Syllabus
1. Fundamentals of the laboratory work Theoretical part: - safety, weights & measures, the fundamentals of pipette handling, introduction of the basic laboratory equipment (centrifuges – balance, RPM/g, flowbox.), sterile work. Practical part: - calculations: unit conversions, volume concentration, molar concentration - preparation of the solutions – dilution, pH adjustment and measurement - preparation of the growth media and agar plates
2. Dry smear preparation and staining, handling the laboratory mouse Theoretical part: - parasitic protozoa transmitted by insects; laboratory animals in parasitology and microbiology Practical part: - blood smear infections of Trypanosoma brucei, Plasmodium berghei - thick drop – diagnostics of Plasmodium berghei - dry smears - Giemsa-Romanowsky staining, Dade Diff-Quik staining - handling of the laboratory mouse, IP inoculation
3. Cloning, quantitative experiments with protozoa Theoretical part: - growth of the microbial cultures, growth curve, calculation of the generation time; cloning methods, colony-forming efficiency Practical part: - usage of the Burker counting chamber - cell counting - handling the liquid media, maintaining cells in culture - cloning by limiting dilution (Trichomonas vaginalis)
4. Cultivation techniques Theoretical part: - types of microbial cultures, cultivation media, procedures in cryopreservation of the protozoa Practical part: - evaluation of the cloning experiment - axenization of the protozoan cultures, making of the migration tube - cryopreservation techniques, cryopreservation of the protozoa, cryobank demonstration
5. Coprological methods Theoretical part: - principles of material collection, hygienic measures, requirements, ocular micrometre calibration, methods overview Practical part: - coprological examination using flotation, sedimentation, larvoscopy
6. ELISA Theoretical part: - principle, usage in diagnostics Practical part: - antibody detection against the Toxoplasma gondii
7. Nucleic acids I Theoretical part: - DNA structure (base pairing), principle of DNA isolation on silicate, principle of isolation of DNA/RNA using TRIzol Reagent, RNA integrity control Practical part: - DNA isolation – silicate columns - DNA precipitation - determination of DNA concentration
8. Nucleic acids II Theoretical part: - PCR principle, PCR modulations (nested PCR, RT-PCR, q-PCR, multiplex PCR) Practical part: - PCR reaction preparation, operating the thermocycler - agarose gel electrophoresis - DNA purification from an agarose gel
9. Rcombinant proteins I Theoretical part: - principle of the expression of foreign protein in bacterial cell, principle of the protein electrophoresis SDS PAGE Practical part: - cryopreservation of the bacterial cultures - assessment of the protein profile of the bacterial colony using of SDS PAGE (protein solubility test)
10. Rcombinant proteins II Theoretical part: - isolation of the recombinant protein in native and denaturing conditions, principle of the isolation of the polyhistidine tagged protein Practical part: - isolation of the recombinant protein using affinity chromatography (His-Tag)
11. Mass spectrometry, immunoblot Theoretical part: - mass spectrometry analysis of biological samples (diagnostics, recombinant proteins identification, proteome analysis), sample preparation Practical part: - immunoblot – identification of His-tagged protein
Annotation
The aim of the course is to introduce students to basic laboratory practice. Students will be acquainted with basic microbiological and parasitological techniques focused on handling the protozoan cultures and diagnostics of protozoan and helminth parasites.
General part of the course is focused on molecular and biochemical methods, where understanding of fundamentals of the methods is emphasized.