In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes

New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes.     

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.     

Individual Adult Human Neurons Display Aneuploidy: Detection by Fluorescence In Situ Hybridization and Single Neuron PCR

Neurons, once committed, exit the cell cycle and undergo maturation that promote specialized activity and are believed to operate upon a stable genome. We used fluorescence in situ hybridization, selective cell microdissection, and loss of heterozygosity analysis to assess degree of aneuploidy in patients with a neurodegenerative disease and in normal controls. We found that aneuploidy occurs in approximately 40% of mature, adult human neurons in health or disease and may be a physiological mechanism that maintains neuronal fate and function; it does not appear to be an unstable state. The fact that neuronal stem cells can be identified in adult humans and that somatic mosaicism may be found in neuronal precursor cells deserves further investigation before using adult neural stem cells to treat human disease.     

Fluorescence In Situ Hybridization in Studying the Human Genome

Physical chromosome mapping by fluorescence in situ hybridization (FISH) is among the major lines of research on the human genome (as well as genomes of numerous other organisms). To localize particular genes or anonymous DNA sequences on individual chromosomes or chromosome regions, FISH was developed in the late 1980s and early 1990s, when the International Human Genome Project and the Russian program Human Genome were launched. Now FISH continues to play a prominent part in studies of the human genome. The review considers the major steps of FISH development in Russia, with special emphasis on the key roles of the Institute of Cytology and Genetics (Novosibirsk) and Engelhardt Institute of Molecular Biology (Moscow). Physical mapping of human chromosomes 3 and 13 by FISH is described in detail. The acquisition of FISH in Russia contributed to the progress in the related fields such as comparative animal genomics (ZOOFISH) and studies of plant chromosomes.     

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.     

Fluorescence In Situ Hybridization and Spectral Imaging of Coral-Associated Bacterial Communities

Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging.     

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.     

Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection

A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.In the past decade, Salmonella spp. have been implicated in multiple food-borne disease outbreaks tied to the consumption of fresh fruits and vegetables (19). In the United States, tomatoes have been the most commonly implicated crop for produce-related salmonellosis, with 12 outbreaks occurring since 1998 (3, 19). Contamination of fresh produce can occur at any point in the farm-to-fork continuum and can result from the use of contaminated irrigation water, runoff from adjacent animal production lots, activities of wild animals in fields, or use of untreated manure as a fertilizer (9, 19). Additional routes may include unsanitary practices by workers in the field or even intentional contamination of crops in the field. Although field environments provide greater opportunities for contamination to occur, contamination of tomatoes with Salmonella also occurs for crops grown in controlled (hydroponic) environments (21). The largest documented fresh-produce-related outbreak of salmonellosis to date in the United States occurred during the summer of 2008. Although tomatoes were initially implicated, the source was difficult to pinpoint, and the outbreak strain was later recovered from jalapeño and serrano peppers grown in Mexico. Methods for detection of Salmonella on fresh produce can play an important role in mitigation of disease from outbreaks such as this by providing decision makers with timely data on the presence of this pathogen in contaminated foods.Adhesive-tape-based sampling methods have been used in clinical, environmental, and food microbiology, beginning in the early 1950s (4, 10, 13, 17), and have recently been combined with an rRNA-targeted whole-cell method for fluorescent labeling of specific microbial cells (fluorescence in situ hybridization [FISH]) for culture-independent analysis of microbial communities present on the surfaces of stone monuments (15). We have extended this approach to the rapid sampling of fresh produce surfaces for detection of Salmonella strains, using tomatoes as a model system. In addition to tomatoes, we found that the method could also be used to sample and detect Salmonella artificially inoculated onto jalapeño pepper, cilantro, and spinach surfaces and that cell-charged tapes could be enriched further on Salmonella-selective agar, or in low-volume (0.5 ml) liquid culture followed by flow cytometric analysis.Tomatoes (red tomatoes on the vine, not waxed or oiled; average weight, 135 g), jalapeño peppers, cilantro, and spinach were obtained from a local grocery store and confirmed to be negative for Salmonella via culture. Square regions (1 cm² each) were drawn on produce surfaces with a fine-tip permanent marker using a sterile paper template. Salmonella strains (overnight cultures of serovars Typhimurium ATCC 14028 and Newport, Salmonella Genetic Stock Centre SARB 36, washed and resuspended in 0.1% peptone water) were spot inoculated within each 1-cm² region. Final cell densities ranged from ∼10⁰ to 10⁷ CFU cm⁻². For tomatoes, inocula were applied to skin at either the top (adjacent to the stem scar) or bottom (adjacent to the blossom scar) of the fruit. For spinach and cilantro leaves, the tops of the leaves (adaxial sides) were used. For some samples, mixtures of individual Salmonella strains and Rhodotorula glutinis ATCC 32765 were also spot inoculated in the same fashion (Fig. ​(Fig.1).1). Microbial inocula were allowed to attach by drying onto the tomato surfaces for ∼3 h at 25°C prior to tape-based sampling. Although preliminary experiments suggested that generic office-grade transparent tape may be suitable in this application, we focused on two commercially available adhesive tapes intended for microbiological use: Fungi-Tape (Scientific Device Laboratory, Des Plaines, IL) and Con-Tact-It sampling tape (Birko Corporation, Denver, CO). Microorganisms were sampled by placing Fungi-Tape or Con-Tact-It tape onto inoculated areas, applying gentle and even pressure to ensure full contact of the sampling tape with the produce surface, and removing the tape-cell complex (Fig. ​(Fig.2A).2A). In some experiments, after lifts of cells from tomato surfaces had been made, tapes were placed onto xylose-lysine-Tergitol 4 agar plates, which were then inverted and incubated for 8 h at 37°C for on-tape formation of microcolonies. Following incubation, adhesive tapes were pressed gently against the agar surface to bind any loosely adherent cells, and the tape-cell complex was removed. Prior to further processing (for fixation, hybridization, and microscopy or on-tape liquid culture), cell-charged tapes were mounted (with generic transparent tape) onto microscope slides, sticky side facing upwards. All inoculation and tape-based sampling experiments were repeated three times, using two Salmonella serovars (Typhimurium and Newport); experiments on recovery efficiency of tape-based tomato sampling using serovar Newport were carried out in duplicate and were repeated three times; cytometry experiments were performed twice.Open in a separate windowFIG. 1.Tape-FISH for detection of Salmonella strains in mixed culture from tomato surfaces. Tomatoes were spiked with a mixture of S. enterica serovar Typhimurium (10⁷ CFU cm⁻²) and R. glutinis (10⁶ CFU cm⁻²) and then sampled with adhesive tape after drying. Tapes were hybridized for 30 min with a combination of probes targeting Salmonella cells (Sal3/Salm-63 cocktail, green label) and eukaryotic cells (EUK 516, red label). These results demonstrate the utility of tape-FISH for simultaneous visualization of the distribution and interactions between multiple phylotypes occurring together on produce surfaces.Open in a separate windowFIG. 2.Tape-based sampling of tomato surfaces and liquid surface miniculture. (A) Microorganisms artificially spiked onto tomato surfaces were sampled using sterile adhesive tape. Tapes were applied with gentle and even pressure, ensuring full contact of the sampling tape with produce surfaces, followed by removal of the tape-cell complex for subsequent processing. (B) Filling of a perfusion chamber prior to enrichment via liquid surface miniculture. The bottom surface of the chamber was comprised of a Salmonella-charged tape, mounted sticky side up. After being filled with 500 μl of nonselective broth (TSB or BPW, as described in the text), chambers were incubated for 5 h, followed by cell harvesting, fixation, hybridization, and analysis via flow cytometry (Fig. ​(Fig.33).Liquid phase enrichment (liquid surface miniculture) was performed by placing a CoverWell perfusion chamber (model PC1R-2.0, nonsterile; Grace Bio-Labs, Bend, OR) on top of a slide-mounted tape and filling the chamber with 500 μl growth medium (Trypticase soy broth [TSB] or buffered peptone water [BPW]), preheated to 37°C (Fig. ​(Fig.2B).2B). The flexible silicone base of this type of chamber allowed formation of a water-tight seal, yielding closed, medium-filled chambers whose bottom surfaces were comprised of Salmonella-charged tapes mounted sticky side up on microscope slides. Perfusion chamber inlet ports were sealed using transparent adhesive tape, and the chambers were incubated at 37°C for 5 h.Prior to FISH, tape-bound cells were fixed for 30 min at 25°C by covering the sample contact area with 500 μl of 10% neutral buffered formalin (Sigma). After fixation, the formalin was discarded, and the tape was washed once in 1× phosphate-buffered saline and then dehydrated in ethanol (a 50%, 80%, and 95% series, exposure for 3 min to 300 μl ethanol at each concentration) prior to hybridization. For fixation of liquid surface cultures, the entire 500-μl volume was transferred into a 1.5-ml microcentrifuge tube, pelleted for 5 min at 2,000 × g, resuspended in 0.5 ml 10% buffered formalin, and fixed for 30 min at 25°C. Fixed samples were harvested via centrifugation (5 min, 2,000 × g), the supernatant was discarded, and cell pellets were resuspended in 0.5 ml of cell storage solution (a 50:50 mix of phosphate-buffered saline-absolute ethanol) and either analyzed directly or stored at −20°C until analyzed.Two oligonucleotide probes previously developed for detection of Salmonella spp., Sal3 (20) and Salm-63 (14), were combined as described by Lantz et al. (18) and applied as a dual probe cocktail at a total concentration of 5 ng/μl probe (2.5 ng/μl each probe). In mixed-flora experiments with R. glutinis, a universal Eucarya probe, EUK 516 (1), was also used at 5 ng/μl. Probes were synthesized and high-pressure liquid chromatography purified by Integrated DNA Technologies (Coralville, IA) and were labeled at the 5′ end with fluorescein or Texas Red (for microscopy work) or with Cy5 (for flow cytometry experiments). For most experiments, samples on tapes were hybridized for 15 min at 55°C using a moisture-sealed slide incubation chamber (Slide Moat model 240000; Boekel Scientific, Feasterville, PA). Briefly, 300-μl volumes of hybridization buffer (0.7 M NaCl, 0.1 M Tris [pH 8.0], 0.1% sodium dodecyl sulfate, 10 mM EDTA, containing probe and preheated to 55°C) were applied to the surface of the tape, and the chamber''s lid was sealed, creating a moist, temperature-controlled environment within the chamber. After 15 min, the lid was removed, and samples were briefly rinsed with probe-free hybridization buffer, which had been preheated to 55°C. Tapes were then processed for microscopy, as described below. In initial tests, and for Fig. ​Fig.1,1, hybridization and washing (30 min each) were carried out in a hybridization oven (Bambino; Boekel Scientific), inside sealed 50-ml polypropylene centrifuge tubes. Due to the limited throughput of this approach, subsequent hybridizations were carried out using the Slide Moat, which allowed analysis of multiple (>20) slides and also provided direct-contact heat transfer. For hybridization of cells grown using liquid surface miniculture, fixed cells (entire 500-μl samples, in cell storage solution) were pelleted (5 min, 2,000 × g) and resuspended in 100 μl of probe-containing hybridization buffer. Samples were hybridized at 55°C on a heat block for 30 min, followed by a 30-min wash step at the same temperature using 500 μl hybridization buffer without probe, and then analyzed via cytometry.Hybridized cells on tapes were counterstained for 10 min in the dark with ∼30 μl mounting medium containing 1.5 μg ml⁻¹ DAPI (Vectashield H-1200; Vector Laboratories, Burlingame, CA) and then mounted with a coverslip and examined using a Leitz LaborLux S microscope equipped with a Canon PowerShot A640 consumer-grade digital camera controlled by Axiovision software (v. 4.6; Carl Zeiss Microimaging, Inc., Thornwood, NY). Raw TIFF outputs from green (fluorescein) and red (Texas Red) channels were adjusted for brightness and contrast to appear as they did via microscopy, and composite images were made using Adobe Photoshop. Flow cytometry of liquid surface miniculture samples was performed on a Becton-Dickinson FACSCanto flow cytometer with red (647-nm) excitation, using bacterial side scatter to trigger event detection. Samples were run for 3 min at a low flow rate (10 μl min⁻¹). Flow cytometry data were analyzed using FlowJo software (v. 8.7.1; Tree Star Inc., Ashland, OR).Since its introduction in 1930, “Scotch”-type adhesive tape has been adopted for a number of “off-label” uses, including use in the household for removal of lint from garments, in forensic science for lifting fingerprints from surfaces, and in the clinic for sampling and detection of intestinal parasites or their eggs via anal tape lifts or for sampling of pathogenic fungi from skin (4, 10). In environmental microbiology, adhesive tape has been used for sampling of microbes from leaf surfaces for subsequent microscopic or cultural analyses (17), and tape-based sampling is an accepted technique in food microbiology for monitoring of food or environmental surfaces (12, 13). For example, the use of Con-Tact-It tape is suggested in the Compendium of Methods for the Microbiological Examination of Foods (12) as an alternative to RODAC plating for estimating the sanitary condition of food processing environmental surfaces (12), and use of this tape has also been combined with acridine orange staining for sampling and analysis of microbial populations on beverage dispenser tips via fluorescence microscopy (16). Extending the approach further, La Cono and Urzì (15) combined tape-based sampling with on-tape FISH for the detection and characterization of microflora present on the surfaces of historic stone monuments and suggested the approach for use on other surfaces, including food contact surfaces. However, in addition to inanimate objects (i.e., cutting boards, countertops, floor tiles, processing equipment, etc.), the surfaces of many foods themselves may become contaminated with human pathogens. In the United States, tomatoes and other fresh produce have been implicated in a number of recent outbreaks of salmonellosis, therefore, we sought to examine the utility of this tape-FISH approach for sampling and direct detection of Salmonella strains on tomato and other fresh produce surfaces.We found that two commercially available microbiological sampling tapes (Fungi-Tape and Con-Tact-It) could be used to remove Salmonella strains and other microorganisms from the surfaces of tomatoes (with greater than 99% recovery efficiency determined for S. enterica serovar Newport at an inoculum level of 10⁷ CFU cm⁻² using Fungi-Tape [data not shown]) and that Salmonella cells could be detected via FISH performed directly on the tape. Use of this tape-FISH approach was also demonstrated for other types of produce considered at risk for contamination with Salmonella spp., including jalapeño peppers, spinach, and cilantro (data not shown). The limit of direct detection via fluorescence microscopy was 10³ CFU cm⁻²—the practical limit of detection for manual microscopy (2)—and all procedures (surface sampling, cell fixation, dehydration, hybridization, counterstaining, and detection) could be carried out within ∼1.5 h. We also found that salmonellae could be enriched at a tape-agar interface by simply laying cell-charged tapes face down on selective agar plates. Substantial microcolony formation was observed after only 8 h at 37°C (data not shown). Alternatively, nonsterile perfusion chambers could be sealed over slide-mounted sampling tapes, allowing liquid surface miniculture-based enrichment of sampled cells in nonselective broths. TSB was superior to BPW, both in its ability to support the growth of Salmonella strains and in promoting release from the tape into liquid miniculture (Fig. ​(Fig.3).3). Although the ultimate level of detection was not determined for the combination of liquid surface microculture and flow cytometry, a relatively small number of cells (10³ cm⁻²) could be detected directly from TSB-washed tapes, and substantial enrichment of Salmonella strains was observed after a brief enrichment in liquid surface miniculture (500-μl volumes, 5 h of enrichment at 37°C), even in the absence of visible turbidity (Fig. ​(Fig.3).3). Our work highlights the potential for tape-FISH to provide rapid and specific detection of Salmonella spp. on fresh produce surfaces, even in the presence of nontarget organisms.Open in a separate windowFIG. 3.Tape-FISH combined with liquid surface miniculture for rapid detection of S. enterica serovar Typhimurium on tomatoes via flow cytometry. Adhesive tape was used to remove serovar Typhimurium from tomato surfaces inoculated with 10³ cells cm⁻². As described for Fig. ​Fig.2B,2B, cell-charged tapes were mounted face-up on microscope slides, and perfusion chambers were placed on top of the tape and filled with nonselective broths. Liquid surface minicultures were incubated for 5 h, mixed via up-and-down pipetting using gel loading tips, processed for FISH, and then analyzed via flow cytometry. When TSB was used, it was possible to detect Salmonella directly from tapes (0 h, TSB). Despite a lack of visible turbidity, substantial enrichment was possible after only 5 h of nonselective pre-enrichment in TSB. These data show the utility of FISH and flow cytometry in combination with adhesive-tape-based sampling for the rapid detection of Salmonella on contaminated tomatoes.As a simple approach for sampling, adhesive-tape methods have a number of potential advantages: they are easy to learn, use, and troubleshoot, and the raw materials or equipment needed are inexpensive and widely available. They are portable enough to facilitate testing in the field or in a food production environment and are nondestructive (15). Because they have the potential to save both time and money, use of such simple methods may free up limited resources, enabling more frequent or extensive testing. Additionally, tape-based sampling comprises elements of both sample preparation and sample presentation. That is, the same action (contact with the food surface) accomplishes both removal of attached organisms from the surface and two-dimensional presentation of the cells on an optically clear film, facilitating downstream processing, such as staining (colorimetric staining, fluorescent staining, and FISH) and direct examination via microscopy. Of special benefit to FISH-based analyses is the fact that microbial cells are removed from the host tissues, which could be a significant source of interference with probe-conferred fluorescence, due to the intense autofluorescence often seen in plant tissues (5, 6, 8).Tape-based detection approaches have long been used in environmental microbiology for examination of plant-associated microorganisms, such as fungi present on leaves (10, 17). A key benefit of this application is that the spatial relationships of the sampled organisms from the leaves are preserved as a “mirror image” in situ on the tape (15, 17). The FISH approach has been used to great advantage in environmental microbiology for cultivation-independent analyses of complex microbial consortia, and FISH has also been a valuable tool for studying the spatial arrangements and physical interactions of specific microbes occurring in foods, such as artisanal cheeses (7, 11). Because FISH is a culture-independent approach, tape-FISH can theoretically be used for in situ examination of target cells on fruit or leaf surfaces, without the need for culture. Because multiple probes can be used, the presence and physical location of more than one phylotype can be determined and monitored simultaneously (Fig. ​(Fig.11).In their study on the colonization of cilantro leaves by S. enterica serovar Thompson, Brandl and Mandrell (5) found that low inocula of this organism were able to reach high cell densities when the leaves were stored under humid conditions. S. enterica serovar Thompson formed distinct microcolonies or large mixed-species aggregates with other enteric species commonly found as epiphytes on cilantro, such as Pantoea agglomerans. In the study of Barak and Liang (3), cocolonization of tomato plants with the plant pathogen Xanthomonas campestris pv. vesicatoria led to significantly higher populations of S. enterica than on plants colonized by S. enterica alone, suggesting cooperative activities of these two organisms during growth on these plants. Metabiotic interactions between proteolytic molds and Salmonella spp. have also been documented for raw, ripe tomatoes, with the metabolic activities of spoilage molds and concomitant physical degradation of tomato surfaces enhancing the growth of S. enterica (23). In light of these studies, culture-independent techniques capable of preserving spatial information on relationships between target cells, competitive or cooperative microflora, and host structures are expected to be of great value to basic research on pathogen-produce interactions. Our tape-FISH protocol may therefore be leveraged as a basic research tool and, when coupled with enrichment, as a rapid and simple approach for sampling and screening for Salmonella on fruit, herb, or leafy greens surfaces in support of routine control measures or as a tool for outbreak investigation.Several factors can potentially impact the efficiency of cell capture or release by the tape, including serovar-dependent differences in cell surface properties, the mode of attachment (i.e., nonspecific adhesion or adhesion mediated by specific structures, such as pili or flagella), the presence of soil on or moisture content of the sample surface, and whether microbial cells are present in a monolayer or in a firmly attached biofilm (6, 13, 17, 22). Because different brands of commercially available tapes are expected to be formulated with different adhesives, they may also vary in their adhesive properties or compatibility with living cells, which could also impact cell recovery, release, or growth. As noted, we were able to recover serovar Newport artificially inoculated at 10⁷ CFU ml⁻¹ onto untreated tomatoes (no waxes or oils) with greater than 99% efficiency using Fungi-Tape, and cells remained culturable, as determined by agar and liquid surface miniculture enrichment.As a sampling method, tape-based removal of microorganisms from vegetable surfaces faces some practical challenges. In principle, FISH is capable of single-cell sensitivity, but, as noted in a review by Amann et al. (2), bringing a single FISH-labeled cell into view under the microscope is technically challenging, with an inverse relationship existing between the number of target cells present and the time needed to find them. Therefore, rapid and reliable detection of fewer than 10³ cells per cm² is not practical using manual microscopy (2), a result that we confirmed for tape-FISH in our work. This is expected to remain a limitation of simple, manual microscopy, but developments in automated microscopy or use of scanning laser cytometry could be effective means for reliable identification of lower levels of target cells occurring on hybridized tapes.One potential limitation of our tape-FISH approach is that salmonellae may be randomly distributed over produce surfaces and might be missed, depending on which surface is tested. In the testing of beef carcasses, sampling is narrowed to well-defined regions (i.e., brisket, flank, rump) previously established to harbor the highest microbial loads. In testing of certain types of produce, it may therefore be possible to focus sampling on well-defined regions of plant surfaces that may preferentially harbor Salmonella spp., such as the vein structures on cilantro leaves or the stem scar of tomatoes (5). The use of such rational sampling approaches may increase the likelihood of detecting Salmonella spp. or other pathogens on the surfaces of some types of produce via tape-FISH.Tape-based sampling methods have long been used in the separate fields of environmental, food, and clinical microbiology. Therefore it is fitting to recognize that tape-FISH, as described here, may have potential applications at various points along the production-to-consumption-to-disease (or farm-to-fork-to-physician) continuum. We have described the use of tape-FISH for detection of Salmonella strains on the surfaces of tomatoes, jalapeños, spinach, and cilantro and have shown for tomatoes that this dual sampling and sample presentation approach can also be combined with brief enrichments using either Salmonella-selective agar (xylose-lysine-Tergitol 4) or nonselective-broth (TSB) culture. In the latter application, we found that because the tape-cell complex is essentially two dimensional, we could perform a liquid surface miniculture step by overlaying a minimal volume of broth on the tape after it was affixed to a microscope slide. In this application, tape-based sampling effectively represents a means for cell concentration prior to enrichment. Enrichment of even relatively few cells in a small volume with subsequent analysis of the entire volume may be a promising means for facilitating earlier detection of target cells, as no subsequent concentration step (filtration, centrifugation, etc.) is needed. In addition to its use for detection, the tape-FISH technique may also be a valuable research tool for exploring events occurring during the colonization of tomatoes by Salmonella, or the interplay between spoilage microflora and Salmonella and the role of such metabiotic interactions on establishment and persistence of infection (3, 23). It is hoped that the established and familiar nature of adhesive-tape-based techniques combined with our simple and streamlined approach for FISH-based staining of target cells will enable more rapid adoption of the tape-FISH approach by food microbiologists who may not be familiar with or currently using whole-cell molecular techniques.     

Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria

We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.     

荧光原位杂交在膀胱癌检测中的应用进展

膀胱癌是我国泌尿系统最常见的恶性肿瘤.目前膀胱癌的治疗效果不容乐观,因此肿瘤的早期诊断、早期治疗显得尤为重要.本文介绍了荧光原位杂交技术在膀胱癌早期诊断及预后监测应用中的研究进展.     

Detection of Viral DNA Sequences in Adenovirus-Transformed Cells by In Situ Hybridization

Cytological preparations of cells transformed by members of three groups of human adenoviruses, adenovirus 12, 7, and 2, were annealed with radioactive complementary RNA (cRNA) (4 x 10(7) to 4.5 x 10(7) dpm/mug) prepared by copying viral DNA with the Escherichia coli DNA-directed RNA polymerase. These in situ hybridizations detected adenovirus-specific DNA sequences in interphase nuclei when transformed cells were annealed with homologous viral cRNA, but not with heterologous viral cRNA. The highest autoradiographic grain counts were found over adenovirus 7-transformed cell nuclei, next over adenovirus 12-, and the lowest over adenovirus 2-transformed cell nuclei. This is the same order as found by reassociation kinetic measurements (K. Fujinaga and M. Green, unpublished data).     

Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4′,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the Cytophaga-Flavobacterium cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.     

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.     

Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.     

Sperm Identification in Maize by Fluorescence in Situ Hybridization

The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm possessing two B chromosomes and the other containing no B chromosomes. The B chromosome-containing sperm most often fertilizes the egg, whereas the sperm nucleus with no B chromosomes fuses with the polar nuclei. Despite the obvious advantages of being able to recognize and then track, separate, and analyze one sperm type from the other, it has not been possible because of the lack of sufficient detectable differences between the two types of sperms. In this study, we used a B chromosome-specific DNA sequence (pZmBs) and in situ hybridization to identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes. Our results are consistent with conclusions from previous genetic studies related to B chromosome behavior during pollen formation. Within pollen tubes, the position in which the B chromosome-containing sperm travels (leading or trailing) in relation to the sperm cell lacking B chromosomes appears to be random.     

荧光原位杂交技术在医学微生物学中的应用

以16S rRNA 为靶序列的寡核苷酸探针荧光原位杂交技术已广泛应用于分析复杂环境中的微生物群落构成,包括监测和鉴定病原微生物以及未被培养微生物．通过对临床样品中微生物细胞的检测能提供微生物在人体中的种类、数量和空间分布等信息．其结果快速准确,较之传统的病原微生物诊断方法具有明显的优越性,在临床应用中有广泛的前景．     

Progresses and Applications of Fluorescence in Situ Hybridization

The techniques of in situ hybridization (ISH) are widely adopted for analyzing the genetic make-up and RNA expression patterns of individual cells. There are four main criterions for evaluating this technique, including detection sensitivity, resolution, capacity and specificity. This review focuses on a number of advances made over the last years in the fluorescence in situ hybridization (FISH). These advances can be catagorized into several branches as follows: (1) Multicolor-FISH (mFISH), including conventional mFISH, combinatorial FISH, ratio labelling FISH, multicolor chromosome painting and comparative genomic hybridization (CGH); (2) Extended DNA fiber-FISH (EDF-FISH), including quantitative DNA fiber mapping (QDFM), molecular combing (MC) and dynamic molecular combing (DMC); (3)In situ PCR-based FISH; (4) Bacterial (or yeast) artificial chromosome-FISH (BAC-FISH or YAC-FISH); (5) Tyramide signal amplification-FISH (TSA-FISH); (6) Polypeptide nucleic acid-FISH (PNA-FISH) and (7) padlock-FISH.     

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes.     

Protocol for Rapid Fluorescence In Situ Hybridization of Bacteria in Cryosections of Microarthropods

A protocol was developed to detect bacteria inhabiting microarthropods by means of small-subunit rRNA-targeted fluorescence in situ hybridization and microscopy. The protocol is based on cryosections of whole specimens. In contrast to more commonly applied paraffin-embedding techniques, the protocol is quicker and reduces the number of manipulations which might damage the microscopic material. The method allowed the study of the bacterial colonization of Folsomia candida (Collembola) and the detection of bacteria in both the gut and tissue.