Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online

Abstract

In microtiter plates, conventional online monitoring of biomass concentration based on optical measurements is limited to transparent media: It also cannot differentiate between dead or viable biomass or suspended particles. To address this limitation, this study introduces and validates a new online monitoring setup based on impedance spectroscopy for detecting only viable biomass in 48- and 96-well microtiter plates. The setup was first validated electronically and characterized by determining the cell constants of the measuring geometry. Defined cell suspensions of Ustilago maydis, Hansenula polymorpha, Escherichia coli and Bacillus licheniformis were characterized to find, among other parameters, the most suitable frequency range and the characteristic frequency of β-dispersion for each organism. Finally, the setup was exemplarily applied to monitor the growth of Hansenula polymorpha online. As reference, three different parallel cultures were performed in established cultivation systems. This new online monitoring setup based on impedance spectroscopy is robust and enables precise measurements of microbial biomass concentration. It is promising for future high-throughput applications.

Progress in methods for the detection of viable Escherichia coli

“…To address the issue that optical measurement-based bioassay methods cannot differentiate between dead and viable biomass, Luchterhand et al developed an online monitoring device that utilized impedance spectroscopy to exclusively detect viable biomass in microtiter plates. 295 The ability to precisely measure microbial biomass concentration using the appropriate frequency range and the characteristic frequency of β-dispersion of organisms, such as E. coli , was envisaged for high-throughput detection.…”

Online 2D Fluorescence Monitoring in Microtiter Plates Allows Prediction of Cultivation Parameters and Considerable Reduction in Sampling Efforts for Parallel Cultivations of Hansenula polymorpha

“…To follow the Process Analytical Technology (PAT) guidance for process analytics and control [ 15 ], these microscale cultivation vessels are often equipped with a miniaturised periphery [ 16 ], enabling at-line and online process monitoring down to droplet-size cultivation volumes [ 17 , 18 ]. Amongst others, spectroscopic measurement methods based on Raman [ 19 , 20 ], impedance [ 21 , 22 , 23 ] or single-wavelength fluorescence [ 24 , 25 ] have successfully been transferred from lab-scale reactors to microscale cultivation systems.…”

Integrating impedance-based growth-rate monitoring into a microfluidic cell culture platform for live-cell microscopy

“…Growth rate measurements in cell cultures using electrical cell-substrate impedance sensing (ECIS) were demonstrated for adherent cells 27,28 . Impedancebased measurements of viable biomass in microtiter plates were also performed for non-adherent cells 29,30 . However, to the best of our knowledge, there is currently no integrated platform, which features continuous and parallel execution of (i) cell culturing under controlled perfusion conditions, (ii) monitoring of the cell growth rate, and (iii) high-resolution imaging of non-adherent or suspended cells in the same single device.…”