LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMOSTRATION. financed by the program

Size: px

Start display at page:

Download "LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMOSTRATION. financed by the program"

Austen Turner
5 years ago
Views:

1 TÁMOP C-13/1/KONV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMOSTRATION financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: NOVEMBER 23. / NOVEMBER 23, 2016 Helyszín / Place: SZBK NÖVÉNYBIOLÓGIAI INT., 104. LAB. / LAB # 104, INST. PLANT BIOLOGY, BRC SZEGED, TEMESVÁRI KRT. 62 Gyakorlati foglalkozás címe / Title of the practical demonstration: Gyakorlatvezető / Demonstrator: BIOMEDICAL POTENTIAL OF CYANOBACTERIA AND ALGAE ISTVÁN-ZOLTÁN VASS Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB

Module #1 Growth conditions of cyanobacteria and microalgae During the Practical you will have an insight into the appearence, cultivation and harvesting of cyanobacterial and algal cultures, and

In the laboratory these cultures can best be maintained in photobioreactors, which serve for small scale cultivation of algae, bacteria or cyanobacteria, and are suited for the monitoring of growth

2 Module #1 Growth conditions of cyanobacteria and microalgae During the Practical you will have an insight into the appearence, cultivation and harvesting of cyanobacterial and algal cultures, and into the way of estimation of growth and protein content of the cells. In the laboratory these cultures can best be maintained in photobioreactors, which serve for small scale cultivation of algae, bacteria or cyanobacteria, and are suited for the monitoring of growth under various conditions. Notes / instructions continued: You will have three Synechocystis cultures grown in a photobioreactor under different light conditions (10 µe, 80 µe, 640 µe) Your task will be to estimate the doubling time of each culture and specify the most optimal light intensity for their growth: o export growth data from the photobioreactor to the computer attached o the softwer will plot the data in a diagram as shown below: o export the data from the program and import it into Excel o using the data in Microsoft Excel determine the doubling rate of the Syenchocystis cells as a function of time page 2

3 Please define the doubling rate/time of Synechocystis under different light conditions: A. 10 µe.. B. 80 µe.. C. 640 µe.. page 3

Module #2 Identification of cyanobacteria and green algae A B You will be given five microbial cultures grown in conical flasks.

Note the hue (colour) difference. In order to identify the microorganisms an optical microscope will be provided.

Synechocystis (small unicellular cyanobacterium of 1µm) B. Nostoc (filamentous cyanobacterium with long straight filaments of uniform cells) C.

4 Module #2 Identification of cyanobacteria and green algae A B You will be given five microbial cultures grown in conical flasks. You need to identify which sample contains one of the following microbes. Note that the appearances of the cultures are similar. Note the hue (colour) difference. In order to identify the microorganisms an optical microscope will be provided. Take 10 µl from each sample with a micropipette, place it on a slide and cover with a cover slip. You will have to differentiate between: C A. Synechocystis (small unicellular cyanobacterium of 1µm) B. Nostoc (filamentous cyanobacterium with long straight filaments of uniform cells) C. Nostoc in combined nitrogen depletion (filamentous with specialized cells called heterocysts for N 2 fixation) D. Spirulina (filamentous cyanobacterium with long spiral filaments of even cells) E. Chlamydomonas (larger unicellular eukaryotic cells 10µm) page 4

5 D E Notes / instructions continued: Please connect the numbers on the conical flasks with the marks of the corresponding cultures! A B C D E page 5

Module #3 Determination of the absorption spectra of photosynthetic pigments in cyanobacteria and green algae In order to release biological molecules from cyanobacterial cells a straightforward

put the tubes on ice immerse the tip of the sonicator ¼-½ of the cell suspension, then: 1. turn on button 1 2. set time (knob 2) to 1-2 minutes 3. set duty cycle (knob 3) to 50% 4.

6 Module #3 Determination of the absorption spectra of photosynthetic pigments in cyanobacteria and green algae In order to release biological molecules from cyanobacterial cells a straightforward method is the sonication of the cells using ultrasound: 5-5 ml Synechocystis and Chlamydomonas suspension will be processed in 15 ml tubes. put the tubes on ice immerse the tip of the sonicator ¼-½ of the cell suspension, then: 1. turn on button 1 2. set time (knob 2) to 1-2 minutes 3. set duty cycle (knob 3) to 50% 4. set the output control (knob 4) to the process should be repeated 3 times with 1 minute pause in between for cooling the tip and the sample on ice 6. put aside 2,5ml (for protein measurement) and add to remaining cell suspension STET-L reagent as follows: 1 ml suspension+100µl STET-L (This reagent contains detergents and Lysozyme for disintegrating the membrane) 7. Incubate the lysate for 5-10 minutes. 8. Centrifuge for 3 minutes at 3000 rpm and save the supernatant for spectrophotometric inspection. Warning: You should always wear a protective headset during the sonication process. page 6

7 Two absorption spectra are shown above: one of them is specific for cyanobacteria (Synechocystis) and the other for green algae (Chlamydomonas). Your task will be to record the spectrums from the above (sonicated and STET-Ltreated) samples Measurements will be set in the range of visible light between nm Specify which spectrum belongs to which organism! A. Red spectrum: B. Black spectrum: page 7

8 Module #4 Estimation of the protein content of cyanobacteria Your task will be to estimate the protein content of Synechocystis cells using the Bradford protein assay Prepare a stock of BSA solution of 1 mg/ml Prepare a series of dilutions containing 0, 1, 10, 20, 30, 40, 50, 60 µg (=µl) BSA and fill them to 100 µl with BG-11 medium Add to each 900 µl 1x Bradford reagent, mix, wait 5-10 minutes and measure the absorbance at 595 nm. Determine the protein content of 1 g (wet weight) Synechocystis cells as follows: Measure the absorbance of your sample: mix 100 µl of above cell extract (sonicated samples) with 900 µl 1x Bradford reagent, wait 5-10 minutes and record the absorbance and estimate the protein content. Using an analytical balance you will measure the weight of a 1.5 ml Eppendorftube. Transfer 1ml Synechocystis cell suspension in the 1.5 ml Eppendorf-tube and centrifuge for 2-3 minutes 12,000 rpm. Discard supernatant completely. Measure again the weight of the tube containing pellet. The difference will be the wet weight of the cells. Using the information acquired in module #1 regarding the doubling rate of the cells, calculate the protein production of 1 gram cells for a period of 1 month. page 8

Module #5 Morphological comparison of plant chloroplasts with cyanobacteria cells Take an Elodea leaf on a slide, add 10 µl Synechocystis culture and examine it under an optical microscope!

9 Module #5 Morphological comparison of plant chloroplasts with cyanobacteria cells Take an Elodea leaf on a slide, add 10 µl Synechocystis culture and examine it under an optical microscope! Note the similarities between cyanobacteria and chloroplasts! Note the movement of the chloroplasts inside the cell cytosol! Compare the size of the cyanobacterial cells and the chloroplasts! Add 10 µl Chlamydomonas culture! Compare the sizes of the cells and chloroplasts! Estimate the number of bacterial cells that would total up to the volume of one Chlamydomonas or plant cell! page 9

ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT. financed by the program

ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT. financed by the program TÁMOP-4.1.1.C-13/1/KONV-2014-0001 projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program