An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study

Abstract

Significance of single cell measurements stems from the substantial temporal fluctuations and cell-cell variability possessed by individual cells. A major difficulty in monitoring surface non-adherent cells such as bacteria and yeast is that these cells tend to aggregate into clumps during growth, obstructing the tracking or identification of single-cells over long time periods. Here, we developed a microfluidic platform for long term single-cell tracking and cultivation with continuous media refreshing and dynamic chemical perturbation capability. The design highlights a simple device-assembly process between PDMS microchannel and agar membrane through conformal contact, and can be easily adapted by microbiologists for their routine laboratory use. The device confines cell growth in monolayer between an agar membrane and a glass surface. Efficient nutrient diffusion through the membrane and reliable temperature maintenance provide optimal growth condition for the cells, which exhibited fast exponential growth and constant distribution of cell sizes. More than 24 h of single-cell tracking was demonstrated on a transcription-metabolism integrated synthetic biological model, the gene-metabolic oscillator. Single cell morphology study under alcohol toxicity allowed us to discover and characterize cell filamentation exhibited by different E. coli isobutanol tolerant strains. We believe this novel device will bring new capabilities to quantitative microbiology, providing a versatile platform for single cell dynamic studies.

Gradient Microfluidics Enables Rapid Bacterial Growth Inhibition Testing

“…15 Therefore this can be used to analyze the growth rates of the bacteria within the colony via the following equation.where S 0 and S are the bacterial colony areas at the initial ( t = 0) time and at time t . For on-chip measurements of bacterial growth, estimations for a given concentration were made using at least 10 randomly selected colonies.…”

Super-Resolution Imaging of Bacteria in a Microfluidics Device

“…The acquisition of a smSRM image: (1) is intrinsically slow (typically 1–40 min), as it requires a density of localization low enough so that a single emitter is activated at any given time in any diffraction-limited area; (2) necessitates a means for correcting sample drift with nanometer precision, which typically involves the use of fiducial marks; (3) needs a high signal-to-noise ratio of detection and low background to achieve high localization precision [9]. Up until now, most fluorescence microscopy experiments of bacterial processes used thin agarose pads to provide a substratum that supports growth or surface motility [10], [11], [12], [13], [14], PDMS-based microfluidics chambers to physically immobilize cells [15], [16], [17], or hybrid microfluidics devices in which thin agarose pads are combined with a custom flow chamber [18], [19], [20], [21]. To date, several super-resolution studies in bacteria have used these methodologies [22], [23], [24], [25], [26] despite some important limitations, such as: (1) poor long-term stability due to drift of the agarose pad produced by desiccation and local melting or by movement of cells in micro-fluidics chambers (due in part to flow); (2) increased background levels; (3) lack of surface flatness; (4) and fluorescence signal bleed-through between channels in multi-color experiments.…”

New Technologies for Rapid Bacterial Identification and Antibiotic Resistance Profiling

“…Recent advances in single-cell monitoring [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] may provide new solutions that can speed bacterial analysis by monitoring changes in growth on a cell-bycell level. Single-cell microbioreactors 30 and microscale incubators 27 provide a means to culture bacteria in very small volumes, and new types of single-cell analysis methods 24,25,34 allow rapid readout of bacterial viability.…”

“…An electrochemical system focused on the analysis of ribosomal RNAs has been shown to be effective for the analysis of urinary pathogens, 36,41,52,67,68,72,75,76,86 and the amenability of electrochemical sensors to incorporation in highly multiplexed arrays makes this approach a good fit with the need for looking a large panels of markers to survey resistance genes comprehensively. Another electrochemical system for bacterial analysis relying on large surface area nanostructured microelectrodes 58,64,71 has been deployed to detect very low levels of bacteria by targeting messenger RNA (mRNA) sequences and other biomarkers 56,60,61,65,66,73,74 and can also be multiplexed to analyze large analyte panels.…”