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Basic parasitological practical course demonstrating general as well as advanced and specific experimental laboratory methods including staining procedures (preparation and staining of blood smears according to Giemsa-Romanowski or using Dade Diff-Quik staining kit), handling of laboratory mice for the purposes of experimental infection or immunization, cultivation techniques (maintenance of axenic cultures, cell counting and cloning, growth and axenization experiments, calculation of growth parameters), immunological methods (ELISA, indirect immunofluorescence), DNA isolation, diagnostic PCR, isolation of recombinant protein, electrophoretic methods, work with inset vectors transmitting diseases, etc.
Last update: Hrdý Ivan, doc. RNDr., Ph.D. (02.09.2024)
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Printed protocols for actual methds and tasks are provided to every student at the course.
http://web.natur.cuni.cz/parasitology/vyuka/para_lab_techniky_Hrdy/Parazitol_lab_tech_Hrdy.htm Last update: Hrdý Ivan, doc. RNDr., Ph.D. (02.09.2024)
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Active presence at the course is required. One justified absence is acceptable. Final test may be required, in relation to the performance of the students during the course. Last update: Hrdý Ivan, doc. RNDr., Ph.D. (02.09.2024)
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Staining methods, manipulation with laboratory mouse
- blood smear, thick drop - staining of dry smears by Giemsy-Romanowski method - fast staining by Dade Diff-Quik - manipulation with laboratory mouse, infection with Trypanosoma brucei, Plasmodium berghei
Basic cultivation techniques
- preparation of antibiotic stock solutions - isolation of the Tritrichomonas foetus axenic culture using a migration tube - passage of axenic cultures, sterility test - freezing techniques, cryopreservation of protozoa
Cloning
- Isolation of clones by dilution method (Tetrahymena pyriformis) - isolation of clones by limiting dilution method (Trichomonas vaginalis) - isolation of clones on agar plates (Crithidia fasciculata)
Quantitative work with protists
- Work with Burker's counting chamber - calculation of cell concentration - evaluation of the growth experiment, calculation of doubling time from growth curves
ELISA
- use in diagnostics - test for the presence of antibodies against Toxoplasma gondii
PCR
- use in diagnostics - PCR test for human pathogens / commensals - DNA isolation methods - DNA electrophoresis, visualization
Preparation and isolation of recombinant protein
- work with bacterial culture, conditions of induction and expression of foreign protein - protein isolation in native and denatured form by affinity chromatography - analysis of protein sample by electrophoresis - electrophoretic methods Fluorescent staining methods
- vital staining with acridine orange - nuclear staining by Hoechst or DAPI - pH sensitive fluorescent probes - FITC labeling - immunofluorescence on wet mounts
Work with insect vectors - experimental blood feeding of mosquitoes Last update: Hrdý Ivan, doc. RNDr., Ph.D. (02.09.2024)
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