Odkrivanje bakterijskih vrst v prebavilih domačih živali z in situ hibridizacijo in epifluorescentno mikroskopijo : magistrsko delo = Detection of bacterial species in the gut of domestic animals with in situ hybridization and epifluorescent microscopy : master of science thesis
Tepšič, Katarina
Rumen bacteria from the genus Prevotella were specifically detected in pure and mixed bacterial cultures and in the rumen fluid samples from a black and white Frisian cow with in situ hybridization ...using broad and species specific 16S rRNA oligonucleotide probes. The probes were labelled with fluorescine isothiocyanate (FITC) or tetramethylrodamine isothiocyanate (TRITC). After the hybridization the fluorescence was detected with epifluorescence microscopy. The specific identification of targeted cells was successful. However, the quantification of in situ hybridized microbial cells under epifluorescent microscope proved to be too demanding and useless as the method for rutine examinations of complex microbial samples without computer assisted digital image analysis system. Therefore a quantitative method for the enumeration of P.bryantii bacterial cells in the rumen fluid samples was developed. The method used is the competitive polymerase chain reaction (c-PCR). PBB1ž4 probe, previously used as a probe in in situ hybridisation experiments, was used as a reverse PCR primer and EUB33B was used as the forward PCR primer. An internal DNA control, based on the analysisof the 16S rDNA sequence of the P. bryantii B1ž4 strain, was constructed. C-PCR products were quantified by capillary electrophoresis and the results were obtained using the regression equation. Using the described system it was estimated that the concentration of P. bryantii celIs, specifically recognised by the PBB1ž4 oligonucleotide, is 2,4 x 10ž7 in a ml of rumen fluid.
Type of material - master's thesis
Publication and manufacture - Ljubljana : [K. Tepšič], 1999
magistrske naloge TEPŠIČ Katarina Odkrivanje IN: 099900556
available - outside loan, loan period: 1 months
National Institute of Biology and BF, Department of Biology, Ljubljana
BF oddelek za biologijo mg 579 TEPŠIČ K. Odkrivanje IN: 0032030
available - reading room
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