Postavljanje Winogradsky stupca

1. 500 ml cilindar
2. CaSO4
3. CaCO3
4. papirnati ručnici
5. blato
Postupak:
1. blato i vodu dopremiti u laboratorij
2. Čajnu žlicu CaCO3, CaSO4 i stare kolumne staviti u stupac i pomiješati s
blatom. Dodati vode.
3. Dodati punu šaku narezanih ručnika – celuloza. Paziti na mjehuriće
zraka!
4. Dodati blato do vrha.
5. Dodati vodu, stupac se ne smije isušiti
6. Inkubirati stupac na sobnog temperaturi, ne na direktnoj sunčevoj
svjetlosti. Kroz 3-6 tjedana trebali bi se vidjeti rezultati.
 PRAKTIKUM IZ
MIKROBIOLOGIJE
EKOSUSTAVA
METODE BROJANJA
STANICA
INDIREKTNO:
• ploče
• MPN

DIREKTNO:
• Svjetlosna mikroskopija
• Fluorescentna mikroskopija
• Protočna citometrija (“flow cytometry”)

Brojanje krupnijih stanica:

U komoricama, hemocitometrom ili elektronskim
brojačem stanica (Coulter-counter, flow cytometer).
Method Advantages Disadvantages
Visual Simple, direct, only need a Tedious, subjective, difficult for small
microscopy basic microscope or rare cells
Brightfield

Epifluorescence Good for small, rare cells, Requires specially equipped

especially good for natural microscope, more complicated
bacteria, picoplankton and sample/slide preparation
heterotrophs

Image analysis Objective, automated No standard system/method, requires

special equipment, software

Fluorometer Simple, direct, minimal Fluorescence per cell not constant,

sample handling, best for absolute cell number requires accurate
quick relative monitoring of cell counts for calibration
cultures

Coulter counter Rapid, precise, objective, Requires special (expensive)

automated, produces size equipment, high maintenance
spectrum, good for cultures (clogging), counts all particles

Flow cytometry Rapid, precise, objective, Expensive instrument, must accurately

automated, counts cell only measure volume analyzed
Bürker-Türk Neubauer

20 x 20 μm 25 x 25 μm

h = 0.1 mm h = 0.1 mm
 ⎛ Σn( fitoplankton) ⎞
⎜⎜ ⎟⎟
⎝ n( komorice) ⎠
Gustoća(fitoplanktona L-1) = /V(procijeđene vode)
V (komorice)
Komorice za sedimentaciju
Biološki odsjek PMF-a
Fazno-kontrasni mikroskop
Biološki odsjek PMF-a
Fluorescentna mikroskopija
(epifluorescence
microscopy)

Synechococcus sp. Mesodinium rubrum

Primarni pigment fikoeritrin fototrofni
cilijat;endosimbiotski
Lijevo – ekscitacija plavim svjetlom cryptophyta
Desno – zeleno svjetlo L-D plavo, UV, zeleno
Photosynthetic picoplankton from the Pacific Ocean (off the Marquesas islands observed by epifluorescence
microscopy (blue exciting light). Orange fluorescing dots correspond to Synechocococus cyanobacteria, red
fluorescing dots to picoeukaryotes. Larger cells (e.g. diatom, upper right) can also be seen

Daniel Vaulot, CNRS, Station Biologique de Roscoff

Workshop, November 2004: “The Role of Flow Cytometry in
Marine Biodiversity and Ecosystem Functioning”, Stazione
Zoologica “Anton Dohrn”, Napulj, Italija
Klut, M. E., T. Bisalputra and N. J. Antia
(1988). The use of fluorochromes in the
cytochemical characterization of some
phytoflagellates. Histochemical J. 20: 35-40.

Klut, M. E., J. Stockner and T. Bisalputra

(1989). Further use of fluorochromes in the
cytochemical characterization of
phytoplankton. Histochem. J. 21: 645-650.
Coulter counter-brojač stanica
Flow cytometry (protočna citometrija)
Dubelaar 2000.
Daniel Vaulot, CNRS, Station Biologique de Roscoff
 Phytoplankton Populations

Cultures

Sargasso Sea

Poulton N. Bigelow Laboratory for Ocean Sciences

Total Particles - York
River
High levels of detritus in estuarine water
show high side scatter relative to forward
scatter

Poulton N. Bigelow Laboratory for Ocean Sciences

 Flow Cytometry
Applications
Bacteria
• Accurate Cell Counts ? Abundance
• Phytoplankton ? Activity
? Population abundances •Respiration
? Size spectra (CTC)
? Growth rates Protozoa
? Cell cycle Viruses
• Particles Functional Probes
? Detritus Invertebrate
? Heterotrophs
? Inorganic
Grazing
Filter feeding
Size selection
http://bioloc.coas.oregonstate.edu/SherrLab/GLOBEC.html
FlowCAM (Flow Cytometer And Microscope)

http://www.bigelow.org/flowcam/
FlowCAM Interactive Scattergram

http://www.bigelow.org/flowcam/