Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry

Abstract

Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria.

Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

“…IFC has been successfully used for cellular characterisation in bacterial and mammalian systems [6,8,9]. Analysing plant cells and in particular the quality of pollen in an efficient and reliable manner is not only of interest of plant breeding and seed production industries, but plays also a role in scientific projects investigating pollen development [30,50], pollen-related heat or cold tolerance [22,23], male-sterility systems [51], the establishment of pollen storage or doubled haploid protocols [13,14].…”

“…The analysis of electric properties in single cells by impedance flow cytometry (IFC) has been described in microorganisms to estimate viability, membrane potential, as well as cell size [6,7], in mammalian cells to characterize culture conditions and apoptosis [8] and to screen for methods to kill breast cancer cells [9]. The IFC system is based on the Coulter system [10] but uses a microfluidic chip which permits measurements in the radio frequency range from 0.1 to 30 MHz with alternating current (AC).…”

Viability and germinability in long term storage of Corylus avellana pollen

“…New applications of microfluidic impedancebased flow cytometry (Cheung et al, 2010;David et al, 2012) are highly promising for the evaluation of pollen viability and have to be considered also for direct applications in agriculture.…”

Single-cell printing based on impedance detection

“…In flow-through impedance spectroscopy, it is common practice to interpret measurements at low frequency AC (e.g., 500 kHz) as a measure of the cell's volume, 18 i.e., jUj pp / cell volume and therefore ffiffiffiffiffiffiffiffiffiffi jUj pp 3 q / cell diameter. Thus, sorting according to cell size becomes feasible.…”