How to quantify bacteria in sediments?

Transcription

1 How to quantify bacteria in sediments? Parkes, R.J., B.A. Cragg and P. Wellsbury, 2000 Phasecontrast microscopy Counting chamber (Thoma, Petroff-Hausser,...) Only feasable for liquid samples. Perry & Staley, Microbiology Dynamics and Diversity 1

2 Filtration of samples for the Epifluorescence-microscopy 2

3 Other methods 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 Flow cytometry Figs: Station Biologique de Roscoff CNRS and Université Pierre et Marie Curie, France 3

4 Colony Forming Units (CFU) Perry & Staley, Microbiology Dynamics and Diversity Most Propable Number Slurry 1 Kontrolle Slurry 2 A B C D E F G H Slurry3 Kontrolle Slurry4 4

5 MPN quantification Detected growth MPN index [cells/ml] Confidence interval (95%) MPN values within a tidal flat sediment column Depth [cm] Anoxic 0.15% 0.06% < 0.01% Oxic 1.35% 0.21% < 0.01% not pasteurised pasteurised Beate Köpke log viable counts [cm -3 ] 5

6 MPN counts depend on incubation conditions - Temperature MPN counts in tidal flat sediment 10 C 4, cm 3 20 C 8, cm 3 30 C 4, cm 3 -Substrate MPN counts in tidal flat sediment Amino acids 1, cm 3 Fatty acids 4, cm 3 - Oxic or anoxic incubations MPN counts in tidal flat sediment Oxic 1, cm 3 Anoxic 4, cm 3 - Supplement of vitamins and other trace elements How many different bacteria do we expect? Validly described species: Prokaryotes (Bakteria und Archaea) Eukaryotes Estimations for the number of bacterial species in 30 g forrest soil (Torsvik et al 1990, Appl Environ Microbiol 58: ) (Dykhuizen 1998, Antonie van Leeuwenhoek 73:25-33) (based on the same set of data) similar for sediments 6

7 Application of molecular probes Hybridisation: Probe (Oligonucleotide) at a target sequence (mostly 16S/23S rrna) Specificity: Strain, family,... up to domain (dependent on target sequence) 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 Dot Blot analysis of bacterial communities Felske et al

8 Analysis of bacterial communities by Fluorescence In-situ Hybridisation, FISH 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) Stronghold of Fluorescence In-situ Hybridisation in Germany is the MPI in Bremen! Anaerobic methane oxidising consortia ANME2 (EelMS932) Desulfosarcina (DSS658) DAPI CARD-FISH 5 µm Boetius, et al. (2000) Nature. 407: detected in gas hydrate bearing sediments Ishi et al detected in tidal flat sediments Archaea (ARCH915) Desulfosarcina (DSS658) 8

9 Fluoresce In Situ Hybridisation Fixation Treatment with fixative, conditioning of cells, filtration natural microbial community Hybridisation Annealing of probes under stringent conditions Washing Detachment of probes that were not bound to the target sequence Specific probes Fluorescent dye 16S rrna Counter staining Staining of all cells by a general fluorescent dye (e.g. DAPI) Visualisation Epifluorescence microscopy DAPI Probe Relation of non specific to specific signals Fig.: B. Rink 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 The Relation of non specific to specific signals can be distorted Interpretation often difficult Especially for samples that show high autofluorescence like sediment, algae, cyanobacteria Optimisation: Signal amplification by CARD-FISH 9

10 CAtalysed Reporter Deposition - FISH Fixation Treatment with fixative, conditioning of cells, filtration natural microbial community Hybridisation Annealing of probes under stringent conditions Specific probes new Horseradishperoxidase, HRP 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 A B Enrichment new Tyramide inactiv Fluorescent dye H 2 O 2 * H 2 O H 2 O 2 Protein (Tyrosin) Peroxidase Activation Peroxidase Washing Molecules that were not converted new Counter staining Staining of all cells by a general fluorescent dye (e.g. DAPI) Visualisation Epifluorescence microscopy DAPI Probe Relation of non specific to specific signals Fig.: B. Rink 10

11 Higher sensitivity by signal amplification Tidal flat sediment FISH CARD-FISH Fig.: M. Mussmann depth (cm) PCR techniques 11

12 SIGnature PCR Amplification of specific PCR products with different length Separation in an agarose gel ca bp 1000 bp 700 bp 650 bp 350 bp Bacteria CFB b-proteobacteria a-proteobacteria Firmicutes, High-GC 100 bp g-proteobacteria SIG-PCR with Mediterranean isolates Reference unknown isolates Süß et al

13 Result of SIG-PCR screening of isolates from Mediterranean sediments indifferent γ-protoebacteria almost exclusively from MPN plates (MKS, AS, Alk) 4 marin phototrophic α-proteobacteria Gram positive high GC α-proteobacteria no growth in AS media 50% from SED Quantitative (real time) PCR SybrGreen I TM -technique No binding at single stranded DNA Intercalation of SybrGreen at double stranded DNA Amplification low fluorescence increasing fluorescence 13

14 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 Rotor and detection units Raw data analysis 14

15 Data analysis - normalised rawdata 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 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 sediments Method: Rhizobium specific real time-pcr with SybrGreen I 16S rrna operons Absolute Relative Results widely distributed in Mediterranean sediments enhanced numbers in sapropels up to 5% of eubacteria typical deep biosphere organisms 15