High efficiency separation of microbial aggregates using capillary electrophoresis

Abstract

Recent advances in the technique of capillary electrophoresis have demonstrated fast, highly efficient separation of mixtures of intact microbes. This paper describes the application of this technique for the separation of microbial aggregates of Micrococcus luteus, Saccharomyces cerevisiae, or Alcaligenes faecalis. The aggregates of these microbes were resolved into several highly efficient peaks with analysis times under 10 min and efficiencies approaching 1000000 plates m(-1) in some cases. A reproducible relationship was found between the electrophoretic mobility and the aggregation number or size of the cluster under a given set of experimental conditions. Often, cellular aggregation was reversible with brief immersion in an ultrasound bath. This reversibility was confirmed by visual microscopy and electrophoretic data.

Determination of pathogenic bacteria by CZE with surface‐modified capillaries

“…Finally, the amplified product was detected by agarose gel electrophoresis [26]. Armstrong and coworkers [27][28][29] utilized two electromigration techniques for determination of different species of bacteria using a very dilute polymer additive [poly(ethylene) oxide] (PEO) to the running buffer. They used CZE for identification of bacterial pathogens, responsible for urinary tract infection (UTI) [30] and for determination of the active bacterial ingredients (Bifidobacterium, Lactobacillus) in tablets (pills) and powder-based commercial products [31].…”

Electrokinetic Detection and Characterization of Intact Microorganisms

“…12,13 On the other hand, bacteria-wall interactions can be reduced or eliminated by dynamic or chemical modification of the active sites on the capillary surface. [6][7][8][9][10][11][12][13][14][15][16] In the case of chemical modification, the modifier is introduced onto the capillary wall by the formation of covalent bonds; for example, a reactive bifunctional silane precursor such as γ-methacryloxypropyltrimethoxysilane can be used. The silane groups may be attached and polymerized by monomers, forming the outer layer on the capillary surface.…”

“…13 Armstrong et al published a series of papers on the mechanistic aspects of the separation process, separating bacterial aggregates, on-line monitoring of bacterial migration, identifying the causative pathogens of urinary tract infections, determining bacterial viability, quantitation of bacteria, and determining live bacterial cells in consumer products. 6,[8][9][10][11]19 The researchers used primarily two electromigration techniques for the separation of several species of microorganisms. The first approach involved CZE of bacteria with a dissolved poly(ethylene oxide) (PEO) solution and UV absorbance detection at λ ) 214 nm.…”

“…19 Most research groups have used a method that provided separations of bacterial cells with very short analysis times and extremely sharp peaks. [6][7][8][9][10][11][12][13][14][15][16][20][21][22] These "apparent efficiencies" were attributed to a very small addition of modifying agents to the running buffer. Most frequently, a very dilute (0.0125-0.025 w/w) solution of PEO was used to decrease the EOF, prevent adsorption of bacteria to the fused-silica surface, and promote consolidation of the cells into a narrow band.…”

Detection of microbial food contaminants and their products by capillary electromigration techniques

“…In a separate report, Armstrong´s group [36] carried out the separation and characterization of cell aggregates using the aforementioned polymer-based BGE in order to gain some insights about the tendency to self-aggregate that microorganims exhibit in aqueous solutions. They demonstrated that, previous sonication treatment of the sample to increase cell dispersion, the separation method was able to distinguish between Micrococcus luteus dispersed cells from cell clusters of the same strain that exhibited a different aggregational degree.…”