Detection of electroporation in microbial cells: Techniques and procedures

Abstract

Application of an electric field to microbial cells can cause electroporation of their membranes, leading to permeabilization and formation of pores in these structures. Assessment of electroporation in microbial cells, such as fungi and bacteria, could be hindered by the presence of additional structures located externally to the cytoplasmic membrane, such as the cell wall and outer membrane. The most common methods to assess the electroporation of these membranes can be divided between those based in the entrance of external compounds into the bacterial cytoplasm (e.g., a fluorescent probe) and those which detect the leakage of intracellular compounds to the environment (e.g., nucleic acids, proteins or ATP). Moreover, other techniques could be used to understand the mechanism of electroporation in microbial membranes, such as direct visualization of damages in cell membranes by electron and atomic force microscopy, evaluation of membrane damages by a selective medium plating technique, measurement of osmotic response, Fourier transform infrared spectroscopy, and molecular tools (including transcriptomics and site-directed mutagenesis). In addition, several methods are proposed in order to avoid interference of the cell wall and/or outer membrane in the evaluation of cytoplasmic membrane permeabilization: (pre)treatment of cells with cationic agents or chelators and preparation of protoplasts or spheroplasts. The described methods to detect electroporation of bacterial cells have their own advantages and disadvantages. Therefore, analysis of results obtained with different methods is recommended to provide a more accurate knowledge in the mechanism of microbial permeabilization by electroporation.