Multi-Frequency Microchannel Electrical Impedance(m-EIS) Method for the Rapid Detection of Proliferating Microorganisms and their Rapid Quantification

Sachidevi Puttaswamy¹, Byung-Doo Lee², Ashley Jurgensmeyer², Anne Baumstummler³, Kathleen Souza⁴ and Shramik Sengupta²*

¹Department of Bioengineering, University of Missouri,1406, E. Rollins Road, 165 AEB, Columbia MO 65211-5200.
²Departmnet of Biological Engineering, University of Missouri,1406, E. Rollins Road, 165 AEB, Columbia MO 6521-5200.
³Department of Predevelopment – Technology – Collaboration, BioMonitoring, Lab Solutions, MilliporeSigma39 Route Industrielle de la Hardt; 67120 Molsheim, France.
⁴Department of Virology and Microbiological Sciences, MilliporeSigma, 80 Ashby Rd, Bedford, MA 01886.

Abstract

Existing culture-based instruments for detecting/quantifying proliferating bacteria in suspensions (BACTEC^TM, BacT/Alert^TM, RABIT^TM etc.) do so based on changes observedin the physical/chemical properties of media(O₂/CO₂ levels, pH etc.) due to bacterial metabolism. Given the limited metabolic-rate of individual bacterium, they have a “threshold-concentration” of ~10⁷-10⁸CFU/ml, and Times to Detection (TTDs) of 12 hours or longer for low initial loads (<100CFU/ml). We recently developed a method that tracks microbial proliferation in suspensions by monitoring the degree of cell polarization of live microorganisms. In the presence of an AC electric field, there occurs a build-up of charge at the microbial membrane, causing them to act like capacitors. As microorganisms multiply, there occurs a corresponding increase in charges stored in the suspension (“bulk-capacitance”), and this increase in bulk-capacitance serves as our “signature” for presence of live microorganisms. In this study, we explain the theory underlying our approach, establish its applicability to a variety of microorganisms,showing that the “Threshold-Concentration” (n_T) for detection is ~10³-10⁴CFU/ml, and TTDs are a function of the initial-load(n₀) and doubling-time(t_D) of the microorganism TTD=1.443*t_D*ln(n_T/n₀) and show that the method can be adapted to obtain the “Most Probable Number” (MPN) of coliforms within 6hrs(vs. >24hrs for existing methods).

Keywords: Viable bacteria; MPN; Rapid Detection; Automated Culture Systems; Bacteria detection; Microfluidics.