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Course, academic year 2019/2020
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Advanced limnological methods - MB160C13
Title in English: Speciální limnologické metody
Czech title: Speciální limnologické metody
Guaranteed by: Department of Ecology (31-162)
Faculty: Faculty of Science
Actual: from 2011
Semester: summer
E-Credits: 3
Examination process: summer s.:
Hours per week, examination: summer s.:0/1 C [weeks/semester]
Capacity: 6
Min. number of students: unlimited
State of the course: taught
Language: Czech
Level: specialized
Guarantor: prof. RNDr. Jaroslav Vrba, CSc.
Annotation -
Last update: RNDr. Veronika Sacherová, Ph.D. (05.05.2011)
The aim of the lecture is: (1) to explain the major principles of selected recent methods frequently used in
limnological research based on fluorescent microscopy and radioisotope labeling approaches, and (2) to practice
them in the laboratory when solving simple tasks in order to determine some selected parameter of population
abundance, biomass, and activity. These methods are mostly very sensitive, simple and not time-consuming,
enabling specific labeling of individuals or populations. We put our emphasis on conducting simple laboratory
experiments and an appropriate data interpretation by students.
ATTENTION: the course is suitable for second year master students and after attending basic Limnological
methods (MO550P86).
Literature - Czech
Last update: VSACH (22.03.2005)

1. Riemann,B., Sondergaard,M. (Eds): Carbon dynamics in eutrophic, temperate lakes, (248 str.). Elsevier, Amsterdam, 1986.

2. Preparation of samples for liquid scintillation counting (a catalog) Nuclear Chicago Corporation, Des Plaines, USA, 1976.

3. Sherr B. F., Sherr E.B. and Fallon R. D., 1987. Use of monodispersed, fluorescently labeled bacteria to estimate in situ protozoan bacterivory. Appl. Environ. Microbiol., 53, 958-965.

4. Porter K. G. and Feig Y.S., 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr., 25, 943-948.

5. Hobbie,J.E., Daley,R.J. and Jasper,S., 1979: Use of Nuclepore filters for counting bacteria by epifluorescence microscopy. Appl. Environ. Microbiol. 33: 1225-1228.

Requirements to the exam - Czech
Last update: RNDr. Veronika Sacherová, Ph.D. (18.11.2011)

Zápočet je udělován za aktivní účast na kurzu a vypracované protokoly ze všech úloh.

Syllabus -
Last update: VSACH (22.03.2005)

L - lecture, P - laboratory practicing

1) L: Fluorescence microscopy, primary and secondary fluorescence, specificity of fluorochrome labeling

P: Microscopic preparations with a low fluorescence background, staining with DTAF and acridine orange, microorganisms containing chloroplasts (aplastidic versus plastidic microbes) in plankton samples

2) L: Staining with DNA-bound fluorochromes, a differentiation of prokaryotic versus eukaryotic microorganisms in a fluorescence microscope

P: Staining with the fluorochrome DAPI - counting of bacteria and protists, an easy differentiation of nuclei of microorganisms; staining with the fluorochrom primulinem - an observation of surface structures of protists (cilia, flagella)

3) L: Fluorochrom double-labeling approaches in microbial ecology, a model predator-prey system - bacteria-protists

P: Labeling of bacteria by the fluorochrome DTAF, DAPI-staining of protists, determination of grazing rates of protists on bacteria

4) L: Major principles for analyzing kinetic data in biochemistry: time-course, saturation kinetics, models for plotting the data, software.

P: Evaluating kinetic data by means of PC

5) L: Spectrofluorometry, major principles and applications

P: Work with fluorometer, its calibration, a calibration curve with methylumbelliferone (MUF).

6) L: A fluorometric assay of extracellular enzyme activities

P: Determining extracellular phosphatase and ß-glucosidase activities with MUF-substrates

7) L: Work with radioisotopes: Major instructions and principles for safety work with radioisotopes, equipment desired for a radioisotope laboratory of the first category

P: How to use the equipment of the radioisotope laboratory. A test of instructions and regulations valid when working with radioisotopes (a prerequisite to be allowed to work with them).

8) L: Measuring sample radioactivity via scintillation spectrometry. Theory, applications, and a calibration of scintillation counter, data evaluation.

P: Practical work with a scintillation counter, the calibration by means of external standards

9) L: 14C-assay, measuring primary production, its proportion released in extracellular products.

P: 14C-assay, measuring primary production, part I.

10) L: A turnover rate of dissolved orthophosphate as assessed by 32P method, incorporation of 32P, an estimation of available orthophosphate according to Rigler

P: 14C-assay, measuring primary production, part II.

11) L: Bacterial production estimated via an uptake of 3H-thymidinu, 3H-leucinu, 35S

P: Bacterial production estimated via an uptake of 3H-thymidine

12) L: Incorporation and oxidation of 14C-substrates (glucose, acetate, etc.), estimating heterotrophic bacterial activity according to Hobbie & Wright (1978).

P: Incorporation rate of 14C-glucose.

13) L: Microautoradiography - principles of the method, possibilities to use differently labeled substrates (3H, 14C, 32P, 33P) for plankton organisms of various size.

P: Microautoradiography with 3H-labeled substrates, interpretation of results.

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