Método de la microgota: usado con agar cromogénico es un procedimiento útil para el monitoreo sanitario en acuicultura

Indexación: Web of Science, Scielo.The microdot method is a downscaling methodology of traditional tenfold serial dilution procedure used in microbiology. The microdot method uses 100 mu L for serial dilution and count colonies in a spot of 10 mu L. In this study we counted colonies directly in a chromogenic agar plate to determine, at the same time, the presence and cell concentration of target bacteria required for sanitary monitoring of Chilean export fishery products. Due to among importers countries the most concerning bacteria included in sanitary monitoring are Escherichia coli, Listeria monocytogenes and Staphylococcus aureus, we used the chromogenic agar; CHROMagar ECC, CHROMagar Listeria and Baird Parker agar, respectively. The results shows no differences between quantitative results obtained with microdot and traditional method during the quantification of a culture of Escherichia coli (1.5 L). The sensitivity and specificity of the microdot method in association with each chromogenic agar was demonstrated in vitro with reference strains. In addition, the usefulness in sanitary monitoring of aquaculture procedures was evaluated in Artemia salina tanks. This method did not detected sanitary problems in surface water. Although other colonies grown in the chromogenic agar plate, their morphological and chromogenic properties not correspond to Escherichia coli, Listeria monocytogenes and Staphylococcus aureus, being identified as Salmonella enterica subsp. enterica, Microbacterium sp., Bacillus sp. and Staphylococcus pasteuri by 16S rRNA gene sequence analysis. Hence, we propose the microdot chromogenic method as a low cost, specific and reliable procedure for sanitary monitoring of aquaculture procedures.El método de la microgota es una versión a menor escala del procedimiento tradicional de dilución en serie en base diez utilizados en microbiología. El método realiza diluciones en 100 µL y cuenta colonias crecidas en una gota de 10 µL. En este estudio se cuentan colonias directamente en placas cromogénicas para determinar densidad celular y presencia de bacterias requeridas en vigilancia sanitaria de productos pesqueros chilenos de exportación. Entre los requisitos de países importadores, la vigilancia sanitaria involucra frecuentemente a Escherichia coli, Listeria monocytogenes y Staphylococcus aureus, por lo que se utilizan los agares cromogénicos; CHROMagar ECC, CHROMagar Listeria y agar Baird Parker para su identificación. La comparación entre el método de microgota y el método tradicional no muestra diferencias al evaluar un cultivo de Escherichia coli (1,5 L). La sensibilidad y especificidad del método de microgota junto a cada agar cromogénico se demostró in vitro con cepas de referencia. Además, en estanques de Artemia salina se evaluó la utilidad de este método para el monitoreo sanitario. Este método no mostró problemas sanitarios en aguas superficiales, aunque otras colonias crecieron en la placa de agar cromogénico. Sus propiedades morfológicas y cromogénicas no corresponden a Escherichia coli, Listeria monocytogenes y Staphylococcus aureus, siendo identificadas según el análisis de la secuencia del gen 16S rRNA como Salmonella enterica subsp. enterica, Microbacterium sp., Bacillus sp. y Staphylococcus pasteuri. Por lo tanto, se propone el método de microgota cromogénico como un procedimiento de bajo costo, específico y fiable para el monitoreo sanitario en acuicultura.http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-560X2016000400009&lng=e

Development of a strategic and tactical game plan for junior mining companies

ABSTRACT This thesis undertakes to present a game plan for junior mining companies. It culminates with a game plan model that incorporates all the key steps and tasks that mining investors and entrepreneurs need to use to establish globally competitive junior mining companies. It is based on the research of several listed junior mining companies. The game of junior mining is defined by rules, the player, the elements of playing, and the definitions of winning and scoring. The rules of the game are those defined by the resources industry and general business concepts. The mining assets around which the junior mining game is played are exploration projects, feasibility studies, mine development projects or operating mines. The player is the junior mining company intent on winning the game. Playing the game is done through executing the steps, tasks and simple models and matrices associated with the four business pillars: strategy development, legal and financial, operations management and risks management. The first pillar is strategic, while the last three pillars are tactical. For each business pillar, databases were developed, for the purposes of creating references and benchmarks for the game plan. The databases have been created from the analysis of twenty randomly selected junior mining companies, the author’s practical experiences and previous research. Scoring the game is undertaken by completing the game score matrix, which scores the mining assets, the business pillars and the financial performance, and provides an overall company score. The total company score highlights the strengths and weaknesses of the company. Undertaking the process of playing the game iteratively will lead to creating a globally competitive junior mining company. Winning the game is defined as creating a sustainable junior mining company, that grows to a mid-tier company, or is bought out by a major mining company. ii In the thesis, the game is played using the hypothetical case study of a of a coal junior mining company, with a marginal coal mine, an attractive feasibility study and an exploration project. Step by step the game is played leading to a company score and exposing the company’s strengths and weaknesses. The research concludes by presenting a holistic game plan model that can be applied to any junior mining company in any commodity in constantly changing resources industry dynamics

Sizing of single fluorescently stained DNA fragments by scanning microscopy

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO‐1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly‐l‐lysine, 3‐aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA‐sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7-14%. The proposed method is straightforward and can be applied to standardized microtiter plate

Untersuchungen zur Pathovar-Prävalenz beim Escherichia coli- bedingten Durchfall neugeborener Saugferkel in ökologisch wirtschaftenden Ferkelerzeugerbetrieben

Saugferkeldurchfall ist eine Faktorenkrankheit, die durch Tierverluste, Wachstumsdepressionen und zusätzlichen therapeutischen Aufwand zu großen wirtschaftlichen Einbußen in der Ferkelproduktion führt. Im Zuge des vorgestellten Forschungsprojekts wurden 699 Kotproben von Saugferkeln mit Durchfall aus 258 Würfen von 18 ökologisch wirtschaftenden Ferkelerzeugerbetrieben auf enterotoxische Escherichia coli (ETEC), Clostridium (Cl.) perfringens, Rotaviren und Kokzidien untersucht. Außerdem wurden 369 Kotproben von Sauen und 419 Proben von gesunden Ferkeln auf Cl. perfringens untersucht. Eine Genotypisierung aller kulturell angezüchteten Cl. perfringens Isolate mittels RAPD-PCR sowie eine MLST von 8 Isolaten schlossen sich an. In 39,5% der erkrankten Würfe wurde Cl. perfringens Typ A nachgewiesen, welches damit der am häufigsten nachgewiesene Durchfallerreger war. 89,7% der Cl. perfringens Typ A Isolate wurden positiv auf das β2-Toxingen getestet. Rotaviren traten in 27,6% und Kokzidien in 20,0% der erkrankten Würfe auf, während ETEC mit 7,7% unerwartet selten diagnostiziert wurden. Cl. perfringens Typ C wurde in keiner Probe nachgewiesen. Auffällig ist, dass die Nachweisrate für Cl. perfringens Typ A bei gesunden Saugferkeln mit 58,9% über der bei erkrankten Saugferkeln liegt und dass nur 8,6% der Cl. perfringens Typ A Isolate von Sauen das β2-Toxingen tragen, welches in 94,2% aller Isolate von Saugferkeln (gesund und erkrankt) nachgewiesen wurde. Diese Erkenntnis deutet darauf hin, dass die Rolle der Sau als Infektionsquelle für die Saugferkel in Hinblick auf Cl. perfringens bisher überschätzt wurde. Die Genotypisierung der Cl. perfringens Typ A Isolate mittels RAPD offenbart eine hohe genetische Diversität der Isolate. Ferner geht aus einer Befragung der Betriebsleiter hervor, dass auf den meisten der teilnehmenden Betriebe erhebliche Defizite bei der Durchführung von Hygienemaßnahmen im Abferkelstall sowie Überwachung von Geburtsverlauf und Kolostrumaufnahme bestehen