Download W.003a SINGLE-BACTERIA CONFOCAL SPECTROSCOPY

Single-bacteria confocal spectroscopy: an ultrasensitive method for real-time
monitoring of bacterial growth
Dong Jin Shin1, Liben Chen2 and Tza-Huei Wang123*
1
Department of Biomedical Engineering, 2Department of Mechanical Engineering, 3Institute of
NanoBioTechnology, Johns Hopkins University, U.S.A.
ABSTRACT
Bacterial growth measurement is routinely performed in both research and clinical microbiology
laboratories. However, current techniques based on bulk growth utilize detection methods with limited
sensitivity, necessitating long incubation times in order to achieve a detectable bacterial concentration.
Single molecule spectroscopy provides a novel and highly sensitive approach to monitoring bacteria
growth. By coupling continuous single-bacteria detection with in-line incubation and growth, we were
able to monitor the growth of bacteria during a 30-minute incubation period, which is substantially
shorter than traditional approaches. This work demonstrates the potential utility of confocal fluorescence
optics for performing rapid bacterial growth assays.
KEYWORDS: Single-molecule spectroscopy, bacterial counting, fluorescence spectroscopy
INTRODUCTION
Bacterial growth monitoring in clinical and research settings are both limited by the sensitivity of the
detection method used. Traditional method using optical density (OD) for measuring growth based on
turbidity is rapid, but has poor sensitivity [1]. Enumeration assays using latest flow cytometers provide
high analytical sensitivity, although this method requires toxic dyes incompatible with live growth
monitoring [2]. On the other hand, culture-based methods such as agar dilutions serve as the standard of
bacterial quantification via direct counting of colony-forming units (CFU), although this method is time
consuming in nature due to incubation time on the scale of days required for conclusive result [3].
Importantly, none of these methods have provisions for monitoring bacterial growth in real-time. In this
work, we propose a method of monitoring bacterial growth in real-time using a combination of confocal
fluorescence spectroscopy and microfluidics (Fig.1A).
THEORY
Bacterial growth is typically monitored in bulk via absorbance. In our work, we utilized a live-cell
fluorescence stain in order to monitor the presence of individual bacterium in growth condition. The confocal fluorescence detection instrument employed in this work (Fig. 1B) is designed such that the observation volume spans the entire cross-section of the microfluidic flow cell (Fig. 1C). As the sample solution is incubated, it is simultaneously flown through a microfluidic flow cell and interrogated with
excitation source. As bacteria undergo binary fission, labeling dye is diluted across daughter cells until
fluorescence subsides below background (Fig. 1D). Growth is thus monitored in real time as a sustained
decrease in number of detectable particles in solution as a function of time (Fig. 1E).
EXPERIMENTAL
A custom-built confocal fluorescence optics utilizing a cylindrical lens is used to shape the
observation volume such that all bacteria flowing through flow cell could be detected [4]. Optical setup
was first characterized and validated using flow cytometer calibration beads. In order to ensure adequate
detection of single bacterium at high flow rates necessary for real-time monitoring of growth, bacterial
labeling condition was subsequently optimized (Fig.2A, 2B). Analytical sensitivity was first
characterized using three methods: 1) OD measurement using NanoDrop 1000 (Thermo Fisher, USA); 2)
Bulk fluorescence monitoring using NanoDrop 3300 (Thermo Fisher, USA); 3) Single-bacteria detection
using the proposed setup. Reference strain of E.coli (ATCC 25922) was labeled in 25μM CellTrace CFSE
978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001
340
19th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 25-29, 2015, Gyeongju, KOREA
dye (Life Technologies, USA), rinsed in 1×PBS and resuspended in Mueller-Hinton broth (BD Biosciences,
USA) prior to each experiment. For growth characterization in confocal fluorescence platform, labeled
E.coli samples were incubated in Muller-Hinten broth for 0, 0.5, 1 and 2 hours. Afterwards, they were
pelleted, rinsed twice in 1x PBS and resuspended in 1xPBS for measurement.
A
B
C
D
E
Figure 1: Overview of real-time bacterial growth assay using single bacteria counting. A) Flow control
and incubation setup. B) Optical setup utilizes a combination of beam expansion and cylindrical lens to
shape the illumination region, enabling low-background and high intensity illumination necessary for
bacteria detection. C) Layout of microfluidic flow cell. Constriction has a cross-section of 5μm×5μm. D)
As bacteria divides (E.coli divides every 20 minutes), labeling dye is also diluted. Over time, labeling dye
is diluted to such an extent that signal is no longer above threshold. E) As a result, the rate of bacterial
growth can be measured as a sustained decrease in number of fully-labeled bacteria in solution.
RESULTS AND DISCUSSION
Analytical sensitivity of the proposed method was compared with existing methods of bacterial detection. Using the standard OD measurement, signal could not be distinguished above background at concentrations below 107 CFU/mL. Bulk fluorescence labeling enabled greater sensitivity with fluorescence signal
showing linear correlation with bacteria concentration down to 105 CFU/mL. Using the proposed method,
bacteria could be monitored at a concentration of 102 CFU/mL with excellent specificity during a 10-minute
acquisition period, surpassing the sensitivity of the standard method of detection by a factor of 105 (Fig. 2).
A
C
D
B
E
Figure 2: Labeling optimization for E.coli as a function of A) incubation time and B) dye concentration.
C) Comparison of analytical sensitivity across 3 detection methods. Optical density (OD) and
fluorescence labeling (FL) yield sensitivity of 107 and 105 CFU/mL respectively. D) The proposed
method yields 102 CFU/mL with a 10-minute measurement time (flow rate 1μL/min). E) Sample trace
obtained from the proposed method at 104 CFU/mL.
341
To monitor the change in fluorescence intensity of bacteria as a function of growth, we first characterized the fluorescence obtained from labeled bacteria population in 1xPBS for detection in low background.
Characterization at various incubation times indicated that bacterial growth was accompanied by a population inversion, indicating the dilution of labeled bacterium due to multiple rounds of binary fission (Fig.
3A). It is of note that extended incubation in growth medium also resulted in a gradual rise in background
fluorescence, suggesting release of fluorescent dye from the interior of bacteria over time (data not shown).
Subsequently, we measured labeled E.coli under growth condition and non-growth condition over 30
minutes. Growth was indicated by a negative exponential decrease in detectable fluorescent bacteria count
(Fig. 3B). As the Hinton-Mueller broth retained a moderate level of background fluorescence, binary fission
resulted in bacteria fluorescence shifting below the background threshold. Applying a threshold above the
background resulted in a gradual loss of peak count as labeled bacteria underwent binary fission.
A
B
Fig. 3: A) Histogram of fluorescence signal obtained from individual bacteria following the specified
duration of incubation in growth condition. Growth is demonstrated as an inversion of the relative
abundance of high-fluorescence peaks to low-fluorescence peaks, indicative of dilution of live-cell
fluorescence stain via binary fission. B) Histogram of peak count as a function of time, monitored under
growth condition (37˚C incubation in Hinton-Mueller broth, green) and no-growth condition (room
temperature, brown).
CONCLUSION
This work demonstrates the concept of in-line bacterial incubation and monitoring for growth measurement with differences that could be visualized in substantially shorter period than typical bacterial
growth assays. The 105-fold advantage in sensitivity of the proposed method over bulk measurement enabled measurement of bacterial growth over the course of 30 minutes. Considering a typical workflow in
clinical microbiology which requires incubation times on the order of days, our method demonstrates an
approach which may drastically shorten the turnaround time of conventional bacterial growth assays.
ACKNOWLEDGEMENTS
We would like to acknowledge the support from National Science Foundation (1033744) and National Institutes of Health (R01AI117032).
REFERENCES
[1] Martens-Habena et al., Appl Environ Microbiol. 72(1): 87-95 (2006).
[2] Schmidt et al, Transfus Med. 16(5) 355-61 (2006).
[3] Espy et al., Clin Microbiol Rev. 19(1): 165-256 (2006).
[4] Liu et al, Biophys J. 95(6): 2964-75 (2008).
CONTACT
* Jeff Tza-Huei Wang, Professor; phone: +1-410-516-7086; [email protected]
342