Electromechanical and elastic probing of bacteria in a cell culture medium

Abstract

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of bacteria of different types in pure water. Here, the BEPFM method is performed for the first time on physiologically relevant electrolyte media, such as Dulbecco's phosphate-buffered saline (DPBS) and Dulbecco's modified Eagle's medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria in DPBS are demonstrated. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media.

Fig. 1

Amplitude-frequency spectra representing electromechanical responses were averaged over the separate areas of voxels for M. lysodeikticus (a,d,g), PLL (b,e,h), and the ratio of ML to PLL (c,f,i) in water (a,b,c), DPBS (d,e,f), and DMEM (g,h,i) during steps in applied bias. The applied voltages are designated by the colors as follows: 100 V – black, 90 V – red, 80 V – blue, 70 V – magenta, 60 V – green, 50 V – dark blue, 40 V – violet, 30 V – cyan, 20 V – brown, 10 V – dark yellow.

Fig. 2

Applied bias versus BEPFM response amplitude at resonance frequency of Gaussian fits of the peaks for M. lysodeikticus in water (■), DMEM ( ) and DPBS ( ) and for PLL in DMEM ( ) and DPBS ( ). The zoomed inset shows a linear relationship for DPBS and DMEM.

Fig. 3

Simple harmonic oscillators (SHO) fit to the spectra of each voxel of BEPFM data form an image displaying contrast between different M. lysodeikticus bacteria and the underlying PLL-coated mica substrate in (a) water, (b) DPBS, and (c) DMEM. Fits were performed on peaks of 54 to 60 kHz at 50 V for water, 60 to 80 kHz at 100 V for DPBS, and 46 to 48 kHz at 100 V for DMEM. The images have dimensions of 2.5 μm by 2.5 μm in water and 3 μm by 3 μm in DPBS and DMEM. AFM images acquired simultaneously with BEPFM data show morphologies as observed in the BEPFM images, and the bacteria are 400 nm at maximum height in water and 600 nm maximum heights in DPBS and DMEM.

Fig. 4

Comparison of electromechanical response spectra in water (a) and DPBS (b) of each bacterium and their PLL substrate is presented by dividing the former by the latter.

Fig. 5

M. lysodeikticus (ML), P. fluorescens (PF), and speculatively damaged or dead PF (D-PF) on PLL in DMEM within the same area and image are shown in the trace (a) height, (b) deflection, and (c) 1^st principal component of the BEPFM amplitude channel. Note that there is a PF cell undergoing cell division in the upper left of the image. The spectra from selected areas on each type of bacterium, ML, PF and D-PF, were averaged and smoothed using adjacent averaging over 75 points. The averaged spectrum for ML (■, black), PF ( ), and D-PF ( ) was divided by the averaged spectrum for PLL (e), all from selected areas as indicated in

Figure 6

Steps in applied force from -2.32 nN to 11.6 nN on ML divided by PLL in DMEM were performed an excitation amplitude of 100 V with slow-scan disabled and result in the spectra shown in (a).

Fig. 7

Force volume maps of M. lysodeikticus (ML), P. fluorescens (PF), and speculatively damaged or dead PF (D-PF) on PLL in DMEM – the same as in Figure 5 – are calculated using the Hertzian model on the approach curve with an force set point of (a) 35 nm or 2 nN and (b) 165 nm or 10 nN. At lower set points, less contrast between bacteria and the PLL surface was observed.