Continuous separation of bacterial cells from large debris using a spiral microfluidic device

Abstract

With the global increase in food exchange, rapid identification and enumeration of bacteria has become crucial for protecting consumers from bacterial contamination. Efficient analysis requires the separation of target particles (e.g., bacterial cells) from food and/or sampling matrices to prevent matrix interference with the detection and analysis of target cells. However, studies on the separation of bacteria-sized particles and defined particles, such as bacterial cells, from heterogeneous debris, such as meat swab suspensions, are limited. In this study, we explore the use of passive-based inertial microfluidics to separate bacterial cells from debris, such as fascia, muscle tissues, and cotton fibers, extracted from ground meat and meat swabs-a novel approach demonstrated for the first time. Our objective is to evaluate the recovery efficiency of bacterial cells from large debris obtained from ground meat and meat swab suspensions using a spiral microfluidic device. In this study, we establish the optimal flow rates and Dean number for continuous bacterial cell and debris separation and a methodology to determine the percentage of debris removed from the sample suspension. Our findings demonstrate an average recovery efficiency of $\sim$80% for bacterial cells separated from debris in meat swab suspensions, while the average recovery efficiency from ground beef suspensions was $\sim$70%. Furthermore, approximately 50% of the debris in the ground meat suspension were separated from bacterial cells.

FIG. 1.

An overview of the separation method and device. The device comprises a sheath flow and sample inlet, as well as two outlets. The separation mechanism relies on the utilization of lift forces and Dean drag to facilitate the inertial migration of particles, which satisfy the focusing criterion ( $a_p/\mathrm{H}\ge 0.07$). In a curved microchannel, the generation of a secondary flow gives rise to the formation of two counter-rotating streams known as the Dean vortices. The particles distributed within the curved microchannel experience the combined effects of the net inertial lift force ( $F_L$) and the Dean drag force ( $F_D$), which determines the particle equilibrium position based on particle size. Near the outer wall of the microchannel, both $F_L$ and $F_D$ act in the same direction, resulting in particles following the path of the Dean vortices irrespective of their size. Near the inner wall, the net lift force and Dean drag force exert opposing effects on the particles, leading to a dynamic equilibrium. “Created with BioRender.com.”

FIG. 2.

(a) Fabricated spiral microfluidic device. (b) Imaging experiment showing the tubing connection to the inner and outer inlets and outlets. (c) Ground meat sample suspension containing bacteria and non-bacterial debris. (d) Beef surface used for the preparation of meat swab suspension.

FIG. 3.

Focusing of the (a) 1.84  $\mu$m fluorescent particles to the outer outlet of the channel, (b) 6.04  $\mu$m fluorescent particles to the inner outlet of the channel, and (c) 10.6  $\mu$m fluorescent particles to the inner outlet of the channel for the optimal sample flow rate of 100  $\mu$l/min and sheath flow rate of 400  $\mu$l/min ( $Re=79.4$ and $De=5.7$). (d) The reduction in Dean number ( $De$) has a notable impact on the focusing behavior of the 10.6  $\mu$m fluorescent particles. By adjusting both the sample and sheath flow rates to 50  $\mu$l/min, the Dean number decreases to 0.8. This reduction in $De$ leads to weaker Dean vortices within the microchannel, subsequently diminishing the influence of lift forces acting on the particles.

FIG. 4.

The intensity plots of the focused stream of the 6.04 and 10.6  $\mu$m fluorescent particles taken across the full channel width of 200  $\mu$m. The 6.04  $\mu$m beads focus in an equilibrium position near the inner wall of the channel but closer to the center of the bifurcation outlet, while the equilibrium position of the 10.6  $\mu$m beads is closer to the inner wall compared to the 6.04  $\mu$m beads.

FIG. 5.

Recovery efficiencies at the outer and inner outlets for (a) $\sim {10}^5$ CFU/ml E. coli MG1655, (b) $\sim {10}^5$ CFU/ml S. aureus 6538, and (c) $\sim {10}^3$ CFU/ml S. aureus 6538. Each bar represents the average of triplicate plate count measurements, and the error bar represents the standard deviation of the triplicate plate count.

FIG. 6.

(a) The recovery efficiency of E. coli MG1655 bacteria from ground meat debris spiked with $\sim {10}^5$ CFU/ml. Each bar represents the average of triplicate plate count measurements, and the error bar represents the standard deviation of the triplicate plate count. Size distribution of (b) the input suspension, which is a mixture of both debris and bacteria, (c) debris in the suspension collected at the inner outlet showing particulate sizes above the critical diameter of the channel, and (d) particulates in the suspension collected at the outer outlet of the microchannel. The binarization of the image is presented for better visualization and size measurements. The scale bars are 50  $\mu$m.

FIG. 7.

(a) The recovery efficiency of E. coli MG1655 bacteria from debris in a meat swabbing suspension spiked with $\sim {10}^3$ CFU/ml concentration. Each bar represents the average of triplicate plate count measurements, and the error bar represents the standard deviation of the triplicate plate count. (b) Distribution of particle sizes in the input suspension, which consists of both debris and bacteria. (c) Particulate sizes above the critical channel diameter were observed in the size distribution of debris in the suspension collected at the inner outlet. (d) The size distribution of particulates in the suspension collected at the microchannel’s outer outlet. The scale bars are 100  $\mu$m.