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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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Kvantitativní zastoupení bakterií rodu Lactobacillus v lidském zažívacím traktu v závislosti na změně stravovacích návyků
Kolísková, Kateřina
This diploma thesis deals with the issue of quantitative representation of the bacterial family in the human digestion system depending on changes in eating habits. The start of literary digest addresses general characteristics of digestion microbiome and its bacterial represen-tation. The most comprehensive part belongs to individual factors, who influenced overall number of bacterial species inhabiting the digestion microbiome. The last chapters of the theoretical part belong to cultivative and molecular biological methods, which are used in examination of the human digestion tract. Experimental part focuses on differences in the number of individual bacteria in the samples of human excrements, whose presence was determined by cultivative methods. Isolation of bacterial DNA was performed, which was subsequently confirmed by polymerase chain reaction. In resulting values, we observed small changes (p < 0,05) in bacterial species L. acidophilus a L. rhamnosus at the end of the second period. All bacterial families, except Bacteroides, had downward tendency (p > 0,05). Based on acquired data from food records, we observed changes in consumption of basic macronutrients (protein, fat, carbohydrates) and fiber. Only in carbohydrates and sim-ple sugars, we observed statistically provable differences (p > 0,05). Final analysis proved that changes in eating habits did not influence the total number of bacterial species inhabiting digestion microbiome.
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Influence of microbial community diversity on the decomposition of organic matter
Vicena, Jakub ; Frouz, Jan (advisor) ; Cajthaml, Tomáš (referee)
Microbial diversity plays an important role in the decomposition of soil organic matter. However, the drivers of this dependence still remain unclear. The work is based on long-term monitoring of soils of different successional stages of different diversity. Soil sampling was conducted on two dumps after brown coal mining in the Sokolov. Soils were X ray sterilized and inoculated by inoculum from both soils in two inoculation density which create gradient of microbial diversity. Then microbial respiration was measured in either supplied or not supplied by litter of Calamagrostis epigejos. Results showed a strong positive correlation between the microbial diversity and decomposition of organic matter if the microbial community is limited by available carbon. If there isn't carbon limit available, the decomposition rate is controlled by the amount of microbial biomass. Results demonstrated positive correlation between the rate of decomposition and the amount of fungal biomass. The soils with the addition of leaf litter showed priming effect in the initial stage of decomposition. In the control samples without addition of litter priming effect wasn't observed. Increasing humidity led to increase of decomposition rate. We can conclude with a clear conscience that similarly conclusive results associated...
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Mobilisation of subglacial microbes during deglaciation and their export to downstream ecosystems
Znamínko, Matěj ; Stibal, Marek (advisor) ; Řeháková, Klára (referee)
Subglacial environments harbor active microbial communities. The composition of these communities is influenced by a number of factors, with glacial hydrology being one of the most important ones. This affects not only formation of subglacial communities but also the mobilisation of microorganisms and their subsequent release to downstream ecosystems. The seasonally evolving drainage system connects supraglacial and subglacial environments, and thus microorganisms from supraglacial environments are exported to proglacial streams along with subglacial microorganisms. The contribution of these two microbial communities to proglacial assemblage is not uniform and may be influenced by the size or geographical location of the glacier or by the phase of the glacier drainage system. Microorganisms from proglacial assemblages can form biofilms in proglacial streams or contribute to the community composition in downstream ecosystems. This work summarizes current knowledge of mobilisation and subsequent export of microorganisms from subglacial environments.
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Influence of microbial community diversity on the decomposition of organic matter
Vicena, Jakub ; Frouz, Jan (advisor) ; Cajthaml, Tomáš (referee)
Microbial diversity plays an important role in the decomposition of soil organic matter. However, the drivers of this dependence still remain unclear. The work is based on long-term monitoring of soils of different successional stages of different diversity. Soil sampling was conducted on two dumps after brown coal mining in the Sokolov. Soils were X ray sterilized and inoculated by inoculum from both soils in two inoculation density which create gradient of microbial diversity. Then microbial respiration was measured in either supplied or not supplied by litter of Calamagrostis epigejos. Results showed a strong positive correlation between the microbial diversity and decomposition of organic matter if the microbial community is limited by available carbon. If there isn't carbon limit available, the decomposition rate is controlled by the amount of microbial biomass. Results demonstrated positive correlation between the rate of decomposition and the amount of fungal biomass. The soils with the addition of leaf litter showed priming effect in the initial stage of decomposition. In the control samples without addition of litter priming effect wasn't observed. Increasing humidity led to increase of decomposition rate. We can conclude with a clear conscience that similarly conclusive results associated...
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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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