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DNA isolation and identification of nonpathogenic species of clostridia isolated from cheeses
Sedláček, Zbyněk ; Eva, Kvasničková (referee) ; Rittich, Bohuslav (advisor)
In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).
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Use of DGGE to analysis and identification of selected microorganisms
Jankeje, Kristína ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on use of DGGE to analysis and identification of selected microorganisms. PCR-DGGE is a method that allows direct characterization of the microbial community in the natural environment without necessity of cultivation. A literature review is devoted to the principle of the method, current applications and its limitations too. In experimental part microbial DNA was isolated and used as a template for PCR reaction. Microbial DNA was then amplified using the universal eukaryotic primers that target the D1/D2 domain of the 26S subunit of ribosomal DNA. To improve specificity and sensitivity of detection nested PCR was chosen using outer and inner primer pairs. Generated amplicons (250 bp) were consequently separated by DGGE. The analysis of selected microorganisms by DGGE technique was performed after optimization of electrophoresis conditions (in particular the denaturing gradient extent and separation time). Despite the optimization, mutual differentiation among individual yeast strains was not possible since each reference strain was represented by several bands in the same positions. In conclusion DGGE profile obtained from wine musts is discussed. Present bands suggest the major presence of non-Saccharomyces yeasts, yeast-like strain A. pullulans is present in the minority and Saccharomyces yeasts are probably present too. The technique remains open for further optimization, particularly as regards the conditions of polymerase chain reaction.
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Effect of available P and phenolics on mineral N release in acidified spruce forest: connection with lignin-degrading enzymes and bacterial and fungal communities
VANĚK, Daniel
Annotation We conducted over four months a shortterm laboratory incubation experiment to find the best prediction parameters (i.e. initial chemical characteristics) to explain differences in microbial respiration rates and mineral N (DIN) release in different litter in an acidified spruce forest. In addition, we wanted to find the link between the activity of key extracellular ligninolytic enzymes, phenoloxidases (PhOx) and peroxidases (PerOx), microbial respiration and composition of fungal and bacterial communities. Samples of spruce needles (Picea abies) and litter of four dominant understorey vegetation; lady fern (Athyrium alpestre), blueberry (Vaccinium myrtillus), reedgrass (Calamagrostis villosa) and hair grass (Avenella flexuosa), were collected in 2005, 2006 and 2007 from six sites located in watersheds of two glacial lakes (Plešné Lake and Čertovo Lake) in the Bohemian Forest, Czech Republic. Litter samples were incubated at 0 and 10 °C in laboratory controlled conditions for 90 days. Activities of PhOx and PerOx, and C mineralization rate were measured regularly each 14 days. Litter quality characteristics and endophytic microbial community structure, based on 16SrDNA-DGGE fingerprint of bacteria and ITS-DGGE of fungi, were determined at the beginning and end of litter incubation. Our results showed a close correlation of phenolics/POX with DIN release (r ? 0.74, p ? 0.001). Using multivariate analyses, POX seems to play an important role in the change of litter fungal and bacterial community composition. At 0 °C the fungal and bacterial communities of reedgrass and blueberry litter changed in relation to POX and PerOx activity, while at 10 °C the fungal communities after the incubation were additionally affected by the phenolics/NTOT and phenolics/PTOT ratios.
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