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How to quantify bacteria in sediments?
Parkes, R.J., B.A. Cragg and P. Wellsbury, 2000
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Perry & Staley, Microbiology –Dynamics and Diversity
Phasecontrast microscopy
Counting 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*10
6base 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
© 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
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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
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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 1,9·10
7cm
3Fatty acids 4,0·10
6cm
3MPN counts in tidal flat sediment
10°C 4,0·10
5cm
320°C 8,2·10
6cm
330°C 4,0·10
5cm
3MPN counts in tidal flat sediment
Oxic 1,0·10
7cm
3Anoxic 4,0·10
5cm
3How 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
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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)
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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 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
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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
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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
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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
2O
Peroxidase
H2O2Activation
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
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Higher sensitivity by signal amplification
FISH CARD-FISH
depth (cm) Fig.: M. Mussmann
Tidal flat sediment
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PCR techniques
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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
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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)
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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
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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
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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