Mechanism of microbial aggregation during capillary electrophoresis

We studied the aggregation of a rod-shaped bacteria, Bifidobacterium infantis, during capillary electrophoresis (CE). A microscope with an intensified CCD camera was employed to monitor the migration and aggregation of bacteria, which are labeled with fluorescent dye Syto 9 and excited with a 488-nm argon ion laser. A collision-based aggregation mechanism is proposed, in which collisions between microbes result from different mobilities and migration directions in the electric field. Individual microbes are aligned differently with respect to the direction of the electric field and exhibit different drag coefficients. The long-range forces include van der Waals attraction and electrostatic repulsion as qualitatively described by DLVO theory. Collisions in CE produce sufficient energy to overcome electrostatic repulsion, thus improving the efficiency of aggregation. This is supported by the fact that higher electric fields always resulted in faster aggregation. Also, when sodium phosphate buffer was used, increasing the ionic strength resulted in faster aggregation. However, when Tris-boric acid-EDTA (TBE, pH 9.1) buffer was used, the aggregation speed decreased when the ionic strength increased. We attribute this to the change of the surface of the bacteria at high borate and EDTA concentration, such as the loss of polysacchatides or the presence of complexation. This reduces the hydrophobicity of the surface and, thus, the short-range attractive forces. The addition of 0.05% poly(ethylene oxide) (PEO) into high ionic strength TBE buffer increased the aggregation rate. This can be attributed to the bridging effect of PEO between microbes. Further increase in the concentration of polymer reduced the aggregation rate, especially when the electric field was low, due in part to the increase in viscosity. The decrease in migration velocity produced lower collision energies and lower aggregation efficiencies as well.