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249. 


Detecting Microorganism Growth with Microfluidic Resonance Detector



Dan Ye

Master Thesis, TU Dresden, October 2012

Quantitatively analyzing the (population) growth of a microorganism is a crucial task in characterizing fast-evolving microbes and in studying the effect of antibiotic agents. Conventionally, the growth of a microbial batch culture is followed by direct or indirect cell enumerating measurements on manually extracted culture aliquots at different times of incubation. Although routinely practiced, the conventional methods have their respective shortcomings such as the labor-intensive nature of direct counting and high intensity saturation for optical methods. A problem common to all current methods is the lack of automated sampling and the disintegration between measurements and incubation which leads to disruption of growth, contamination and low throughput. In this project, we have introduced a novel microfluidic resonance detector (MRD) consisting of a newly developed resonance detector integrated to a droplet-based microfluidic platform. The resonance detector is able to detect fluids flowing through the core of an inductive coil, allowing highly selective, fast, contact- and label-free detection without screening problem. The microfluidic platform reduced the reagent volume needed by encapsulating microbial culture into droplets and allowed automatic sampling, thus realizing seamless measurement incubation integration. The performance of this novel detection platform in quantitative microorganism growth analysis was investigated with Escherichia coli strain MC4100-YFP batch cultures in Luria Bertani (LB) broth and M9 minimal medium and compared to growth curves obtained by conventional direct cell counting, dry mass and turbidity (OD600) measurements. While automatic sampling, measurement incubation integration, high throughput and sample volume reduction were indeed realized, it was found that the MRD signal ratios of a bacterial culture were very subtle. Instead of relating directly to cell concentrations, the signal ratio was found to be contributed mainly by the medium, the composition of which changed along the course of growth. LB broth culture showed a stronger MRD response than M9 medium culture. Increase of alkaline pH in LB broth culture and decrease in ammonium concentration in M9 medium culture contributed to an increase of signal in the exponential phases. The increased cell concentration might lead to a decrease in the stationary phase of both cultures, while the increasing ammonium concentration in LB broth culture contributed to a stronger decrease. Examining the MRD responses of individual changing factors in the bacterial culture (cell concentration, glucose concentration, pH, highly dissociable salt concentration, acetate and ammonium concentrations) has led to the proposition that the MRD detects the power loss caused by eddy current formation in non-magnetic fluids, a quantity that is proportional to the fluid conductivity and thus the ionic strength. The subtlety of the MRD responses of bacterial cultures follows from the fact that its overall ionic strength remained roughly the same throughout the growth. Although bacterial cultures responded to the MRD weakly and it was difficult to draw a straightforward relation between the MRD profile and the classical growth curve, the MRD would find itself extremely useful in monitoring a wide range of other biochemical reactions and processes in which ionic strength is a crucial factor.



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