Bacterial surface charge density is a defining characteristic of the cell’s interfacial physiology and largely determines how the cell interacts with its local environment. Directly measuring the surface charge density of a bacterial cell, however, is an experimentally non-trivial task. Nonetheless, an experimental technique capable of accurately quantifying surface charge is needed in order to better understand molecular adaptations in interfacial physiology which arise in response to acute environmental changes. Herein, we demonstrate application of the surface-sensitive nonlinear optical technique, second harmonic light scattering (SHS), as a means of directly quantifying the surface charge density of individual living bacteria. Specifically, this can be achieved by measuring the number of molecular ions electrostatically adsorbed on the exterior cellular surface as counter charges. As an initial proof-of-principle, we monitor the time- and concentration-dependent interactions of the small molecular cation, malachite green (MG), with representative strains of Gram-positive (L. rhamnosus) and Gram-negative (E. coli) bacteria. Surprisingly, despite numerous differences in the composition of the cellular ultra-structures of these two bacterial strains, the SHS-deduced passive uptake rates of the MG ion were shown to be virtually identical. This can be understood by noting that the cellular ultra-structures are topologically similar. However, the adsorption densities of cationic MG (in the limit of saturation) on the exterior surfaces of the two bacteria were shown to be characteristically distinct. The charge density of the polyanionic lipopolysaccharide coated outer surface of the Gram-negative strain (6.6±1.3 nm<sup>-2</sup>) was deduced to be seven times larger than that of the comparatively flat protein surface layer of the Gram-positive strain (1.0±0.2 nm<sup>-2</sup>). The feasibility of exploiting this disparity of surface charge density (as deduced with SHS) as a basis for Gram-classification of bacteria will be presented.

Determination of cell membrane surface charge density by second harmonic light scattering

ACS Spring Meeting 2021

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