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© Bert Engelen www.icbm.de/pmbio

How to quantify bacteria in sediments?

Parkes, R.J., B.A. Cragg and P. Wellsbury, 2000

© Bert Engelen www.icbm.de/pmbio

Perry & Staley, MicrobiologyDynamics and Diversity

Phasecontrast microscopy

Counting chamber (Thoma, Petroff-Hausser, ...)

Only feasable for liquid samples.

(2)

© Bert Engelen www.icbm.de/pmbio

Filtration of samples for the Epifluorescence-microscopy

(3)

© Bert Engelen www.icbm.de/pmbio

Amount of DNA

(2-4*10

6

base pairs per procaryotic genome) Amount of ATP

ATP * 250 BioC (in g) Flowcytometer

Direct counts in a capillary system

Other methods

© Bert Engelen www.icbm.de/pmbio Figs: Station Biologique de Roscoff CNRS and Université Pierre et Marie Curie, France

Flow cytometry

(4)

© Bert Engelen www.icbm.de/pmbio Perry & Staley, Microbiology – Dynamics and Diversity

C olony F orming U nits (CFU)

Slurry 3

Slurry 4 Kontrolle A

B C D E F G H

1 2 3 4 5 6 7 8 9 10 11 12

10-1 10-3 10-5

10-2 10-4 10-6

10-6 10-4 10-2 10-5 10-3 10-1

Slurry 1

Slurry 2 Kontrolle

M ost P ropable N umber

(5)

© Bert Engelen www.icbm.de/pmbio

MPN quantification

Detectedgrowth MPN index[cells/ml] Confidence interval (95%)

© Bert Engelen www.icbm.de/pmbio

Anoxic Oxic

not pasteurised pasteurised 1.35%

0.15%

0.21%

0.06%

< 0.01%

< 0.01%

Depth [cm]

0 100 200 300 400 500

0 1 2 3 4 5 6 7 600

0 1 2 3 4 5 6 7 8

log viable counts [cm

-3

]

MPN values within a tidal flat sediment column

Beate Köpke

(6)

© Bert Engelen www.icbm.de/pmbio

MPN counts depend on incubation conditions

- Temperature

- Substrate

- Oxic or anoxic incubations

- Supplement of vitamins and other trace elements

MPN counts in tidal flat sediment Amino acids 1,9·10

7

cm

3

Fatty acids 4,0·10

6

cm

3

MPN counts in tidal flat sediment

10°C 4,0·10

5

cm

3

20°C 8,2·10

6

cm

3

30°C 4,0·10

5

cm

3

MPN counts in tidal flat sediment

Oxic 1,0·10

7

cm

3

Anoxic 4,0·10

5

cm

3

How many different bacteria do we expect?

Validly described species:

5 000 Prokaryotes (Bakteria und Archaea) 1 700 000 Eukaryotes

Estimations for the number of bacterial species in 30 g forrest soil

3 000

(Torsvik et al 1990, Appl Environ Microbiol 58:782-787)

500 000

(Dykhuizen 1998, Antonie van Leeuwenhoek 73:25-33) (based on the same set of data)

similar for sediments

(7)

© Bert Engelen www.icbm.de/pmbio

Application of molecular probes

Techniques:

Membrane- hybridisation

Extracted, immobilised RNA/DNA (Dot Blot, DNA-Chips)

Fluorescence In-situ Hybridisation, FISH:

Fixed cells (binding at ribosomes)

Signal enhancement by higher ribosome content

Specificity:

Strain, family, ... up to domain (dependent on target sequence)

Hybridisation:

Probe (Oligonucleotide) at a target sequence (mostly 16S/23S rRNA)

© Bert Engelen www.icbm.de/pmbio

Dot Blot analysis of bacterial communities

Felske et al. 1996

(8)

© Bert Engelen www.icbm.de/pmbio

Analysis of bacterial communities by Fluorescence In-situ Hybridisation, FISH

Stronghold of Fluorescence In-situ Hybridisation in Germany is the MPI in Bremen!

Coupling of molecular „probes“

with fluorescence dyes

Speciffic annealing at regions of the rRNA

Staining of cells on different phylogenetic levels

Detection under a microscopic slide (In-situ)

Anaerobic methane oxidising consortia

ANME2 (EelMS932)

Desulfosarcina (DSS658)

Boetius, et al. (2000) Nature. 407:623-626

5 µm

detected in gas hydrate bearing sediments

DAPI CARD-FISH

Archaea (ARCH915) Desulfosarcina (DSS658) detected in

tidal flat sediments

Ishi et al. 2005

(9)

© Bert Engelen www.icbm.de/pmbio

Fluoresce In Situ Hybridisation

natural microbial community

Fixation

Treatment with fixative, conditioning of cells, filtration

Washing

Detachment of probes that were not bound to the target sequence

Hybridisation

Annealing of probes under stringent conditions

Fluorescent dye Specific

probes

16S rRNA

Counter staining

Staining of all cells by a general fluorescent dye (e.g. DAPI)

Visualisation

Epifluorescence microscopy

Probe DAPI

Relation of non specific to specific signals

Fig.: B. Rink

© Bert Engelen www.icbm.de/pmbio

Problems

Probe signal depends on the amount of ribosomal RNA, and therefore on the physiological state of the cells!

DAPI counter stain includesinactive cells and even spores

Interpretation often difficult

Optimisation : Signal amplification by CARD-FISH Especially for samples that show high autofluorescence like sediment, algae, cyanobacteria

The Relation of non specific to specific signals can be distorted

(10)

© Bert Engelen www.icbm.de/pmbio

CAtalysed Reporter Deposition - FISH

natural microbial community

Fixation

Treatment with fixative, conditioning of cells, filtration

Hybridisation

Annealing of probes under stringent conditions

Horseradish- peroxidase, HRP Specific

probes

new

16S rRNA

Washing

Detachment of probes that were not

bound to the target sequence

Fig.: B

. Rink

Tyramide signal amplification (TSA)

marked substrate (Tyramide) is enriched within the cell by chemical reaction and binding to proteines

B

Protein (Tyrosin) H

2

O

Peroxidase

H2O2

Activation

Enrichment

* Peroxidase

H2O2

Fluorescent dye Tyramide

inactiv A

new

Washing new

Molecules that were not converted

Counter staining

Staining of all cells by a general fluorescent dye (e.g. DAPI)

Visualisation

Probe DAPI

Fig.: B. Rink

(11)

© Bert Engelen www.icbm.de/pmbio

Higher sensitivity by signal amplification

FISH CARD-FISH

depth (cm) Fig.: M. Mussmann

Tidal flat sediment

© Bert Engelen www.icbm.de/pmbio

PCR techniques

(12)

© Bert Engelen www.icbm.de/pmbio

SIGnature PCR

Separation in an agarose gel

g-Proteobacteria Firmicutes, High-GC a-Proteobacteria b-Proteobacteria CFB

Bacteria ca. 1500 bp

1000 bp 700 bp 650 bp 350 bp

100 bp

Amplification of specific PCR products with different length

SIG-PCR with Mediterranean isolates

Reference unknown isolates

Süß et al. 2004

(13)

© Bert Engelen www.icbm.de/pmbio

indifferent

4 marin phototrophic α-Proteobacteria

α-Proteobacteria Gram positive high GC γ-Protoebacteria

31

38 13 32

Result of SIG-PCR screening of isolates from Mediterranean sediments

no growth in AS media

50% from SED almost exclusively

from MPN plates (MKS, AS, Alk)

© Bert Engelen www.icbm.de/pmbio

Quantitative (real time) PCR

SybrGreen I

TM

-technique

⇒ low fluorescence ⇒ increasing fluorescence Amplification

No binding

at single stranded DNA Intercalation of SybrGreen

at double stranded DNA

(14)

© Bert Engelen www.icbm.de/pmbio

qPCR protocol

PCR reaction components Temperature program

- Stainless polymerase - SybrGreenI

- 10µl of DNA template

- Detection of fluorescence after every elongation step

- Melting curve analysis

The maschine

Rotor-Gene 2000/3000 Corbett Research, Australia

Raw data analysis

Rotor and

detection units

(15)

© Bert Engelen www.icbm.de/pmbio

Data analysis

Threshold value:

-Level of highest amplificaton rate Ct -values:

- Number of cycles that are needed to reach the threshold - in direct relation to copy number

of the original sample -„normalised“ rawdata

Standard curve

- Calculation of DNA copies in the original sample

-Number of organisms calculated by genome size and 16S rRNA copy number

© Bert Engelen www.icbm.de/pmbio

Application of the qPCR on Mediterranean sediments Method: Rhizobium specific real time -PCR with SybrGreen I

Results

⇒ widely distributed in Mediterranean sediments

⇒ enhanced numbers in sapropels ⇒ up to 5% of eubacteria

⇒ typical deep biosphere organisms 16S rRNA operons

Absolute Relative

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