Optimized droplet digital CFU assay (ddCFU) provides precise quantification of bacteria over a dynamic range of 6 logs and beyond

Abstract

Standard digital assays need a large number of compartments for precise quantification of a sample over a broad dynamic range. We address this issue with an optimized droplet digital approach that uses a drastically reduced number of compartments for quantification. We generate serial logarithmic dilutions of an initial bacterial sample as an array of microliter-sized droplet plugs. In a subsequent step, these droplets are split into libraries of nanoliter droplets and pooled together for incubation and analysis. We show that our technology is at par with traditional dilution plate count for quantification of bacteria, but has the advantage of simplifying the experimental setup and reducing the manual workload. The method also has the potential to reduce the assay time significantly.

“Antibiotic inhibition of bacteria growth in droplets reveals heteroresistance pattern at the single cell level”

“…One bacteria per 2nL droplet is equivalent to 5x10 5 CFU/mL which is the standard for MIC tests 11,13 . In principle, the antibiotic screening in droplets is 'digital MIC assay' 14,15 where outcome of each discrete experiment in droplet is binary: '1-positive' with detectable bacteria growth or '0-negative' without growth. For single cell analysis it is necessary to avoid encapsulating two or more bacteria as much as possible.…”

“…[We calculated initial inoculum density in virtual array with digital counting algorithm and equations 14,22 ] We set a threshold to distinguish between positive and negative droplets based on their fluorescence ( Fig 3D). Next we investigated separately each library in virtual array to calculate their positive droplet fractions ( Fig 3E).…”

Droplet microfluidics for high‐sensitivity and high‐throughput detection and screening of disease biomarkers

“…Droplet platforms have shown to be effective for single-cell growth assays. Researchers have cultured cells as diverse as yeast (Boitard et al, 2012), mammalian cell lines (Clausell-Tormos et al, 2008;Köster et al, 2008), bacterial cells (Boedicker et al, 2008;Kaushik et al, 2017;O. Scheler et al, 2017;Ott Scheler et al, 2016), as well as multicellular organisms like Caenorhabditis elegans (Clausell-Tormos et al, 2008), and zebrafish (Baret, Beck, Billas-Massobrio, Moras, & Griffiths, 2010) in droplets.…”

“…O. Scheler et al (2017) presented a platform that utilizes 1-nL volume droplets in order to confine single bacterial cells (that have been pre-exposed to antibiotics) along with the viability dye C12-resazurin (Figure 8ai). Following 5 hr offchip incubation (Figure 8aii), the droplets were reinjected into a detection device in order to count the number of fluorescent FIGURE 8 Droplet based bacterial growth quantification and antibiotic susceptibility testing (AST).…”

CellProfiler: A fit tool for image analysis in droplet microfluidics

“…In microbiology, droplet microfluidics has opened up several new experimental possibilities 1 . Examples of microfluidics applications include antibiotic susceptibility studies on population and single cell level 2 , investigation of microbial interactions 3 , and quantification of viable bacteria 4 . Due to its higher precision, sensitivity and robust automatic calibration, droplet digital PCR (ddPCR) has become a strong alternative to traditional quantitative PCR (qPCR) in quantifying nucleic acids in both diagnostics and laboratory research 5,6 .…”