Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification

Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7% ± 18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.     

Changes in community composition during dilution cultures of marine bacterioplankton as assessed by flow cytometric and molecular biological techniques

Dilution cultures are a common technique for measuring the growth of bacterioplankton communities. In this study, the taxonomic composition of marine bacterioplankton dilution cultures was followed in water samples from Plymouth Sound and the English Channel (UK). Bacterial abundances as well as protein and DNA content were closely monitored by flow cytometry. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments and fluorescence in situ hybridization (FISH) were applied directly to the water samples and to cells sorted from the dilution cultures based on their protein and DNA content. As expected, a rapid activation of bacteria occurred. However, molecular techniques showed that the community developed in the dilution culture within 1 day was significantly different from that in the original water samples. Whereas in the original samples, cells detectable by FISH were dominated by members of the C ytophaga / Flavobacterium (CF) cluster, in dilution cultures, gamma-proteobacteria accounted for the majority of cells detected, followed by alpha-proteobacteria. An actively growing and an apparently non-growing population with average cellular protein contents of 24 and 4.5 fg respectively, were sorted by flow cytometry. FISH indicated mostly gamma- (64%) and alpha-proteobacteria (33%) in the first active fraction and 78% members of the CF cluster in the second fraction. Sequencing of DGGE bands confirmed the FISH assignments of the latter two groups. The data presented clearly show that even relatively short-term dilution experiments do not measure in situ growth, but rather growth patterns of an enrichment. Furthermore, it was demonstrated that the combination of flow cytometric analysis and sorting combined with FISH and DGGE analysis presented a fairly rapid method of analysing the taxonomic composition of marine bacterioplankton.     

Direct image-based correlative microscopy technique for coupling identification and structural investigation of bacterial symbionts associated with metazoans

Coupling prokaryote identification with ultrastructural investigation of bacterial communities has proven difficult in environmental samples. Prokaryotes can be identified by using specific probes and fluorescence in situ hybridization (FISH), but resolution achieved by light microscopes does not allow ultrastructural investigation. In the case of symbioses involving bacteria associated with metazoan tissues, FISH-based studies often indicate the co-occurrence of several bacterial types within a single host species. The ultrastructure is then relevant to address host and bacterial morphology and the intra- or extracellular localization of symbionts. A simple protocol for correlative light and electron microscopy (CLEM) is presented here which allows FISH-based identification of specific 16S rRNA phylotypes and transmission electron microscopy to be performed on a same sample. Image analysis tools are provided to superimpose images obtained and generate overlays. This procedure has been applied to two symbiont-bearing metazoans, namely, aphids and deep-sea mussels. The FISH protocol was modified to take into account constraints associated with the use of electron microscopy grids, and intense and specific signals were obtained. FISH signals were successfully overlaid with bacterial morphotypes in aphids. We thus used the method to address the question of symbiont morphology and localization in a deep-sea mussel. Signals from a type I methanotroph-related phylotype were associated with morphotypes displaying the stacked internal membranes typical for this group and three-dimensional electron tomography was performed, confirming for the first time the correspondence between morphology and phylotype. CLEM is thus feasible and reliable and could emerge as a potent tool for the study of prokaryotic communities.     

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.     

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°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.     

Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton

An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior amplification by polymerase chain reaction (PCR). The results obtained were compared with those of quantitative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially available kits was established. A set of redundant and hierarchically structured probes was applied, and specificity of hybridization was assessed by additional control oligonucleotides comprising single central mismatches. The protocol was initially tested by microarray analysis of bacterial pure cultures. Complete discrimination of all control oligonucleotides was achieved, indicating a high degree of hybridization specificity. In a co-culture, abundant members were detected by microarray analysis, but signal ratios of positive probes did not correlate well with quantitative data from FISH experiments. A marine picoplankton sample from the German Bight was analysed. Bacterial populations with relative abundances of at least 5% were detected by hybridizing 0.1 microg of total RNA extracted from a sample of 375 ml equivalent to 4.1 x 10(8) cells. Our results demonstrate that major populations of marine bacterioplankton can be identified by microarray analysis in a fast and reliable way, even in relatively low volumes of sea water.     

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.     

A survey of the relative abundance of specific groups of cellulose degrading bacteria in anaerobic environments using fluorescence in situ hybridization

AIMS: The utility of fluorescence in situ hybridization (FISH) for detecting uncultured micro-organisms in environmental samples has been shown in numerous habitats. In this study a suite of three FISH probes for cellulolytic bacteria is described and their efficacy is demonstrated by quantifying the relative abundance of the target micro-organisms in a range of industrial biomass samples. METHODS AND RESULTS: The probes were designed from data derived from an artificial landfill leachate reactor study and 16S rRNA gene databases. The original biomass sample proved to be well described by the three probes targeting a total of 51% of the bacterial (EUBMIX targeted) cells in quantitative FISH experiments. CONCLUSIONS: Three probes were developed and applied to samples from a range of industrial digesters. The CSTG1244 probe, specific for organisms closely related to Clostridium stercorarium, were observed in the widest range of samples (7 of the 19 samples tested). The CTH216a FISH probe, specific for organisms closely related to Clostridium thermocellum, described the highest proportion of the bacterial population within any one sample (46% in an anaerobically digested sludge sample). Finally, the BCE216a probe, specific for organisms closely related to Bacteroides cellulosolvens, achieved the lowest level of hybridisation of the three probes tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the three groups of anaerobic cellulolytic micro-organisms were present in different bioreactors but at variable abundances ranging from low (where other organisms would have been responsible for cellulolysis) to high. We showed the potential of using group specific FISH probes and quantitative FISH in environmental studies. The utility of using newly designed FISH probes was demonstrated by their ability to detect and quantify the target bacterial groups in samples from a range of industrial wastewater digesters.     

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.     

Using light scatter signal to estimate bacterial biovolume by flow cytometry.

BACKGROUND: In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. METHODS: The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. RESULTS: With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. CONCLUSIONS: Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.     

Exploring the taxonomic and functional diversity of marine benthic micro-Eukaryotes along the Red Sea coast of Jeddah city

BackgroundsDiverse marine habitats along Jeddah's Red Sea coast support rich biodiversity. Few studies have been done on its diverse communities, especially its microbial counterparts. Metagenomic analysis of marine benthic micro-eukaryotic communities was performed for the first time on the Red Sea coast of Jeddah. This research looks into their community structure and metabolic potential.MethodsNext-generation sequencing was used to examine the micro-eukaryotic communities of seven sedimentary soil samples from four Jeddah coast locations. After isolating DNA from seven benthic sedimentary soil samples, the 18S rDNA V4 regions were amplified and sequenced on the Illumina MiSeq. It was also verified using an Agilent Technologies 2100 Bioanalyzer with a DNA 1000 chip (Agilent Technologies, Fisher Scientific). A standard curve of fluorescence readings generated by qPCR quantification using the Illumina library was achieved using the GS FLX library. Metagenomic data analysis was used to evaluate the microbial communities' biochemical and enzymatic allocations in studied samples.ResultsBlast analysis showed that the top ten phyla were Annelida, Eukaryota, Diatomea, Porifera, Phragmoplastophyta, Arthropoda, Dinoflagellata, Xenacoelomorpha Nematoda, and uncultured. Annelida was also found in the highest percentage (93%), in the sample M followed by Porifera (64%), the most abundant in the control sample then Eukaryotes (61%), Phragmatoplastophyta (55%), Arthropoda, and Diatomea (the least common) (32%). community diversity analysis: using Shannon and inverse Simpson indices showed sediment composition to be effective. Also, PICRUST2 indicated that the most abundant pathways were pyruvate fermentation to isobutanol, pyrimidine deoxyribonucleotide phosphorylation, adenosine ribonucleotide de novo biosynthesis, guanosine ribonucleotide de novo biosynthesis, NAD salvage pathway I, the super pathway of glyoxylate bypass and aerobic respiration I (cytochrome c).ConclusionResults showed that high throughput metagenomics could reveal species diversity and estimate gene profiles. Environmental factors appear to be more important than geographic variation in determining the structure of these microbial communities. This study provides the first report of marine benthic micro-eukaryotic communities found on the Red Sea coast of Jeddah and will serve as a good platform for future research.     

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.     

High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Routine fluorescence in situ hybridization in soil

The use of fluorescence in situ hybridization (FISH) to identify and enumerate soil bacteria has long been hampered by the autofluorescence of soil particles masking the bacterial signals and because the need of counting hundreds of bacteria in order to achieve statistically reliable data is time consuming. Recently, it was demonstrated that Nycodenz facilitates FISH in soil by concentrating bacteria on membrane filters and avoiding autofluorescent soil particles. We present a routine protocol for FISH in soil including the use of Nycodenz. The protocol allows fast and easy enumeration of hundreds of bacteria. We propose the use of silicon grease coated slides to treat in parallel seven samples per hybridization. Further, we developed a semi-automated approach for the enumeration of bacteria by implementing macros concatenating all steps of the image analyzes in the Image J software. Using Nycodenz, software-assisted bacterial counts statistically matched eye-counts of the same images and it was possible to count 880 DAPI stained bacteria per ten images. Fifty-five percent of these bacteria were co-labelled with the FISH probe specific for the Domain Bacteria, in accordance with recent FISH studies of bacterial populations in bulk soil. With a soil slurry protocol used for comparison, soil particles impaired automatic counts of the bacteria and FISH analysis, and only 88 DAPI stained bacteria per ten images could be counted by eye. With the Nycodenz protocol, 5 mM Na(2)EDTA used as an extractant increased the number of bacteria observed by 49%. In contrast, Tween 20 (1% or 5%) had no significant effect and increased the variability between the samples. Overall, the proposed procedure allows to process a high number of samples and to achieve a time efficient FISH characterization of soil bacterial communities.     

Spatial structure in the “Plastisphere”: Molecular resources for imaging microscopic communities on plastic marine debris

Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the “Plastisphere”—the thin layer of life on the surface of PMD—has been technology‐limited. Research into microbe–microbe and microbe–substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI‐FISH (combinatorial labelling and spectral imaging – fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha‐, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell‐count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro‐metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales.     

FISH methods in phycology: Phototrophic biofilm and phytoplankton applications

Abstract

Many photosynthetic microorganisms, living attached to immersed substrates or free in the water column, lack distinct morphological details, are small in size and often unculturable. Thus, whole-cell fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has become a valuable and widely used technique to identify bacteria and protists within their natural communities. FISH methods not only allow direct, cultivation independent determination of community composition, but provide spatio-temporal quantification of microorganisms in the environment. Coupling of FISH techniques to Confocal Laser Scanning Microscopy (CLSM) has become essential for the assessment of diversity and structural integrity in three-dimensional complex biofilm samples. Combining FISH with microautoradiography (FISH-MAR) and microsensors also opens new perspectives in microbial ecology by providing new tools for revealing physiological properties of organisms with single-cell resolution. This paper briefly summarizes the application of FISH methods to phototrophic biofilm and phytoplankton research. The potential of DNA microarray technology in phycological research is highlighted, especially for the fast and accurate identification of HAB (Harmful Algal Bloom) species in marine phytoplankton. Some CLSM and FISH data from phototrophic biofilms from an Italian wastewater treatment plant are shown.     

Detection of bacterial contamination in starch and resin-based papermaking chemicals using fluorescence techniques

Rapid fluorescence techniques were evaluated for the detection of bacterial contaminants in papermaking chemicals including starch and the resin-based sizes and starch slurries used in the paper industry. Viable and non-viable bacterial cells were visualised by fluorescent probes and detected by epifluorescence microscopy and flow cytometry. The best discrimination ability was obtained with the fluorescent probes LIVE/DEAD and SYBR Green, based on the staining of cellular nucleic acid, and ChemChrome V3, which demonstrated cellular enzymatic activity. The process samples had to be diluted and filtered before fluorescence staining and analysis because they were viscous and contained solid particles. Fluorescence microscopic counts of bacteria in highly contaminated process samples were similar to plate counts, but flow cytometric enumeration of bacterial cells in process samples yielded 2- to 10-fold lower counts compared with plate counts, depending on the consistency of the sample. The detection limits in flow cytometric analysis and in epifluorescence microscopy were 10³–10⁶ cells ml⁻¹ and 10⁵–10⁶ cells ml⁻¹, respectively. Intrinsic bacterial contamination was detectable with fluorescence techniques and highly contaminated process samples could be analysed with fluorescence methods. Electronic Publication     

Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis.

BACKGROUND: Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. METHODS: A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements. RESULTS: Three clusters could be detected from the bacterial assemblage. Protozoan grazing had a strong impact on the bacterial community, which could be analyzed in detail at the level of individual subgroups. CONCLUSIONS: Investigation at the level of bacterial subgroups allowed a more detailed analysis than whole-community statistics and delivered essential and ecologically meaningful information. Image analysis proved to be an adequate tool to detect the subgroups without a priori knowledge.     

Visualization and enumeration of marine planktonic archaea and bacteria by using polyribonucleotide probes and fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4',6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50-56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 10(5)/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or "ghosts," as was suggested in a previous report.     

A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.