Whole Slide Imaging for High-Throughput Monitoring of Microbial Growth and Its Application to Rapid Antimicrobial Susceptibility Testing

Archival abstract submitte

Laser and optical based methods for detecting and characterising microorganisms

This work investigated novel optical methods of characterizing the activity of microorganisms. Two different systems are studied in detail in this work. The possibility of using line scan speckle systems and imaging systems to understand the microbial behaviour, growth and motility was investigated. Conventionally, the growth and viability of microorganisms are determined by swabbing, plating and incubation, typically at 37degreesC for at least 24 hours. The proposed system allows real-time quantification of morphology and population changes of the microorganisms. An important aspect of the line scan system is the dynamic biospeckle. Dynamic speckle can be obtained from the movement of particles suspended in liquids. The speckle patterns show fluctuations in space and time which may be correlated with the activity of the constituents in the suspension. Initially the speckle parameters were standardized to non-motile and inert specimens such as polystyrene microspheres and suspensions of Staphylococcus aureus. The same optical systems and parameters were later tested on motile, active and live organisms of Escherichia coli. The experimental results that are presented describe the time history of the dynamic speckle pattern. A number of algorithms were used to analyse the intensity data. A 2D-FFT algorithm was used to evaluate the space and time-varying autocorrelation. Analysis of the speckle data in the Fourier domain provided insight into the motility of the organisms in broth. The mathematical analysis also gave further insight into the culture broth evaporation and its particle sedimentation characteristics at 37degreesC. These features correlated with the periodic motions associated with the organism and may therefore provide a signature for the organism and a means of monitoring. These results aided the developemnt of imaging bacterial detection systems which were discussed in the second half of the work. The second experimental system focuses on quantifying the morphology and population dynamics of Euglena gracilis under ambient conditions through image processing. Unlike many other cell systems, Euglena cells change from round to long to round cell shape and these different cell shapes were analyzed over time. In the morphological studies of single Euglena cells, image processing tools and filtering techniques were used and different parameters identified and their efficiency at determining cell shape compared. The best parameter for processing the images and its effectiveness in detecting even the interior motions of constituents within a dead cell was found. The efficiency of the measurement parameters in following sequences of shape changes of the Euglena cell was compared with the visual assessment tests from 12 volunteers and other simple measurement methods including parameters relating to the cells eccentricity, and image processing in the space and frequency domains. One of the major advantages of this system is that living cells can be examined in their natural state without being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes in live cells can be observed and recorded in high contrast and sharp clarity. The population statistics of Euglena gracilis was done in liquid culture. A custom built microscopy system was employed and the laser beam was coupled with a dark field illumination system to enhance the contrast of the images. Different image filters were employed for extracting useful information on the population statistics. Similarly as with the shape study of the Euglena cell, different parameters were identified and the best parameter was selected. The population study of the Euglena cells provided a detection system that indicated the activity of the population

Development of a model to assess cleaning and disinfection of complex root canal systems

The remaining debris and biofilm in the anatomical complexities of root canal systems can affect treatment outcomes. Files with asymmetric cross-section design may improve debris and biofilm removal from these difficult spaces during canal preparation. Tooth opacity and different densities of the remaining materials prevent the direct systematic assessment of the preparation process. The present study assessed remaining debris and biofilm using a novel transparent root canal model with novel approaches. Natural and simulated root canal samples with isthmus space were evaluated. Canal preparation by ProTaper Next and Revo-S asymmetric systems was evaluated in comparison to the standard ProTaper Universal symmetric system. The root canals were investigated by microcomputed tomography (micro-CTL confocal laser scanning microscopy (CLSML and optical coherence tomography (OCT) imaging tools. Data analysis was undertaken with SPSS (V. 24). Files with asymmetric cross-section and constant taper removed more debris and biofilm from the complex root canal system. The model allowed direct assessment of remaining materials and confirmed the novel imaging approach with the OCT. In conclusion, the asymmetric design improves debris and biofilm removal especially when used with a constant taper. The model was verified as an ideal system for assessing root canal treatment in vitro

Rapid Antimicrobial Susceptibility Testing Based on Bacterial Motion Tracking

abstract: Antibiotic resistant bacteria are a worldwide epidemic threatening human survival. Antimicrobial susceptibility tests (ASTs) are important for confirming susceptibility to empirical antibiotics and detecting resistance in bacterial isolates. Current ASTs are based on bacterial culturing, which take 2-14 days to complete depending on the microbial growth rate. Considering the high mortality and morbidity rates for most acute infections, such long time frames are clinically impractical and pose a huge risk to a patient's life. A faster AST will reduce morbidity and mortality rates, as well as help healthcare providers, administer narrow spectrum antibiotics at the earliest possible treatment stage. In this dissertation, I developed a nonculture-based AST using an imaging and cell tracking technology. I track individual Escherichia coli O157:H7 (E. coli O157:H7) Uropathogenic Escherichia Coli (UPEC) cells, widely implicated in food-poisoning outbreaks and urinary tract infections respectively. Cells tethered to a surface are tracked on the nanometer scale, and phenotypic motion is correlated with bacterial metabolism. Antibiotic action significantly slows down motion of tethered bacterial cells, which is used to perform antibiotic susceptibility testing. Using this technology, the clinical minimum bactericidal concentration of an antibiotic against UPEC pathogens was calculated within 2 hours directly in urine samples as compared to 3 days using current gold standard tools. Such technologies can make a tremendous impact to improve the efficacy and efficiency of infectious disease treatment. This has the potential to reduce the antibiotic mis-prescription steeply, which can drastically decrease the annual 2M+ hospitalizations and 23,000+ deaths caused due to antibiotic resistance bacteria along with saving billions of dollars to payers, patients, and hospitals.Dissertation/ThesisDoctoral Dissertation Bioengineering 201

Critical review on biofilm methods

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.The authors would like to acknowledge the support from the EU COST Action BacFoodNet FA1202

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Single-Molecule Detection of Unique Genome Signatures: Applications in Molecular Diagnostics and Homeland Security

Single-molecule detection (SMD) offers an attractive approach for identifying the presence of certain markers that can be used for in vitro molecular diagnostics in a near real-time format. The ability to eliminate sample processing steps afforded by the ultra-high sensitivity associated with SMD yields an increased sampling pipeline. When SMD and microfluidics are used in conjunction with nucleic acid-based assays such as the ligase detection reaction coupled with single-pair fluorescent resonance energy transfer (LDR-spFRET), complete molecular profiling and screening of certain cancers, pathogenic bacteria, and other biomarkers becomes possible at remarkable speeds and sensitivities with high specificity. The merging of these technologies and techniques into two different novel instrument formats has been investigated. (1) The use of a charge-coupled device (CCD) in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement by simultaneously tracking and detecting single-molecules in multiple microfluidic channels was demonstrated. The CCD/TDI approach allowed increasing the sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s-1 per channel and 2208 molecules s-1 for an eight channel microfluidic system was achieved. A cyclic olefin copolymer (COC) waveguide was designed and fabricated in a pre-cast poly(dimethylsiloxane) stencil to increase the SNR by controlling the excitation geometry. The waveguide showed an attenuation of 0.67 dB/cm and the launch angle was optimized to increase the depth of penetration of the evanescent wave. (2) A compact SMD (cSMD) instrument was designed and built for the reporting of molecular signatures associated with bacteria. The optical waveguides were poised within the fluidic chip at orientation of 90° with respect to each other for the interrogation of single-molecule events. Molecular beacons (MB) were designed to probe bacteria for the classification of Gram +. MBs were mixed with bacterial cells and pumped though the cSMD which allowed S. aureus to be classified with 2,000 cells in 1 min. Finally, the integration of the LDR-spFRET assay on the cSMD was explored with the future direction of designing a molecular screening approach for stroke diagnostics

3D Microfluidics for Environmental Pathogen Detection and Single-cell Phenotype-to-Genotype Analysis

The emergence of microfluidic technologies has enabled the miniaturization of cell analysis processes, including nucleic acid analysis, single cell phenotypic analysis, single cell DNA and RNA sequencing, etc. Traditional chip fabrication via soft lithography cost thousands of dollars just in personnel training and capital cost. The design of these systems is also confined to two dimensions limited by their fabrication. To address the needs of smooth transition from technology to adoption by end-users, less complexity is urgently needed for microfluidics to be applied in pathogen detection under low-resource settings and more powerful integration of analyses to understand single cells. This dissertation presents my explorations in 3D microfluidics involving simulation-aided design of pretreatment devices for pathogen detection, fabrication through 3D printing, utilization of alternative commercial parts, and the combination with hydrogel material to link phenotypic analysis with in situ molecular detection for single cells. The main outputs of this dissertation are as follows: 1) COMSOL Multiphysics® was used to aid the design and understanding of microfluidic systems for environmental pathogen detection. In the development of an asymmetric membrane for concentration and digital detection of bacteria, the quantification requires Poisson distribution of cells into membrane pores; the flow field and particle trajectories were simulated to validate the cell distribution in capturing pores. In electrochemical bacterial DNA extraction, the hydroxide ion generation, species diffusion, and cation exchange were modeled to understand the pH gradient within the chamber. To address the overestimated risk by polymerase chain reactions (PCR) that detects all target nucleic acids regardless of cell viability, we developed a microfluidic device to carry out on-chip propidium monoazide (PMA) pretreatment. The design utilizes split-and-recombine (SAR) mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. Ten SAR mixers were employed based on fluid flow and diffusion simulation. High-resolution 3D printing was used for prototyping. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was experimentally demonstrated. 2) Water-in-oil droplet-based microfluidic platforms for digital nucleic acid analysis eliminates the need for calibration that is required for qPCR-based environmental pathogen detection. However, utilizing droplet microfluidics generally requires fabrication of sub-100 µm channels and complicated operation of multiple syringe pumps, thus hindering the wide adoption of this powerful tool. We designed a disposable centrifugal droplet generation device made simply from needles and microcentrifuge tubes. The aqueous phase was added into the Luer-Lock of the commercial needle, with the oil at the bottom of the tube. The average droplet size was tunable from 96 μm to 334 μm and the coefficient of variance (CV) was minimized to 5%. For droplets of a diameter of 175 μm, each standard 20 μL reaction could produce ~10⁴ droplets. Based on this calculated compartmentalization, the dynamic range is theoretically from 0.5 to 3×10³ target copies or cells per μL, and the detection limit is 0.1 copies or cells per μL. 3) Based on the disposable droplet generation device, we further developed a novel platform that enables both high-throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-sized biocompatible polyethylene glycol (PEG) hydrogel beads. The crosslinked hydrogel network in aqueous phase adds additional robustness to droplet microfluidics by allowing reagent exchange. The hydrogel beads demonstrated enhanced thermal stability, and achieved uncompromised efficiencies in digital PCR, digital loop-mediated isothermal amplification (dLAMP), and single cell phenotyping. The crosslinked hydrogel network highlights the prospective linkage of various subsequent molecular analyses to address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. This platform has the potential to advance the understanding of single cell genotype-to-phenotype correlations. 4) For effective sorting of the hydrogel beads after single cell phenotyping, a gravity-driven acoustic fluorescence-based hydrogel beads sorter was developed. The design involves a 3D-printed microfluidic tube, two sequential photodetectors, acoustic actuator, and a control system. Instead of bulky syringe pumps used in traditional cell or droplet sorting, this invention drives beads suspended in heavier fluorinated oil simply by buoyancy force to have the beads float through a vertical channel. Along the channel, sequential photodetectors quantify the bead acceleration and inform the action of downstream acoustic actuator. Hydrogel beads with different fluorescence intensity level were led into different collection chambers. The developed sorter promises cheap instrumentation, easy operation, and low contamination for beads sorting, and thus the full establishment of the single cell phenotype-genotype link. In summary, the work in this dissertation established a) the simulation-aided design and 3D printing to reduce the complexity of microfluidics, and thus lowered its barrier for environmental applications, b) a simple and disposable device using cheap commercial components to produce monodispersed water-in-oil droplets to enable easy adoption of droplet microfluidics by non-specialized labs, c) a hydrogel bead-based analysis platform that links single-cell phenotype and genotype to open new research avenues, and d) a gravity-driven portable bead sorting system that may extend to a broader application of hydrogel microfluidics to point of care and point of sample collection. These simple-for-end-user solutions are envisioned to open new research avenues to tackle problems in antibiotic heteroresistance, environmental microbial ecology, and other related fundamental problems.</p