Fig 1.
Workflow diagram highlighting inputs, output, sample preparation, and fluorescence procedure.
A previously published diagram is utilized paired with real-time, high-speed photography of droplet formation (4) and collection at output (5).
Table 1.
Generation timea and lag phase durationb of the tested bacteria in different culture broth medium (TSB, BPW and RV broth) at 37°C or 41.5°C (RV broth only), with an initial bacterial concentration of 103 CFU/ml medium.
Fig 2.
Medium and FITC-Ab were encapsulated in 50–70 μm diameter droplets (1x medium and 0.83 μg/ml FITC-Ab in-droplet) to visually assess medium contribution to fluorescence.
Fluorescence exposure and look-up-table (LUT) settings are standardized across all four media evaluated. The LUT values can be found in the upper left-hand corner of each image. Media under test included phosphate buffered saline (a), Rappaport-Vassiliadis (b), buffered peptone water (c), and tryptic soy broth (d).
Table 2.
Fluorescence and pH of medium commonly used for traditional culture of S. Typhimurium.
Fig 3.
Fluorescence of bacteria relative to background fluorescence in-droplet of six bacterial species encapsulated in droplet with 0.83 μg/ml FITC-Ab per droplet in phosphate buffered saline.
Five fluorescence measurement replicates were performed per bacterial species/strain. Error bars represent the 95% confidence interval.
Fig 4.
Bacterial species (a: S. Typhimurium; b: E. aerogens; c: C. freundii) with 0.83 μg/ml FITC-Ab in phosphate buffered saline.) each droplet is approximately 50–70 μm in diameter (scale on image is 50 μm). Bright field images are presented complimentary to FITC images and visually show bacterial concentration at an ideal focal plane (5 μm interval stacking and merging of images into two dimensional images is not optimal for bright field images). FITC images are merged 5 μm interval focal plane images and represent a two-dimensional image of the entire droplet.
Fig 5.
Fluorescent images of S. Typhimurium incubation in-droplet with sterile deionized water, and 10 μg/ml FITC-Ab in 1x RV broth at 37°C.
Fig 6.
Bright field images of S. Typhimurium incubation in-droplet with sterile deionized water, and 10 μg/ml FITC-Ab in 1x RV broth at 37°C.
Fig 7.
Relative fluorescence of encapsulated S. Typhimurium and E. aerogens in shredded lettuce wash water (0.5x in-droplet) and incubated in Rappaport-Vassiliadis broth (1x in-droplet) for 5 h with a FITC-Ab concentration of 10 μg/ml in-droplet.
A potential detection threshold region is identified on the figure. No measurable relative fluorescence was identified in time points 0 through 2 for both E. aerogens. Five replicates were measured for each bacterial species at each time point.
Fig 8.
Fluorescent images of S. Typhimurium incubation in-droplet with 0.5x shredded lettuce wash water, and 10 μg/ml FITC-Ab in 1x RV broth at 37°C.
Fig 9.
Bright field images of S. Typhimurium incubation in-droplet with 0.5x shredded lettuce wash water, and 10 μg/ml FITC-Ab in 1x RV broth at 37°C.
Table 3.
Comparison of lag phase duration and generation times for S. Typhimurium and E. aerogens outside and inside droplet.
Table 4.
Bacterial strains utilized in this study.