Rapid microbiological detection method based on ultrasonic instrumentation

Los métodos de detección rápida de microorganismos se están convirtiendo en una herramienta esencial para el control de calidad en el área de la biotecnología, como es el caso de las industrias de alimentos y productos farmacéuticos y bioquímicos. En este escenario, el objetivo de esta tesis doctoral es desarrollar una técnica de inspección rápida de microoganismos basada en ultrasonidos. La hipótesis propuesta es que la combinación de un dispositivo ultrasónico de medida y un medio líquido diseñado específicamente para producir y atrapar burbujas, pueden constituir la base de un método sensible y rápido de detección de contaminaciones microbianas. La técnica presentada es efectiva para bacterias catalasa-positivas y se basa en la hidrólisis del peróxido de hidrógeno inducida por la catalasa. El resultado de esta reacción es un medio con una creciente concentración de burbujas. Tal medio ha sido estudiado y modelado desde el punto de vista de la propagación ultrasónica. Las propiedades deducidas a partir del análisis cinemático de la enzima se han utilizado para evaluar el método como técnica de inspección microbiana. En esta tesis, se han investigado aspectos teóricos y experimentales de la hidrólisis del peróxido de hidrógeno. Ello ha permitido describir cuantitativamente y comprender el fenómeno de la detección de microorganismos catalasa-positivos mediante la medida de parámetros ultrasónicos. Más concretamente, los experimentos realizados muestran cómo el oxígeno que aparece en forma de burbujas queda atrapado mediante el uso de un gel sobre base de agar. Este gel fue diseñado y preparado especialmente para esta aplicación. A lo largo del proceso de hidrólisis del peróxido de hidrógeno, se midió la atenuación de la onda y el “backscattering” producidos por las burbujas, utilizando una técnica de pulso-eco. Ha sido posible detectar una actividad de la catalasa de hasta 0.001 unidades/ml. Por otra parte, este estudio muestra que por medio del método propuesto, se puede lograr una detección microbiana para concentraciones de 105 células/ml en un periodo de tiempo corto, del orden de unos pocos minutos. Estos resultados suponen una mejora significativa de tres órdenes de magnitud en comparación con otros métodos de detección por ultrasonidos. Además, la sensibilidad es competitiva con modernos y rápidos métodos microbiológicos como la detección de ATP por bioluminiscencia. Pero sobre todo, este trabajo muestra una metodología para el desarrollo de nuevas técnicas de detección rápida de bacterias basadas en ultrasonidos. ABSTRACT In an industrial scenario where rapid microbiological methods are becoming essential tools for quality control in the biotechnological area such as food, pharmaceutical and biochemical; the objective of the work presented in this doctoral thesis is to develop a rapid microorganism inspection technique based on ultrasounds. It is proposed that the combination of an ultrasonic measuring device with a specially designed liquid medium, able to produce and trap bubbles could constitute the basis of a sensitive and rapid detection method for microbial contaminations. The proposed technique is effective on catalase positive microorganisms. Well-known catalase induced hydrogen peroxide hydrolysis is the fundamental of the developed method. The physical consequence of the catalase induced hydrogen peroxide hydrolysis is an increasingly bubbly liquid medium. Such medium has been studied and modeled from the point of view of ultrasonic propagation. Properties deduced from enzyme kinematics analysis have been extrapolated to investigate the method as a microbial inspection technique. In this thesis, theoretical and experimental aspects of the hydrogen peroxide hydrolysis were analyzed in order to quantitatively describe and understand the catalase positive microorganism detection by means of ultrasonic measurements. More concretely, experiments performed show how the produced oxygen in form of bubbles is trapped using the new gel medium based on agar, which was specially designed for this application. Ultrasonic attenuation and backscattering is measured in this medium using a pulse-echo technique along the hydrogen peroxide hydrolysis process. Catalase enzymatic activity was detected down to 0.001 units/ml. Moreover, this study shows that by means of the proposed method, microbial detection can be achieved down to 105 cells/ml in a short time period of the order of few minutes. These results suppose a significant improvement of three orders of magnitude compared to other ultrasonic detection methods for microorganisms. In addition, the sensitivity reached is competitive with modern rapid microbiological methods such as ATP detection by bioluminescence. But above all, this work points out a way to proceed for developing new rapid microbial detection techniques based on ultrasound.

Detection and Tracking of Traffic Signs Using a Recursive Bayesian Decision Framework

In this paper we propose a new method for the automatic detection and tracking of road traffic signs using an on-board single camera. This method aims to increase the reliability of the detections such that it can boost the performance of any traffic sign recognition scheme. The proposed approach exploits a combination of different features, such as color, appearance, and tracking information. This information is introduced into a recursive Bayesian decision framework, in which prior probabilities are dynamically adapted to tracking results. This decision scheme obtains a number of candidate regions in the image, according to their HS (Hue-Saturation). Finally, a Kalman filter with an adaptive noise tuning provides the required time and spatial coherence to the estimates. Results have shown that the proposed method achieves high detection rates in challenging scenarios, including illumination changes, rapid motion and significant perspective distortion

A new ultrasonic signal amplification method for detection bacteria

A new method is presented that increases the sensitivity of ultrasound-based techniques for detection of bacteria. The technique was developed for the detection of catalase-positive microorganisms. It uses a bubble trapping medium containing hydrogen peroxide that is mixed with the sample for microbiological evaluation. The enzyme catalase is present in catalase-positive bacteria, which induces a rapid hydrolysis of hydrogen peroxide, forming bubbles which remain in the medium. This reaction results in the amplification of the mechanical changes that the microorganisms produce in the medium. The effect can be detected by means of ultrasonic wave amplitude continuous measurement since the bubbles increase the ultrasonic attenuation significantly. It is shown that microorganism concentrations of the order of 105 cells ml−1 can be detected using this method. This allows an improvement of three orders of magnitude in the ultrasonic detection threshold of microorganisms in conventional culture media, and is competitive with modern rapid microbiological methods. It can also be used for the characterization of the enzymatic activity.

Efficient hybrid monocular-stereo approach to on-board, video-based traffic sign detection and tracking

In this paper we propose an innovative method for the automatic detection and tracking of road traffic signs using an onboard stereo camera. It involves a combination of monocular and stereo analysis strategies to increase the reliability of the detections such that it can boost the performance of any traffic sign recognition scheme. Firstly, an adaptive color and appearance based detection is applied at single camera level to generate a set of traffic sign hypotheses. In turn, stereo information allows for sparse 3D reconstruction of potential traffic signs through a SURF-based matching strategy. Namely, the plane that best fits the cloud of 3D points traced back from feature matches is estimated using a RANSAC based approach to improve robustness to outliers. Temporal consistency of the 3D information is ensured through a Kalman-based tracking stage. This also allows for the generation of a predicted 3D traffic sign model, which is in turn used to enhance the previously mentioned color-based detector through a feedback loop, thus improving detection accuracy. The proposed solution has been tested with real sequences under several illumination conditions and in both urban areas and highways, achieving very high detection rates in challenging environments, including rapid motion and significant perspective distortion

Mealiness Detection in Agricultural Crops: Destructive and Nondestructive Tests: A Review

Mealiness is known as an important internal quality attribute of fruits/vegetables, which has significant influence on consumer purchasing decisions. Mealiness has been a topic of research interest over the past several decades. A number of destructive and nondestructive techniques are introduced for mealiness detection. Nondestructive methods are more interesting because they are rapid, noninvasive, and suitable for real-time purposes. In this review, the concept of mealiness is presented for potato, apple, and peach, followed by an in-depth discussion about applications of destructive and nondestructive techniques developed for mealiness detection. The results suggest the potential of electromagnetic-based techniques for nondestructive mealiness evaluation. Further investigations are in progress to find more appropriate nondestructive techniques as well as cost and performance.