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).     

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.     

Catalyzed reporter deposition-fluorescence in situ hybridization allows for enrichment-independent detection of microcolony-forming soil bacteria

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition.     

In situ detection of bacteria in calcified biofilms using FISH and CARD-FISH

Modified protocols of fluorescence in situ hybridization (FISH) and catalyze reporter deposition fluorescence in situ hybridization (CARD-FISH) were developed in order to detect bacteria in situ in calcified stromatolite biofilms. Smooth, well-preserved thin sections of calcified biofilms (~5 microm thin, vertical sectioning of ~1 cm deep) were obtained by cryo-sectioning using the adhesive tape-stabilization technique. A modified hybridization buffer was applied during hybridization to prevent calcite dissolution as well as false binding of oligonucleotide probes to the charged mineral surfaces. Particularly, bright and specific CARD-FISH signals allowed the detection of bacteria in intensively calcified biofilms even at low magnification, which is suitable for investigating millimeter- to centimeter-scale vertical distribution patterns of bacteria.     

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.     

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.     

Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization Allows for Enrichment-Independent Detection of Microcolony-Forming Soil Bacteria

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition.     

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.     

Bacterial communities associated with benthic organic matter in headwater stream microhabitats

Bacterial communities associated with a variety of benthic detritus types were studied in three streams in the context of the chemical characteristics of the sediment material and the stream water. A cell purification assay was developed for a quantitative microscopic evaluation of bacterial community structure in detritus samples by fluorescence in situ hybridization (FISH). The efficiency of FISH with fluorescently monolabelled probes was compared with FISH with signal amplification by catalysed reporter deposition (CARD-FISH). In detritus types poor in organic carbon and nitrogen, the numbers of prokaryotes were related to the chemical characteristics of the stream water column, whereas no such relationship was found for detritus types rich in organic carbon and nitrogen. These results might help to provide criteria for the selection of detritus types for river ecosystem assessment and monitoring. The percentage of bacteria detected by FISH with monolabelled probes was correlated with the detritus total organic matter (OM). This is likely attributed to a higher ribosome content of microbial cells on substrates rich in OM. Cell detection by CARD-FISH did not show any correlation with OM content, indicating that this technique renders the results more independent from the activity state of cells. Fluorescence in situ hybridization with four group-specific probes suggested a relationship between substrate quality and the composition of the microbial assemblages on the various types of detritus. The improved protocol for cell purification and CARD-FISH may facilitate future investigations on the relationship between the riverine benthic detritus quality and microbial community composition.     

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.     

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.     

Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes.

Fluorescence in situ hybridization (FISH) with chromosome-specific probes has been applied to detection of numerical aberrations involving chromosomes 13, 18, and 21 in metaphase and interphase amniocytes. High-complexity, composite probes for chromosomes 13, 18, and 21 were used as hybridization probes for this study. These probes were constructed as chromosome-specific libraries in Bluescribe plasmids and are designated pBS-13, pBS-18, and pBS-21. Elements of these probes bind at numerous sites along the target chromosome and, when detected fluorescently, stain essentially the entire long arm of the target chromosome. The target chromosome number (i.e., the number of chromosomes of the type for which the probe was specific) was correctly determined in 20 of 20 samples in which metaphase spreads were analyzed and in 43 of 43 samples in which interphase nuclei were analyzed; all of these studies were conducted in blind fashion. These results suggest the utility of FISH with composite probes for rapid detection of numerical aberrations in metaphase and interphase amniotic cells.     

Improved quantification of Dehalococcoides species by fluorescence in situ hybridization and catalyzed reporter deposition

Chlorinated ethenes contamination of soil and groundwater is a widespread problem in most industrialized countries. To date, there is a general consensus in the literature that members of the genus Dehalococcoides are required for complete dechlorination of these compounds. The availability of specific identification tools to track their distribution in the field is therefore a topic of particular relevance in environmental studies. These microorganisms have been successfully visualized by fluorescence in situ hybridization (FISH) in highly active dechlorinating cultures. However, FISH detection of Dehalococcoides under low activity conditions can be strongly hampered by their small cell size and low ribosome content. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative detection method. In a trichloroethene (TCE) dechlorinating enrichment culture, CARD-FISH, using proteinase K as a permeabilization pre-treatment, was found to be significantly superior to conventional FISH in terms of both microscopic visualization and quantification efficiency (about 30%). An application of this method on contaminated aquifer samples is also presented and discussed.     

Differentiation of two very similar glaucomid ciliate morphospecies (Ciliophora, Tetrahymenida) by fluorescence in situ hybridization with 18S rRNA targeted oligonucleotide probes

Conventional, morphological identification of ciliates and other protozoa needs considerable experience and often is difficult as various staining methods must be applied. New molecular techniques, such as fluorescence in situ hybridization (FISH) with gene probes, are powerful means to overcome this problem. As a test case, the morphology of two very similar, and thus difficult to differentiate ciliate morphospecies, Glaucoma scintillans and Glaucomides bromelicola, were compared. They were then distinguished by applying the Ciliate-FISH technique with a set of eight 18S rRNA targeted oligonucleotide probes, four of which have been developed for specific detection of G. scintillans. The remaining four probes were designed to detect G. bromelicola in order to prove probe specificities by binding to the homologous target region of the probes mentioned before. The tests resulted in a clear and easy differentiation of the two species by fluorescence signals of three of the four tested probe pairs. Thus, FISH techniques are very useful for the identification and detection of protozoa and might be of great help studying geographical distributions of known taxa.     

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores.Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.     

Specific detection of Dehalococcoides species by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.     

High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: Rapid chromosome identification by directly fluorescently labeled alphoid DNA probes

We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accurate identification of human chromosomes in metaphase and interphase cells. Cy3-labeled probes had several advantages: (1) a high level of fluorescence (5–10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection of target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high energy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we successfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid probes for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid chromosome detection during a simple 15-min FISH procedure, and can be recommended for basic research and clinical studies, utilizing FISH.     

Prenatal detection of aneuploidies using fluorescencein situ hybridization: A preliminary experience in an Indian set up

Fluorescencein situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used. Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes 13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics     

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.