Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Summary

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.

Abstract

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.

Introduction

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 reversibility^1-3. 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 done^4,....

Protocol

1. Preparation of Glassware and Cultures

1. Before proceeding with this experiment, prepare 5% sheep’s blood agar (SBA) plates for growing MRSA. Incubate SBA plates for 24 hr at 37 °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 °C for 1 - 2 months.
   NOTE: Sheep’s blood agar plates come in pre-made packs containing between 20 - 25 plates based on the manufacturer, and typically consist of a tryptic soy agar base with the addition of 5% w/v sheep’s blood.
2. Make 500 ml of tryptic soy broth....

Results

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 µm to 10 x 10 µm. 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 lines to scan.......

Discussion

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.......

Materials

List of materials used in this article

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