[Show abstract][Hide abstract] ABSTRACT: The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label-free forward light-scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter-image signatures were acquired using a CCD (charge-coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light-scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light-scatter information provided classification in 1-2 min with an accuracy of 99%. The light-scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non-culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light-scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates.
[Show abstract][Hide abstract] ABSTRACT: Light scattering is one of the most fundamental optical processes whereby electromagnetic waves are forced to deviate from a straight trajectory by non-uniformities in the medium that they traverse. This presentation summarizes our recent research on application of light-scatter measurements paired with machine learning and pattern recognition methodologies for label-free classification of bioparticles. Two separate examples of light scatter-based techniques are discussed: forward-scatter measurements of bacterial colonies in an imaging system, and flow cytometry measurements of scatter signals formed by individual bacterial particles. Recently, we have reported a first practical implementation of a system capable of label-free classification and recognition of pathogenic species of Listeria, Salmonella, Vibrio, Staphylococcus, and E. coli using forward-scatter patterns produced by bacterial colonies irradiated with laser light. Individual bacteria in flow also form complex patterns dependent on particle size, shape, refraction index, density, and morphology. Although commercial flow cytometers allow scatter measurement at two angles this rudimentary approach cannot be used to separate populations of bioparticles of similar shape, size, or structure. The custom-built system used in the presented work collects axial light-loss and scatter signals at five carefully chosen angles. Experimental results obtained from colony scanner, as well from the extended cytometry instrument, were used to train the pattern-recognition algorithm. The results demonstrate that information provided by scatter alone may be sufficient to recognize various bioparticles with 90-99% success rate, both in flow and in imaging systems.
[Show abstract][Hide abstract] ABSTRACT: Technologies for rapid detection and classification of bacterial pathogens are crucial for securing the food supply. This report describes a light-scattering sensor capable of real-time detection and identification of colonies of multiple pathogens without the need for a labeling reagent or biochemical processing. Bacterial colonies consisting of the progeny of a single parent cell scatter light at 635 nm to produce unique forward-scatter signatures. Zernike moment invariants and Haralick descriptors aid in feature extraction and construction of the scatter-signature image library. The method is able to distinguish bacterial cultures at the genus and species level for Listeria, Staphylococcus, Salmonella, Vibrio, and Escherichia with an accuracy of 90-99% for samples derived from food or experimentally infected animal. Varied amounts of exopolysaccharide produced by the bacteria causes changes in phase modulation distributions, resulting in strikingly different scatter signatures. With the aid of a robust database the method can potentially detect and identify any bacteria colony essentially instantaneously. Unlike other methods, it does not destroy the sample, but leaves it intact for other confirmatory testing, if needed, for forensic or outbreak investigations.
[Show abstract][Hide abstract] ABSTRACT: The formation of bacterial colonies and biofilms requires coordinated gene expression, regulated cell differentiation, autoaggregation, and intercellular communication. Therefore colonies of bacteria have been recognized as multicellular organisms or "superorganisms." It has consequently been postulated that the phenotype of colonies formed by microorganisms can be automatically recognized and classified using optical systems capable of collecting information related to cellular pattern formation and morphology of colonies. Recently we have reported a first practical implementation of such a system, capable of noninvasive, label-free classification and recognition of pathogenic Listeria species. The design employed computer-vision and pattern-recognition techniques to classify scatter patterns produced by bacterial colonies irradiated with laser light. Herein we report our efforts to extend this system to other genera of bacteria such as Salmonella, Vibrio, Staphylococcus, and E. coli. Application of orthogonal moments, as well as texture descriptors for image feature extraction, provides high robustness in the presence of noise. An improved pattern classification scheme based on an SVM algorithm provides better results than the previously employed neural network system. Low error rates determined by cross-validation, reproducibility of the measurements, and overall robustness of the recognition system prove that the proposed technology can be implemented in automated devices for bacterial detection.
[Show abstract][Hide abstract] ABSTRACT: We investigate the relationship of incubation time and forward-scattering signature for bacterial colonies grown on solid nutrient surfaces. The aim of this research is to understand the colony growth characteristics and the corresponding evolution of the scattering patterns for a variety of pathogenic bacteria relevant to food safety. In particular, we characterized time-varying macroscopic and microscopic morphological properties of the growing colonies and modeled their optical properties in terms of two-dimensional (2-D) amplitude and phase modulation distributions. These distributions, in turn, serve as input to scalar diffraction theory, which is, in turn, used to predict forward-scattering signatures. For the present work, three different species of Listeria were considered: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. The baseline experiments involved the growth of cultures on brain heart infusion (BHI) agar and the capture of scatter images every 6 h over a total incubation period of 42 h. The micro- and macroscopic morphologies of the colonies were studied by phase contrast microscopy. Growth curves, represented by colony diameter as a function of time, were compared with the measured time-evolution of the scattering signatures.
Journal of Biomedical Optics 01/2008; 13(1):014010. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A model for forward scattering from bacterial colonies is presented. The colonies of interest consist of approximately 10(12) - 10(13) individual bacteria densely packed in a configuration several millimeters in diameter and approximately 0.1-0.2 mm in thickness. The model is based on scalar diffraction theory and accounts for amplitude and phase modulation created by three macroscopic properties of the colonies: phase modulation due to the surface topography, phase modulation due to the radial structure observed from some strains and species, and diffraction from the outline of the colony. Phase contrast and confocal microscopy were performed to provide quantitative information on the shape and internal structure of the colonies. The computed results showed excellent agreement with the experimental scattering data for three different Listeria species: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. The results provide a physical explanation for the unique and distinctive scattering signatures produced by colonies of closely related Listeria species and support the efficacy of forward scattering for rapid detection and classification of pathogens without tagging.
[Show abstract][Hide abstract] ABSTRACT: Bacterial contamination of food products puts the public at risk and also generates a substantial cost for the food-processing industry. One of the greatest challenges in the response to these incidents is rapid recognition of the bacterial agents involved. Only a few currently available technologies allow testing to be performed outside of specialized microbiological laboratories. Most current systems are based on the use of expensive PCR or antibody-based techniques, and require complicated sample preparation for reliable results. Herein, we report our efforts to develop a noninvasive optical forward-scattering system for rapid, automated identification of bacterial colonies grown on solid surfaces. The presented system employs computer-vision and pattern-recognition techniques to classify scatter patterns produced by bacterial colonies irradiated with laser light. Application of Zernike and Chebyshev moments, as well as Haralick texture descriptors for image feature extraction, allows for a very high recognition rate. An SVM algorithm was used for classification of patterns. Low error rates determined by cross-validation, reproducibility of the measurements, and robustness of the system prove that the proposed technology can be implemented in automated devices for bacterial detection.
[Show abstract][Hide abstract] ABSTRACT: Polyclonal antibodies are typically produced in rabbits. The rabbit's health plays an important role in the quality of antibodies produced. Therefore, recommendations have been made by organizations on which bacteria and how frequently to test rabbit colonies. Since it is well known that rabbits may contain cross-reactive antibodies in their preimmune serum, it is common to test the rabbits for reactivity prior to immunization. Here preimmune sera from 19 different rabbits were tested with ELISA against 27 pathogenic and nonpathogenic bacterial cultures. ELISA results showed that Salmonella enterica serovar Typhimurium and Bacillus cereus AS4-12 had the highest average absorbance values (0.60 and 0.54, respectively) and the most preimmune serum samples testing positive was 17. Pseudomonas putrefaciens and B. subtilis had the lowest absorbance values (< 0.1) and did not test positive in any of the preimmune serum samples. Fourteen of the 27 cultures showed positive reactions with 50% or more of the preimmune serum samples tested. Fifty-three percent of the rabbit preimmune sera showed positive reactions with 10 or more bacterial cultures. In Western blot analyses, selected serum samples showing the highest ELISA values reacted with bands in the 97, 36, and 29 kDa regions or with bands in the 63 kDa and 32 kDa regions. Data suggest that the presence of cross-reactive antibodies in the preimmune serum is a common problem amongst the samples tested. Extensive preimmune serum testing should be implemented when polyclonal antibodies are intended for diagnostic testing.
Journal of Immunoassay and Immunochemistry 02/2006; 27(4):351-61. · 0.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial contamination by Listeria monocytogenes puts the public at risk and is also costly for the food-processing industry. Traditional methods for pathogen identification require complicated sample preparation for reliable results. Previously, we have reported development of a noninvasive optical forward-scattering system for rapid identification of Listeria colonies grown on solid surfaces. The presented system included application of computer-vision and patternrecognition techniques to classify scatter pattern formed by bacterial colonies irradiated with laser light. This report shows an extension of the proposed method. A new scatterometer equipped with a high-resolution CCD chip and application of two additional sets of image features for classification allow for higher accuracy and lower error rates. Features based on Zernike moments are supplemented by Tchebichef moments, and Haralick texture descriptors in the new version of the algorithm. Fisher's criterion has been used for feature selection to decrease the training time of machine learning systems. An algorithm based on support vector machines was used for classification of patterns. Low error rates determined by cross-validation, reproducibility of the measurements, and robustness of the system prove that the proposed technology can be implemented in automated devices for detection and classification of pathogenic bacteria.