Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

Prokaryotic community analysis with CARD-FISH in comparison with FISH in ultra-oligotrophic ground- and drinking water

AIMS: We compared the applicability of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and FISH to enumerate prokaryotic populations in ultra-oligotrophic alpine groundwaters and bottled mineral water METHODS AND RESULTS: Fluorescent oligonucleotide probes EUB338 and EUB338mix (EUB338/EUB338-II/EUB338-III) were used to enumerate bacteria and probes EURY806 and CREN537 for Euryarchaea and Crenarchaea, respectively. Improved detection of Planctomycetales by probe EUB338-II was tested using a different permeabilization step (proteinase K instead of lysozyme). Total detection efficiency of cells in spring water of four different alpine karst aquifers was on average 83% for CARD-FISH and only 15% for FISH. Applying CARD-FISH on bottled natural mineral waters resulted in an average total hybridization efficiency of 89%, with 78% (range 77-96%) bacteria and 11% (range 3-22%) identified as Archaea. CONCLUSIONS: CARD-FISH resulted in substantially higher recovery efficiency than FISH. Hence, CARD-FISH appears very suitable for the enumeration of specific prokaryotic groups in ground- and drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first evaluation of CARD-FISH on ultra-oligotrophic ground- and drinking water. Results are relevant for basic research and drinking water distributors. Archaea can comprise a significant fraction of the prokaryotic community in bottled mineral water.     

Flow sorting of marine bacterioplankton after fluorescence in situ hybridization

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.     

Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the β-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of β-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the β-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.     

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml(-1)) followed by achromopeptidase (60 U ml(-1)) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

FISH and chips: marine bacterial communities analyzed by flow cytometry based on microfluidics

To unveil the structure of natural marine pelagic bacterial communities, PCR-based techniques as well as fluorescence in situ hybridizations (FISH) were successfully performed in the past. Using fluorescence microscopes or confocal laser scanning microscopes (CLSM) for the analysis of FISH experiments, it was possible to differentiate bacterial communities, but most attempts to combine flow cytometry and FISH for this purpose have failed till now. Here we present a successful analysis of FISH experiments of natural marine pelagic bacterial communities using a flow cytometer based on microfluidics (Agilent 2100 bioanalyzer). Marine water samples were enriched on polycarbonate filters and hybridized with Cy5 labeled gene probes of different phylogenetic depth. Bacteria were detached from the filters and subsequently analyzed in the Cell Chip of the Agilent 2100 Bioanalyzer. Samples were counter-stained using SYTOX. In all samples the EUB338 positive signals could be clearly differentiated from those of the NON probe. Furthermore a dominance of alpha-protebacteria (as indicated by the probes ALF968 and G rB) could be observed. Microfluidics based flow cytometry is a promising technique for the analysis of natural bacterial communities from the marine environment.     

An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml⁻¹) followed by achromopeptidase (60 U ml⁻¹) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).     

Diversity and abundance of “Pelagibacterales” (SAR11) in the Baltic Sea salinity gradient

The candidate order “Pelagibacterales” (SAR11) is one of the most abundant bacterial orders in ocean surface waters and, periodically, in freshwater lakes. The presence of several stable phylogenetic lineages comprising “Pelagibacterales” correlates with the physico-chemical parameters in aquatic environments. A previous amplicon sequencing study covering the bacterial community in the salinity gradient of the Baltic Sea suggested that pelagibacteral subclade SAR11-I was replaced by SAR11-IIIa in the mesohaline region of the Baltic Sea. In this current study, we investigated the cellular abundances of “Pelagibacterales” subclades along the Baltic Sea salinity gradient using catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). The results obtained with a newly designed probe, which exclusively detected SAR11-IIIa, were compared to CARD-FISH abundances of the marine SAR11-I/II subclade and the freshwater lineage SAR11-IIIb (LD12). The results showed that SAR11-IIIa was abundant in oligohaline–mesohaline conditions (salinities 2.7–13.3), with maximal abundances at a salinity of 7 (up to 35% of total Bacteria, quantified with a universal bacterial probe EUB). As expected, SAR11-I/II was abundant (27% of EUB) in the marine parts of the Baltic Sea, whereas counts of the freshwater lineage SAR11-IIIb were below the detection limit at all stations. The shift from SAR11-IIIa to SAR11-I/II was confirmed in the vertical salinity gradient in the deeper basins of the Baltic Sea. These findings were consistent with an overlapping but defined distribution of SAR11-I/II and SAR11-IIIa in the salinity gradient of the Baltic Sea and suggested the adaptation of SAR11-IIIa for growth and survival in mesohaline conditions.     

Seasonal dynamics of particle-associated and free-living marine Proteobacteria in a salt marsh tidal creek as determined using fluorescence in situ hybridization

The seasonal distributions of salt marsh free-living and particle-associated bacteria belonging to three subdivisions of the Proteobacteria were determined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). More than 66% (median = 78%) of total bacterial cells that were stainable with the fluorescent DNA stain Yo-Pro-1 were also detected using the bacterial probe EUB338. The alpha-Proteobacteria, especially those from the marine Rhodobacter group, were abundant on suspended particles and as free-living cells all year round. The marine Rhodobacter group constituted more than 25% of the particle-associated bacteria and more than 18% of the free-living bacteria. Probes specific for three subgroups within the marine Rhodobacter group detected more than 49% of the total marine Rhodobacter group cells. These subgroups displayed different seasonal dynamics. The marine Rhodobacter group is clearly a widespread, diverse and important bacterial lineage in bacterioplankton and particle-associated assemblages in south-eastern United States salt marshes at all times of the year.     

Counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rRNA oligonucleotide probe

A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 micrometer). A nonsense probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the probes and for binding by the rhodamine part of the probe, control experiments in which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 x 10(8) active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 x 10(8) active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 micrometer), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4', 6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic probes and for automatic counting of soil bacteria.     

Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content

We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of (3)H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to (3)H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems.     

Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.     

The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set.

In situ hybridization with rRNA-targeted oligonucleotide probes has become a widely applied tool for direct analysis of microbial population structures of complex natural and engineered systems. In such studies probe EUB338 (AMANN et al., 1990) is routinely used to quantify members of the domain Bacteria with a sufficiently high cellular ribosome content. Recent reevaluations of probe EUB338 coverage based on all publicly available 16S rRNA sequences, however, indicated that important bacterial phyla, most notably the Planctomycetales and Verrucomicrobia, are missed by this probe. We therefore designed and evaluated two supplementary versions (EUB338-II and EUB338-III) of probe EUB338 for in situ detection of most of those phyla not detected with probe EUB338. In situ dissociation curves with target and non-target organisms were recorded under increasing stringency to optimize hybridization conditions. For that purpose a digital image software routine was developed. In situ hybridization of a complex biofilm community with the three EUB338 probes demonstrated the presence of significant numbers of probe EUB338-II and EUB338-III target organisms. The application of EUB338, EUB338-II and EUB338-III should allow a more accurate quantification of members of the domain Bacteria in future molecular ecological studies.     

Comparison of Fluorescently Labeled Oligonucleotide and Polynucleotide Probes for the Detection of Pelagic Marine Bacteria and Archaea

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections ≥70% of total cell counts, e.g., coastal surface waters during spring and summer.     

Comparison of fluorescently labeled oligonucleotide and polynucleotide probes for the detection of pelagic marine bacteria and archaea

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections > or =70% of total cell counts, e.g., coastal surface waters during spring and summer.     

Phylogenetic analysis and in situ identification of bacteria community composition in an acidic Sphagnum peat bog

The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (>or=95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3x10(7) and 1.1x10(7) cells g-1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.     

Influence of phosphate and disinfection on the composition of biofilms produced from drinking water, as measured by fluorescence in situ hybridization

Biofilms were grown in annular reactors supplied with drinking water enriched with 235 microg C/L. Changes in the biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization. EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (alpha), beta (beta), and gamma (gamma) subclasses of Proteobacteria, respectively. The stability of biofilm composition was checked after the onset of colonization between T = 42 days and T = 113 days. From 56.0% to 75.9% of the cells detected through total direct counts with DAPI (4'-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNA of most bacteria. Among these cells, 16.9%-24.7% were targeted with the BET42a probe, 1.8%-18.3% with the ALF1b probe, and <2.5% with the GAM42a probe. Phosphate treatment induced a significant enhancement to the proportion of gamma-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria. Disinfection with monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cells that hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of the ALF1b, BET42a, and GAM42a probes. The percentage of cells detected by any of the three probes (ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells. Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targeted by any of these three probes was noted, which reached up to 87%. However, it should be noted that the global bacterial densities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment. Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initial biofilm community, up to the point at which the other bacterial groups were destroyed by chlorination. The results suggest that there were variations in the kinetics of inactivation by disinfectant, depending on the bacterial populations involved.     

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.     

Numerical dominance and phylotype diversity of marine Rhodobacter species during early colonization of submerged surfaces in coastal marine waters as determined by 16S ribosomal DNA sequence analysis and fluorescence in situ hybridization

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the alpha-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.