have been previously dried or immobilized onto surfaces20. Humid conditions have also been shown to affect KPFM imaging of surfaces6.
In this study, we employed FM-KPFM and AFM to examine the role of SP on the attachment of methicillin-resistant Staphylococcus aureus USA100 (MRSA) to poly-L-lysine functionalized stainless steel and gold surfaces. MRSA has recently garnered status as a multi-drug resistant (MDR) &#8220;superbug&#8221; due to its naturally selected resistance to many &#946;-lactam antibiotics and cephalosporins21. MRSA infections are now more challenging, difficult, and formidable to treat, leading to the use of harsher antimicrobials such as vancomycin or oxazolidinones which have higher toxicity levels in humans, and therefore cannot be used as long-term treatments22. Stainless steel was chosen due to its medical relevance and common use as a material in hypodermic needles, bedpans, door handles, sinks, etc. Gold was used as a comparative metal. FM-KPFM was utilized to examine if microbial membrane SP changes upon attachment to the substrates.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5500ILM Atomic Force Microscope</td>
			<td style="width:71px;">Agilent Technologies</td>
			<td style="width:119px;">#N9435S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">AFM/STM Metal Specimen Discs</td>
			<td style="width:71px;">TED PELLA, INC.</td>
			<td style="width:119px;">#16219</td>
			<td>Stainless steel sample discs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DPE (Low-Noise) Conductive SPM Probes</td>
			<td style="width:71px;">Mikromasch</td>
			<td style="width:119px;">#HQ:DPE-XSC11</td>
			<td>There are 4 Pt-coated cantilevers per chip.&#160; We utilized cantilever B for experiments.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PELCO Gold Coated AFM/STM Metal Specimen Discs</td>
			<td style="width:71px;">TED PELLA, INC.</td>
			<td style="width:119px;">#16218-G</td>
			<td>Gold sample discs</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PicoView Software</td>
			<td style="width:71px;">Agilent Technologies</td>
			<td style="width:119px;">#N9797B</td>
			<td>5500ILM Atomic Force Microscope imaging software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pico Image Software (Pico Image Basic)</td>
			<td style="width:71px;">Agilent Technologies</td>
			<td style="width:119px;">#N9797AU-1FP</td>
			<td>5500 ILM Atomic Force Microscope post-image processing software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scilogex D3024 High Speed Micro-Centrifuge</td>
			<td style="width:71px;">Thomas Scientific</td>
			<td style="width:119px;">#91201513</td>
			<td>Centrifuge used in cell-washing steps to separate cells (pellet) from media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypticase Soy Agar with 5% Sheep Blood</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">#221261</td>
			<td>Pre-made plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptic Soy Broth</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">#257107</td>
			<td>Comes as a dry powder.&#160; Instruction on how to make come on the container.</td>
		</tr>
	</tbody>
</table>

surface potential measurement of bacteria using kelvin probe force microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

The ability to measure SP using KPFM relies on the principle that both the sample surface and cantilever tip are conductive to some extent. Stainless steel and gold acted as conductive surfaces to which MRSA were attached. KPFM images were taken of 15 MRSA cells on both surfaces with 512 x 512 resolutions, and with scan areas ranging from 5 x 5 &#181;m to 10 x 10 &#181;m. Scanning was carried out with line speeds ranging from 0.02 lines/sec to 0.05 lines/sec. Therefore, with 512 data points collected per line and 512 lin...

Watch this Scientific Journal Video about Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy at JoVE.com

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

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21.6K 조회수.  University of Guelph. Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces. 