FAST QUANTIFICATION OF AEROBIC BACTERIA USING DROPLET
MICROFLUIDICS
Ott Scheler1*, Tomasz S. Kaminski1, Artur Ruszczak1, Pawel Debski1 and Piotr Garstecki1*
1
Microfluidics and Complex Fluids Research Group
Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224
Warsaw, Poland
ABSTRACT
This manuscript describes droplet microfluidic technology for fast culturing and detection of aerobic
bacteria using metabolic marker dye dodecylresorufin (C12R).
KEYWORDS: Droplets, Microfluidics, Bacteria, Resorufin
INTRODUCTION
Precise quantification of bacteria is an important part in many different fields of microbiology. For
example to enumerate the number of pathogens in a sample [1-2] or to analyze antibiotic susceptibility
[3]. Traditionally, bacteria have been quantified by the plate count method [4], but this gold standard
approach demands at least an overnight incubation, or even longer in the case of slowly growing bacteria.
Droplet digital microfluidics has been demonstrated as a substantial improvement for detection and
quantification of bacteria [5-7]. There has been a great challenge in finding a suitable and universal
marker dye for bacteria in droplets as the commonly used resorufin dye leaks between the droplets. There
have been suggestions on adjusting the media composition or using only certain types of oils and
surfactants [8-11], but there is still a need for more efficient technology that would allow stable culturing
and credible detection of viable bacteria in droplets.
This paper explores fast culturing and quantification of aerobic bacteria using a marker dye
dodecylresorufin (C12R) that is a derivative of commonly used resorufin, but has been shown to be more
stably maintained in droplets [12].
EXPERIMENTAL
Escherichia coli DH5α with eGFP and AMP resistance genes in plasmid was used as test organism in
this study. LB-Lennox media (Roth Gmbh, Germany) with 100μg/mL AMP and 1mM IPTG was used in
incubation experiments. C12-Resazurin (Life Technologies, USA) and resazurin (Sigma-Aldrich, USA)
were used as fluorescent dye substrates for dodecylresorufin and resorufin, respectively. Microfluidic
chips with different flow-focusing geometries for droplet generation and post-incubation fluorescence
readout were both made from PDMS (Sylgard 184, Dow Corning, USA). Volume of the droplets in
described experiments was 1.1 nL. Fluorescence of droplets was measured using A1R confocal
microscope (Nikon, Japan), with excitation at 488 (eGFP) and 561 nm (C12R and resorufin). NovecTM
HFE 7500 fluorinated oil (3M, USA) was used with 1% PFPE-PEG-PFPE triblock surfactant synthesized
according to protocol by Holtze et.al [13]. Droplets were collected in 250µL tube and incubated @ 37°C.
Data analysis was carried out using MS Office Excel (Microsoft, USA).
978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001
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19th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 25-29, 2015, Gyeongju, KOREA
RESULTS AND DISCUSSION
In our experiments the dye substrate was converted into detectable C12R fluorescent signals in subnanoliter droplets (Fig1a). Fluorescence of both eGFP and metabolic marker was measured in individual
droplets using confocal microscope and microfluidic chip (Fig1b).
Figure 1: Conversion of dye substrate into fluorescent signal. a) confocal microscope image of droplet emulsion, b)
droplet counting using microfluidic reading chip and confocal microscope, c) exemplary trace of relative fluorescence raw data (eGFP and C12R) that includes 32 droplets measured with ~400Hz frequency.
Increased fluorescence of metabolic marker correlates with increased eGFP signal as shown on droplet
signal histogram on Fig 1c and on fluorescence signal plot Fig2a. The increased fluorescence of droplets is
detected faster with conventional resorufin, but C12R signal is more stably maintained in droplets over
longer intervals (Fig2b). More stable maintenance of fluorescent signal allows implementation of our technology for broader range of bacteria including the slow-growing species.
Figure 2: Marker dye fluorescence analysis in droplets. a) eGFP and C12R signal plot of droplet emulsion (each
dot represents one droplet), b) C12R and resorufin signal intensities of empty (colorless) and bacteria containing
droplets (red and purple) at different timepoints.
CONCLUSION
This paper demonstrates fast culturing and quantification of bacteria with C12R marker dye that is
more stably maintained in droplets compared to traditional resorufin often used in the field. C12R has
been used before in droplet microfluidics [12], but not for digital counting of viable bacteria. Resorufinand dodecylresorufin-based assays can basically be adapted for all aerobic organisms and are therefore
suitable for vast number of pathogens and model microorganisms. Future applications of our technology
may include, but is not limited to: droplet digital quantification of bacteria, antibiotic susceptibility tests
on single CFU initiated populations and investigation of antibiotic action mechanisms.
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ACKNOWLEDGEMENTS
Project financed by the ERC Starting Grant 279647.
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CONTACT
* [email protected], [email protected]
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