Portable bacterial identification system based on elastic light scatter patterns
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Conventional diagnosis and identification of bacteria requires shipment of samples to a laboratory for genetic and biochemical analysis. This process can take days and imposes significant delay to action in situations where timely intervention can save lives and reduce associated costs. To enable faster response to an outbreak, a low-cost, small-footprint, portable microbial-identification instrument using forward scatterometry has been developed.
This device, weighing 9 lb and measuring 12 × 6 × 10.5 in., utilizes elastic light scatter (ELS) patterns to accurately capture bacterial colony characteristics and delivers the classification results via wireless access. The overall system consists of two CCD cameras, one rotational and one translational stage, and a 635-nm laser diode. Various software algorithms such as Hough transform, 2-D geometric moments, and the traveling salesman problem (TSP) have been implemented to provide colony count and circularity, centering process, and minimized travel time among colonies.
Experiments were conducted with four bacteria genera using pure and mixed plate and as proof of principle a field test was conducted in four different locations where the average classification rate ranged between 95 and 100%.
KeywordsPortable bacterial identification Forward scattering Colony count Automation
Microbial identification is essential in various bioscience-related areas such as biosecurity, food safety, and monitoring and prevention of nosocomial infections. In general, three steps are needed to deliver correct identification of a species: sample acquisition/preparation, microbe detection, and microbe identification. Throughout the years, most effort in instrument development using optical technology has been focused on the development of a colony counter, which is essentially a detection device; further testing is required for identification. To identify and classify bacterial colonies, morphological methods, to observe characteristics of the bacterial colony via visual inspection, are widely studied and applied [1, 2, 3]. Previous studies employed a number of image-processing tools [4, 5, 6, 7, 8] to realize the optical colony counting process, including distance transforms, adaptive thresholds, modified Hough transforms, etc. In recent years, prototype systems and software for simple and rapid automatic counting of bacterial colonies have also been developed. Liu et al.  adapted an imaging system originally developed for ELISPOT assays to count colonies automatically and used their system to measure serum bactericidal activity (SBA) in human sera. Later, Putman et al.  showed that automated colony counters can process images of plated bacterial colonies obtained with digital cameras or document scanners, so that small laboratories without a colony counter can send an image of a plate via the Internet and receive an accurate colony count from a remote laboratory that own a dedicated counting software. Recently, Clarke et al.  presented a counting system equipped with a software called NICE (NIST’s Integrated Colony Enumerator), which combines extended minima and threshold algorithms to locate colonies and distinguishes touching colonies. Chen and Zhang  proposed an automatic bacterial colony counter that can detect dish/plate regions, identify colonies, separate aggregated colonies, and report colony counts. In their paper, they used a typical morphology-based system, a one-class support vector machine (SVM) with radial basis function (RBF) as the colony classifier. However, in the proposed instrument, the incident laser beam is capable of extracting the microscopic morphological differences between bacterial colonies that are visually indistinguishable.
Most previous research focused only on counting the bacterial colony and consequently required further steps to identify the actual bacterial species. Our proposed instrument not only counts the colonies but also captures forward-scatter patterns to identify them; this is the crucial advantage over those systems already mentioned. The reported system embodies the design concept which employs Elastic Light Scatter (ESL) patterns for bacterial identification. Early work by Guo  and Banada et al.  inspired the development of a forward scatterometer–based detection and identification technology. The biophysical understanding of the elastic light scattering was reported by treating the individual bacterial colonies as biological spatial light modulators [15, 16, 17]. Briefly, the incoming wavefront of the light source interacts with the microscopic features of the bacterial colony and propagates to the imaging plane to provide a unique scatter pattern. No labeling agent is required to discriminate among different types of bacteria. The first reported forward scatterometer system required manual positioning of the incident beam and motion control to record the forward-scattering signatures, which is tedious, laborious, and inefficient. To increase the efficiency of the data acquisition process, Bae et al.  further developed a new version of the instruments that was fully automated.
In this report, we describe a portable forward scatterometer that is lighter in weight, is smaller in footprint, and provides wireless data transmission capability. These characteristics make the portable forward scatterometer a perfect instrument for field-deployable microbial identification as suggested by Robinson et al. . To simulate this situation, a field test was conducted where ELS patterns captured remotely were electronically transmitted for classification and identification. Compared to the previous instrument, this system has a number of improvements. First, the instrument is designed with rotation-linear movements rather than two linear movements, resulting in lighter weight and smaller instrument footprint. Second, several image-processing schemes have been added or improved (Hough transforms and R-θ centering algorithm). Third, wireless communication capability enabling access to the scatter-pattern database was incorporated to enable detection and identification from remote locations.
Results and discussions
The forward scatterometer consists of a circular-beam laser module (Coherent Inc, Santa Clara, CA, USA), a 45 mirror, and a monochrome camera that is positioned just below the stage. The laser module has a wavelength of 635 nm and an output power of 0.95 mW. The camera used in the scatter module is the same as the colony imaging camera except for an added neutral-density filter (OD 2.5, Edmund Optics, Barrington, NJ) to prevent saturation of the CCD detector. The captured scatter patterns are imported to the colony-centering software, which performs automatic fine adjustment of the culture dish position to obtain circularly symmetric scatter signatures.
Motion control includes two stepper motors (PK243-01AA, Oriental Motor U.S.A. Corp, Torrance, CA, USA) driven by stepper-motor driver (MMD-17, Advanced Micro Systems, Inc, Essex Junction, VT, USA); the drivers are controlled by a four-axis motion-control chip (UMX-26, Pro-Dex Inc, Beaverton, OR), which produces a half-duty cycle square-wave step pulse to control the motors under various operation speeds. Two motors control the movement of the stage in the R direction and rotation of the petri dish in the θ direction, respectively. Both stepper motors have a step angle of 1.8°. For rotation, the motor is attached to a gear with 24 teeth and drives a larger gear below the petri dish having 192 teeth, providing a resolution of 0.225° per step. Equivalently, for a petri dish with a diameter of 88 mm, the linear resolution is about 180 microns at the edge of the dish. For linear movement of the stage, the motor is connected to a lead screw having a pitch of 0.25 in. per revolution, resulting in a resolution of 0.00125 in., or 31.75 μm per step. The UMX-26 motion controller is also connected to a PC via a USB 2.0 interface, and is controlled by passing ASCII command strings to the chip. The controller provides a range of velocities from 0 to 1.044 × 106 step pulses per second and a range of accelerations from 0 to 8 × 106 pulses per second, where 400 pulses constitute a single revolution. A universal power supply (Smart-UPS 1400, American Power Conversion, W. Kingston, RI, USA) with 1400 VA capacity was used as a system power source when a field test was performed.
As shown in Figure 2, a custom-built package was developed for automatic detection of scatter signatures. The software operates in of three phases: stage initialization, colony locating, and colony centering.
1) Stage initialization In the first phase, the stage is centered with respect to the imaging camera for acquisition of the whole petri dish image. Since the radius of the dish is fixed, it is possible to pick the circular outline of the dish rim in the image as a reference object. Application of the Hough transform [20, 21, 22] enables calculation of the distance between the center of the image and the reference outline.
where neven is the number of even codes in the chain-code  representation of the colony edge and nodd is the number of odd codes in the chain code. After locating the colony and obtaining the morphometric descriptors, the system performs a colony selection process, as colonies with diameters smaller than the predefined values or with low roundness values are excluded from the further scanning. Non-circular colonies are generally formed by the intersection of two or more separate colonies.
Elastic light scattering
ELS is defined as an optical measurement technique that utilizes the characteristics of the spatial distribution of the scattered light. ELS signal strength is very high compared to other spectroscopic and inelastic scattering techniques. By analyzing the ELS signal, it is possible to solve an inverse-scattering problem without any specific labeling reagent such as nucleic acid (DNA or RNA) or antibody probes, fluorophore molecules, or enzymes. The explanation of the ELS pattern differentiation originates from considering the bacterial colony as an optical amplitude/phase modulator [16, 17]. When the photons from the laser interact with the micro structures of the colonies, the 2-D spatial distribution of the incoming wavefront is modified depending on the morphological differences of the individual colony. Therefore, upon leaving the far side of the colonies, the departing wavefront is now encoded with different amplitude and phase modulation in 2-D space which can be theoretically modeled via the Huygens-Fresnel principle in rectangular coordinate [16, 17]. When this disturbed wavefront is propagated to the imaging plane, the characteristic scattering pattern is generated via spatial interference.
Compared to optical microcopy, ELS provides high throughput results and correlates well with the three dimensional structures. In addition, ELS does not require any types of label or stain to interrogate biophysical characteristics. Microscopy provides the real image of the object wheras ELS provides a transformed image that requires a solution of inverse problem to actually image an object. However, if the purpose is to discriminate among minute morphological changes, ELS identifies the sample in a fast, accurate, and label-free way.
Pure and mixed plate experiments
Four different bacterial genera were scanned for single-species testing. Bacterial colonies were prepared as outlined in the material and methods section and three steps (stage initialization, colony locating, and colony centering) were performed automatically.
Comparison with forward scatterometer
Field-test data: GPS location, types of bacteria, and their classification accuracy
No. of colonies
The great advantage of the proposed instrument is shown by the field-testing results. Even though the experiment was performed locally around the campus, we can easily imagine the impact of using the portable device for resource-scarce areas. The screening result can be delivered wirelessly to the local instrument by accessing thousands of reference ELS images using feature extraction. The only drawback of the proposed system is that the system requires the bacterial colony to be grown on an agar medium. However, compared to the other reagents and expensive instruments needed for other types of screening, the petri-dishes and media can be considered as cost-effective consumables. In addition, numerous rapid methods require the plating of the bacteria anyway for final confirmation of the identification.
For hardware designs, since the UPS can provide 1400 VA of power and the consumption rate of the overall system is high, a single UPS can sustain about 15 min of continuous operation for the portable scatterometer and its accessories. This means that we can scan two plates of bacterial samples with an average number of 15-20 colonies per plate. In the future, both a higher-capacity power source and greater energy efficiency will be incorporated into the overall design of the system to maximize the measurement capability along with a modified optics to reduce the number of CCD cameras. Finally, the laser pointing stability is a critical issue when centering between the incoming laser and the colony center; measurements performed in a moving vehicle resulted in an increased number of centering steps or in poor scatter images. Therefore, all scanning and image acquisition were performed when the vehicle was parked.
A field-deployable bacterial identification system that uses elastic light scatter images has been introduced. The proposed device can provide more rapid identification of the source of an outbreak for resource-poor areas via wireless access capability to a central database, which can play an important role in constructing a national/international surveillance network. The portable device weighs 9 lb, is 12 × 6 × 10.5 inches, and is designed to access a scatter-fingerprint database using a commercial cellular wireless network. The hardware design includes a linear and rotational mechanism to provide a small footprint and incorporates various algorithms for colony locating and centering. Four representative bacterial genera, L. innocua F4248, E. coli stbl, E. faecium, and S. aureus, were used for single-species testing and comparison with the reference forward scatterometer for database compatibility. Finally, a field test was conducted at four different locations and resulted in classification rates of 95-100%.
Four different bacterial genera were selected as representative samples: Listeria innocua F4248, Escherichia coli stbl, Enterococcus faecium, and Staphylococcus aureus. Trypticase soy agar (BD, catalog #211043) was used for sample preparation. 40 mg agar powder was suspended in 1 L Millipore water, boiled for 1 min, and allowed to cool. Twenty-five ml cooled agar was dispensed onto sterile 88-mm round petri dishes. Samples were serially diluted and 25 μl bacterial cultures was dispensed into the center of each petri dish. An L-shaped spreader (RPI Corp. #247660) was used to spread the bacterial dilution on the agar; the plate was then placed in a 37°C incubator.
This research was supported through National Institute of Health Grant number 1R56AI089511-10.
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