Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

The overall goal of the following experiment is to observe changes in cell membrane charge after adherence to different material substrates. This is achieved by culturing the microorganisms in nutrient rich medium to increase their numbers. Next material surfaces that the microorganisms will adhere to are cleaned and functionalized for optimal microbial attachment rates.

Then the microorganisms are plated on the various material substrates to allow for Kelvin probe force microscopy or KPFM imaging. The results show that material substrate type and its inherent charge have an effect on cell activity as seen by changes in cell membrane charge and cell adherence. The main advantage of this technique over existing methods like electrophoretic, light scattering and isoelectric point determination is that KPFM allows for the examination of the charge of individual cells and biofilms instead of entire cultures or colonies.

This is beneficial when wanting to compare cell to cell as well as biofilm electrical characteristics with high accuracy and precision. To begin, prepare 5%sheep's blood agar plates streak the MRSA bacteria onto the prepared plates and incubate for 24 hours at 37 degrees Celsius to generate single well isolated colonies. Make 500 milliliters of triptych soy broth, supplemented with 1%glucose and 0.1%sodium chloride autoclave, along with two clean glass test tubes and one box of one milliliter pipette sips.

After allowing the medium and test tubes to cool under a biosafety cabinet or near a buns and burner, pipette six milliliters of the TSB into each test tube. Using one of the previously prepared sheeps blood agar plates, inoculate single colony of MRSA into one of the tubes. Use the second tube as a negative control to ensure sterility incubate the tubes in a reciprocal incubator for 24 hours at 37 degrees Celsius and 200 RPM.

The MRSA tube should show growth or nothing should have grown in the control test tube. Next, create a subculture by using one milliliter from the 24 hour culture to inoculate six milliliters of fresh tryptic soy broth and incubate at 37 degrees Celsius and 200 RPM for another six hours. Then pipette one milliliter of the culture into afus tube and centrifuge at 850 times G for three minutes.

Remove the supinate and use deionized water to wash the pellet two times with forceps in the dominant hand. Hold a 20 millimeter stainless steel a FM sample disc and use five milliliters of deionized water to clean each side. Place the clean discs into a beaker of deionized water and sonicate for one minute following sonication.

Use the forceps to remove the sample discs from the beaker and lean them at an approximate 60 degree angle against the edge of an open Petri dish on a paper towel To dry completely use the lid of the dish to cover the drying discs. Once dry, transfer the disc into a Petri dish to functionalize the surface of the disc. Pipette 200 to 400 microliters of 0.1%polyol lysine onto the discs in a biosafety cabinet and incubated room Temperature for one hour using forceps to hold the discs.

Use deionized water to wash them as demonstrated earlier in this video. Next plate, 200 to 400 microliters of resuspended cells that have been washed twice onto the coated sample discs. After incubating the room temperature for 30 minutes, gently use one milliliter of deionized water to wash the discs.

Allow the discs to dry overnight before imaging to carry out Kelvin Probe Force microscopy imaging. Turn on the computer along with the atomic force microscope or AFMs, HEB and MAC three controller. Open an A FM imaging software such as Ulence P ovu overview.

Select A-C-A-F-M or the correct designation for imaging in intermittent contact mode. Using a FM forceps in the dominant hand and holding the spring loaded clip holder open in the other hand carefully place A-K-P-F-M cantilever into the headpiece. Carefully load the headpiece into the A FM and connect the laser light and stage lift motor wires.

Then align the laser onto the tip of the cantilever. To align the laser onto the tip of the cantilever. Rotate the front to back knob clockwise a few times to move the laser on top of the cantilever chip.

When the laser reaches the chip, it will be blocked and will no longer be visible. Then rotate the front to back knob counterclockwise until the laser spot reappears. The laser is now on the chip's edge.

Rotate the left to right knob to position the laser on the cantilever. As the laser passes over the cantilever, it will disappear and reappear in rapid succession. The laser spot should be visible in the frosted glass cover where the photo diode unit will later sit.

Turn the front to back knob counterclockwise to move the spot down the cantilever towards the tip until the spot on the frosted glass disappears. Now turn the front to back knob clockwise only slightly in order to position the laser so that it sits just on the cantilever tip, the laser spot will reappear on the frosted glass. Once the laser is aligned, hold the stage lift motor in the open position for 10 seconds to ensure enough space between the cantilever and sample.

When attaching the sample to the A FM, now place the sample on the KPFM stage. Use copper wire to ground the sample and connect the appropriate wires from the A FM to the sample stage. Then connect the stage to the A FM in the A FM software.

Ensure that the setting time is at no more than 10 milliseconds and that the approach speeds do not exceed two microns per second. In order to avoid tip damage, then tune the cantilever and begin moving the cantilever tip towards the sample. Once the cantilever tip reaches a sample surface, select an imaging window size and ideal imaging area, optimize eye and P gains along with the cantilever set point so that topographical imaging is optimized.

When topographical imaging is optimized, turn on the KPFM module in FM mode. Set the imaging window to five microns by five microns with five 12 by five 12 resolution or slow scan speeds. For FM KPFM settings, set the drive between five and 10%Set the drive frequency to between one and five kilohertz with a bandwidth of two kilohertz.

Set the iron NP gains for the F-M-K-P-F-M to 0.3%After acquiring KPFM images, use post imaging processing software to analyze the images and to collect SP data using the settings outlined in this video Demonstration. KPFM images were taken on 15 MRSA cells on both stainless steel and gold surfaces. Shown here are the types of images collected.

Because KPFM operates using dual frequencies, topographic images and electrical data of the cell surface were simultaneously collected. Thermal filters were applied to surface potential maps in order to more easily discern small changes in surface potential. In this graph, images were analyzed to determine microbial membrane surface potential, and then used to compare for growth on different substrates.

Surface potential scans of the bare stainless steel and bare gold substrates showed overall negative surface potentials. Polyol lysine coated stainless steel and gold discs both exhibited a shift to positive surface potentials. MRSA cell membrane potentials varied between the stainless steel and gold surfaces.

With the stainless steel exhibiting a significantly larger positive membrane surface potential for cells compared to MRSA cells on the gold substrate that showed negative surface potentials. To demonstrate the full capabilities of KPFM, an example of a step height graph of MRSA on stainless steel is seen here. This shows the difference in surface potential between the substrate surface and the microbial membrane.

After watching this video, you should have a good understanding of how to use Kelvin probe force microscopy to examine the effects of electrical charge on microbial cell attachment and activity on different material substrate surfaces.