Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy

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Surface Viscoelasticity of Individual Gram-Negative bacterial Cells Measured Using Atomic Force Microscopy

V. Vadillo-Rodriguez, T J. Beveridge, and T R Dutcher Journal of Bacteriology, Vol. 190, No. 12, June 2008, p. 4225-4232

Keywords: Viscoelasticity, creep

Summary

Bacteria can be divided into two types: Gram-negative and Gram-positive, depending on the characteristics of their cell wall. Gram-negative bacteria, like the Pseudomonas aeruginosa used in this study, have two cell membranes separated by a thin layer called a periplasm. This flexible layer is a peptidoglycan, which consists of stiff polysaccharides connected by flexible protein cross-linkers. The qualitative mechanical properties of large numbers of cells and the elastic behavior of single cells have both been previously investigated, but this is one of the first works to look at the full viscoelastic properties of an individual bacterium. To accomplish this, the authors examine the force-displacement curves of an AFM-tip pushed into the cell surface, as well as the deformation over time at a constant force. The authors find that the time-scales for these processes, on the order of a couple seconds, is likely optimal for the cells. It allows the cell to maintain its integrity while growing, while still preventing the magnification of potentially hazardous local stresses.

Soft Matter Concepts

The main feature of this paper is its focus on viscoelastic, rather than purely elastic behavior. An elastic material will deform proportional to an applied stress, whereas a viscous material will flow at a rate proportional to the stress. In the case of the cells, there is an immediate deformation of the cells, followed by a slow approach to some equilibrium strain. With an atomic force microscope, the authors were able to apply these stresses to a single cell and measure the viscoelastic response, as illustrated below:

DutcherFig1.jpg

From A to C, the tip of the AFM is advanced towards the substrate at a rate of 1.98 microns per second. After the tip reaches the cell surface at time B, the slope of the force-displacement line can be used to determine the elastic constant. Once a predetermined force is attained, the force is held fixed and the resulting displacement <math>\delta z</math> is measured over the course of a time <math>\delta t</math>. This dynamics of this process are determined by the viscous properties of the cell.

The researchers took images the cell over a range of forces to observe how the cell distorted under high stress. Interestingly, the cell's cross-section became smaller at higher forces, most likely due to a redistribution of the cell's contents to the ends of the cells. However, this process was completely reversible (see right), likely due to the high turgor pressure of the cell.

DutcherFig4.jpg

To test the validity of this novel AFM technique, the authors add glutaraldehyde to the cells, which increases the covalent bonding at the cells surface and results in a roughened appearance (note: scan size is 2 microns x 2 microns). The authors suggest that this is due to increased cross-linking at the surface, but don't pursue the issue further. However, glutaraldehyde is used in many biophysical studies, so it would be worthwhile to study its effect in more detail.

DutcherFig3.jpg

The mechanical properties of the cell support the theory of glutaraldehyde stiffening the cell wall. At a constant force, the untreated cell deforms more and reaches equilibrium faster than its treated counterpart. For comparison, practically no deformation is observed if the experiment is repeated directly on the glass substrate.

DutcherFig7.jpg

The authors suggest some possible industrial applications for these viscoelastic studies, such as looking at the effect of antibiotics and comparing the results to glutaraldehyde treatment.