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Postavljanje Winogradsky stupca1. 500 ml cilindar 

2. CaSO4

3. CaCO3

4. papirnati ručnici

5. blatoPostupak:

1. blato i vodu dopremiti u laboratorij

2.Č

ajnu žlicu CaCO3, CaSO4 i stare kolumne staviti u stupac i pomiješati sblatom. 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.

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PRAKTIKUM IZ

MIKROBIOLOGIJE

EKOSUSTAVA

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METODE BROJANJAETODE BROJANJA

STANICATANICAINDIREKTNO:

• ploče

• MPN

DIREKTNO:

• Svjetlosna mikroskopija

• Fluorescentna mikroskopija

• Protočna citometrija (“flow cytometry”)

Brojanje krupnijih stanica:

U komoricama, hemocitometrom ili elektronskimbrojačem stanica (Coulter-counter, flow cytometer).

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Expensive instrument, must accurately

measure volume analyzed

Rapid, precise, objective,

automated, counts cell only

Flow cytometry

Requires special (expensive)equipment, high maintenance

(clogging), counts all particles

Rapid, precise, objective,automated, produces size

spectrum, good for cultures

Coulter counter

Fluorescence per cell not constant,

absolute cell number requires accurate

cell counts for calibration

Simple, direct, minimal

sample handling, best for

quick relative monitoring of 

cultures

Fluorometer

No standard system/method, requires

special equipment, software

Objective, automatedImage analysis

Requires specially equipped

microscope, more complicated

sample/slide preparation

Good for small, rare cells,

especially good for natural

bacteria, picoplankton andheterotrophs

Epifluorescence

Tedious, subjective, difficult for small

or rare cells

Simple, direct, only need a

basic microscope

Visual

microscopy

Brightfield

DisadvantagesAdvantagesMethod

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Bürker-Türk

20 x 20 μm

h = 0.1 mm

Neubauer 

25 x 25 μm

h = 0.1 mm

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Gustoća(fitoplanktona L-1) =(komorice)V

)(

)(⎟⎟

 ⎠

 ⎞⎜⎜

⎝ 

⎛ Σ

komoricen

on fitoplanktn

/V(procijeđene vode)

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Komorice za sedimentaciju

Biološki odsjek PMF-a

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Fazno-kontrasni mikroskop

Biološki odsjek PMF-a

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Fluorescentna mikroskopija

(epifluorescencemicroscopy)

Synechococcus sp.

Primarni pigment fikoeritrin

Lijevo – ekscitacija plavim svjetlom

Desno – zeleno svjetlo

Mesodinium rubrum fototrofni

cilijat;endosimbiotski

cryptophyta

L-D plavo, UV, zeleno

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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 

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Workshop, November 2004: “The Role of Flow Cytometry in

Marine Biodiversity and Ecosystem Functioning”, Stazione

Zoologica “Anton Dohrn”, Napulj, Italija

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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.

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Coulter counter-brojač stanica

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Flow cytometry (protočna citometrija)

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Dubelaar 2000.

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Daniel Vaulot, CNRS, Station Biologique de Roscoff 

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Phytoplankton Populations

Cultures

Sargasso Sea

Poulton N. Bigelow Laboratory for Ocean Sciences

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Total Particles - York

RiverHigh levels of detritus in estuarine water 

show high side scatter relative to forwardscatter 

Poulton N. Bigelow Laboratory for Ocean Sciences

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Flow Cytometry

Applications

• Accurate Cell Counts

• Phytoplankton Population abundances

Size spectra

Growth rates

Cell cycle

• Particles

Detritus Heterotrophs

Inorganic

Bacteria Abundance

Activity•Respiration

(CTC)

ProtozoaViruses

Functional Probes

InvertebrateGrazing

Filter feeding

Size selection

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http://bioloc.coas.oregonstate.edu/SherrLab/GLOBEC.html

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FlowCAM (Flow Cytometer And Microscope)

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

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FlowCAM Interactive Scattergram

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