How to quantify bacteria in sediments?

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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


Perr

y &

Stal

ey, M

icro

biol

ogy

–D

ynam

ics

and

Div

ersi

ty

Phasecontrast microscopyCounting chamber (Thoma, Petroff-Hausser, ...)Only feasable for liquid samples.

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Filtration of samples for the Epifluorescence-microscopy


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Amount of DNA

(2-4*106 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/pmbioFigs: Station Biologique de Roscoff CNRS and Université Pierre et Marie Curie, France

Flow cytometry

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Perry & Staley, Microbiology – Dynamics and Diversity

Colony Forming Units (CFU)


Slurry3

Slurry4

Kontrolle

ABCDEFGH

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

Most Propable Number

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MPN quantification Detectedgrowth

MPN index[cells/ml]

Confidence interval (95%)


Anoxic Oxic

not pasteurised

pasteurised

1.35%0.15%0.21%0.06%

< 0.01%< 0.01%

Dep

th [cm

]

0

100

200

300

400

500

0 1 2 3 4 5 6 7600

0 1 2 3 4 5 6 7 8

log viable counts [cm-3]

MPN values within a tidal flat sediment column

Beate Köpke

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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 1,9·107 cm3

Fatty acids 4,0·106 cm3

MPN counts in tidal flat sediment10°C 4,0·105 cm3

20°C 8,2·106 cm3

30°C 4,0·105 cm3

MPN counts in tidal flat sedimentOxic 1,0·107 cm3

Anoxic 4,0·105 cm3


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 speciesin 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

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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)


Dot Blot analysis of bacterial communities

Felske et al. 1996

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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 differentphylogenetic levels

Detection under a microscopicslide (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. 2

005

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Fluoresce In Situ Hybridisationnatural microbial

communityFixation

Treatment with fixative, conditioning of cells,filtration

WashingDetachment of probes that were not bound to the target sequence

HybridisationAnnealing of probes under stringent conditions

Fluorescentdye

Specificprobes

16S rRNA

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

VisualisationEpifluorescence microscopy

ProbeDAPI

Relation of non specific to specific signals

Fig.

: B. R

ink


ProblemsProbe 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

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CAtalysed Reporter Deposition - FISH

natural microbial community

FixationTreatment with fixative, conditioning of cells,filtration

HybridisationAnnealing of probes under stringent conditions

Horseradish-peroxidase, HRP

Specificprobes

new

16S rRNA

WashingDetachment of probes that were not bound to the target sequence Fi

g.: B

. Rin

k


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

B

Protein(Tyrosin)H2O

Peroxidase

H2O2

Activation

Enrichment

*

Peroxidase

H2O2

FluorescentdyeTyramide

inactiv

Anew

newWashing

Molecules that were not converted

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

VisualisationEpifluorescence microscopy

ProbeDAPI

Relation of non specific to specific signals

Fig.

: B. R

ink

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Higher sensitivity by signal amplification

FISHCARD-FISH

dept

h (c

m)

Fig.

: M. M

ussm

ann

Tidal flat sediment


PCR techniques

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SIGnature PCR

Separation in an agarose gel

g-Proteobacteria

Firmicutes, High-GC

a-Proteobacteriab-Proteobacteria

CFB

Bacteriaca. 1500 bp

1000 bp

700 bp650 bp

350 bp

100 bp

Amplification of specificPCR products with different length


SIG-PCR with Mediterranean isolates

Reference unknown isolates

Süß et al. 2004

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indifferent

4 marin phototrophic α-Proteobacteria

α-Proteobacteria

Gram positive high GC

γ-Protoebacteria31

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)


Quantitative (real time) PCR

SybrGreen ITM-technique

⇒ low fluorescence ⇒ increasing fluorescence

Amplification

No binding at single stranded DNA

Intercalation of SybrGreen at double stranded DNA

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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

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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


Application of the qPCR on Mediterranean sedimentsMethod: 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 operonsAbsoluteRelative

How to quantify bacteria in sediments?

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