Capillary electrophoresis of microbial aggregates

Abstract

Uncontrolled aggregation of bacterial cells is a significant disadvantage of electrophoretic separations. Various aspects of the electrophoretic behavior of different strains of Gram-positive Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus(1), and Micrococcus luteus bacteria and Gram-negative Escherichia coli bacteria were investigated in this study. Our findings indicate that bacteria can be rapidly analyzed by CZE with surface charge modification by calcium ions (Ca(2+)). Bound Ca(2+) ions increase zeta potential to more than 2.0 mV and significantly reduce repulsive forces. Under the above conditions, bacterial cells create compact aggregates, and fewer high-intensity signals are observed in electropherograms. The above can be attributed to the bridging effect of Ca(2+) between bacterial cells. CE was performed to analyze bacterial aggregates in an isotachophoretic mode. A single peak was observed in the electropherogram.

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

“…The barrier of electrostatic repulsion between the surface and the cells should be the lowest in this case (Rouissi et al 2011;Dziubakiewicz and Buszewski 2014). However, the results presented in Figs.…”

Evaluation of Intact Cell Matrix-Assisted Laser Desorption/Ionization Timeof- Flight Mass Spectrometry for Capillary Electrophoresis Detection of Controlled Bacterial Clumping

“…Bacterial cells were rinsed with water twice, then suspended in 0.005M Ca(NO 3 ) 2 solution in order to modify the surface charges, and after 1 h the pellet with bacterial cells was washed again to remove free Ca 2+ aq ions [19]. The final bacterial sample was suspended in an outlet buffer.…”

“…The electrophoretic analysis was performed in a nonlinear system, namely: outlet buffer: TB (C TRIS =4.5 mM, C B =50 mM, pH 8.3), inlet buffer: TB-HCl (C TRIS =4.5 mM, C B =50 mM, C HCl =4.4 pH 7.15), I max =100 μA, U=15 kV, t=23°C, λ=214 nm, and injection in the pressure mode at 50 mbar for 25 s. Between runs, the capillaries were washed with 1.0M NaOH, deionized water -for 2 min each, and a running buffer for 4 min. A total volume of 0.5 mL stock bacterial suspension was used for electrophoretic measurements [19]. The focused fractions of bacterial clumping were collected in CE-MS mode, diluted and transferred to a MALDI target according to the above-mentioned procedure.…”

“…Modification of bacteria with calcium ions determines the change in their electrophoretic mobility and reduces repulsive forces. This results in creation of controlled clumping and signal amplification [19]. Application of buffers with different ions mobilities (an isotachophoretic mode) and without bacterial surface modification with Ca 2+ allowed focusing the zones [19].…”

“…This results in creation of controlled clumping and signal amplification [19]. Application of buffers with different ions mobilities (an isotachophoretic mode) and without bacterial surface modification with Ca 2+ allowed focusing the zones [19]. Therefore, it was possible to collect focused microbial cells and to detect their IC MALDI spectra [34].…”