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Les bactéries se répandent aux interfaces en formant des colonies, qui peuvent être considérées comme des suspensions denses actives. L'objet de cette thèse est le développement et l'analyse de modèles simples pour élucider le rôle des phénomènes physico-chimiques et passifs - tels que l'osmose, la tension de surface et le mouillage - dans l'expansion des colonies bactériennes aux interfaces solide/air. Les modèles sont basés sur une description hydrodynamique des couches minces de suspensions liquides, qui est complété par des processus bioactifs.Dans un premier temps, nous nous sommes intéressés à l'expansion osmotique des biofilms. Dans ce mécanisme, la bactérie sécrète une matrice polymérique qui agit comme un osmolyte et entraîne un afflux d'eau, riche en nutriments, du substrat humide vers le biofilm. Nous avons constaté que la mouillabilité du substrat est une des déterminantes principales de la vitesse d'expansion du biofilm. En-dessous d'une mouillabilité critique l'expansion s'interrompt, bien que la colonie soit biologiquement active. Cependant, une légère réduction de la tension de surface et une amélioration de la mouillabilité qui en résulte suffisent à induire un étalement continu. Les bactéries peuvent activement contrôler la tension de surface par la production de bio-surfactants.Dans un deuxième temps, nous avons étudié l'expansion de colonies bactériennes aidée par des molécules biologiques tensioactives auto-produites. Dans ce mécanisme, des flux de Marangoni résultant d'une concentration non uniforme de molécules tensioactives aux bords de la colonie peuvent favoriser l'expansion coopérative et provoquer une instabilité de la forme axi-symétrique des colonies bactériennes. Notre modèle nous a permis de reproduire quatre modes de développement différentes, à savoir l'étalement arrêté et continu des colonies circulaires, l'étalement des colonies avec des bords légèrement modulées et la formation de doigts prononcés.Dans la dernière partie, nous avons fait un premier pas vers l'incorporation de la motilité actif des bactéries dans notre modèle et présentons donc un modèle phénoménologique pour un film mince active. / Bacteria colonise interfaces by the formation of dense aggregates. In this thesis, we develop and analyse simple models to clarify the role of passive physico-chemical forces and processes - such as osmosis, surface tension effects and wettability - in the spreading of bacterial colonies at solid-air interfaces. The models are based on a hydrodynamic description for thin films of liquid suspensions that is supplemented by bioactive processes.We first focus on the osmotic spreading mechanism of bacterial colonies that relies on the generation of osmotic pressure gradients. The bacteria secrete a polymeric matrix which acts as an osmolyte and triggers the influx of nutrient-rich water from the moist substrate into the colony. We find that wettability crucially affects the spreading dynamics. At low wettability, the lateral expansion of the colony is arrested, albeit the colony is biologically active. However, a small reduction of the surface tension and the resulting improvement of the wettability suffices to induce continuous spreading. This can, e.g., result from the production of bio-surfactants by the bacteria.Next, we study passive liquid films covered by insoluble surfactants before developing a model for the surfactant-driven spreading of bacterial colonies. In this spreading mechanism, Marangoni fluxes arising due to a non-uniform surfactant concentration at the edges of the colony drive cooperative spreading and may cause an instability of the circular colony shape. We find that variations in wettability and surfactant production suffice to reproduce four different types of colony growth, namely, arrested and continuous spreading of circular colonies, slightly modulated front lines and the formation of pronounced fingers.In the final part, we take a first step towards the incorporation of active collective bacterial motion in the employed thin-film framework and present a phenomenologically derived model for active polar films.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Silicon-based electric DNA chip arrays were utilized to fast identify pathogenic microorganisms with respect to the capacity to produce toxins involved in foodborne poisoning and infections. Bacteria of the B. cereus and the enterohemorrhagic E. coli (EHEC) groups contain different set-ups of various virulence factors that are encoded by the corresponding genes. The purpose of this work was to develop a fast and simple method for determination of the presence of these virulence genes in a colony from primary enrichment cultures. A target gene is detected through hybridization to a surface-immobilized specific capture probe and biotin-labeled detection probe. Following binding of an enzyme conjugate to this sandwich hybrid complex, a current signal is generated by electronic redox recycling of the enzymatic product paminophenol (pAP). Two versions of the assay were developed. In the first version the capture probes were immobilized on magnetic beads, which carried out all reactions until the pAP generation, while the final electric signal was created by transferring pAP to a single-electrode chip surface. In the second version a silicon chip array with 16 parallel sensing electrode positions each of them functionalized by capture probes, carried out all assay steps on the chip surface. This instrument can realize automatic and multiplexed gene detection. The kinetics of bacterial cell disruption and impact of DNA fragmentation by ultrasound were determined. The experimental data suggested that the increased signal after first minutes of ultrasonication were due to the accumulation of released DNA amount, while the further signal increase resulted from the improved hybridization with the shortened target DNA strands. Studies on probe localization on the 16-electrode chip assays indicated that the probe-targeting site, which was located at the 5’-end of strands, gave rise to the highest signal level due to the efficient targetprobes hybridization and the following enzyme binding. When these functionalized chip arrays were exposed to the cell homogenates, the sensing electrodes were fouled by cellular proteins and therefore led to dramatically decreased redox-recycling current. To circumvent this, samples were treated by DNA extraction after the 1st sonication and then DNA fragmentation by a 2nd time sonication. The DNA extract removed most of the interfering components from bacterial cell. This sample treatment was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the chip arrays functionalized by eight DNA probes. The signal patterns of eight virulence genes from chip assays agreed well with PCR control analyses for both strains. By simply adding the SDS detergent to cell homogenates, chip surface blocking effect can be significantly reduced even without DNA extraction treatment. After optimization of some critical factors, the 16-electrode DNA chips with the improved sensing performance can directly detect multiple virulence genes from a single E. coli colony in 25 min after the introduction of supernatant of ultrasonicated cell lysate. / QC 20100824

<p>Silicon-based electric chip arrays were developed for characterization of Bacillus</p><p>cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all</p><p>chemical reactions until and including the enzymatic reaction took place in an</p><p>eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control</p><p>cells of B. subtilis. The 16 positions of the silicon chip array were activated by</p><p>immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation.</p>

Silicon-based electric chip arrays were developed for characterization of Bacillus cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all chemical reactions until and including the enzymatic reaction took place in an eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control cells of B. subtilis. The 16 positions of the silicon chip array were activated by immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation. / QC 20101111

This master thesis deals with image processing of Petri dishes. The first part is about terms and procedures in laboratory practice. The following part is about devices and algorithms for image analysis of Petri dishes. The second part is about draft and implementation of own colony counting application, which was developed in cooperation with the microbiological laboratory. The application is developed for mobile devices running Android OS. The master thesis also includes the design and creation of two lighting units, which uses as an accessory for better results in the analysis of Petri dishes. In conclusion are presented results, compared lighting units and various types of mobile devices, on which the application was tested.