Quantifying Substrate Uptake by Individual Cells of Marine Bacterioplankton by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Microautoradiography

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up ³H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.     

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.     

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).     

Rapid Quantification of the Toxic Alga Prymnesium parvum in Natural Samples by Use of a Specific Monoclonal Antibody and Solid-Phase Cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Use of Microautoradiography Combined with Fluorescence In Situ Hybridization To Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa

The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [³⁵S]DMSP ranged between 27 and 51% of 4′,6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [³H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [³⁵S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [³H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (α-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [³⁵S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the γ-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and γ-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating ³⁵S from DMSP (around 50%). Altogether, the application of AU with [³⁵S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.     

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.     

Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.     

Simultaneous Quantification of Active Carbon- and Nitrogen-Fixing Communities and Estimation of Fixation Rates Using Fluorescence In Situ Hybridization and Flow Cytometry

Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and ¹⁴C/¹⁵N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean.     

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.     

Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Increase in Fluorescence Intensity of 16S rRNA In Situ Hybridization in Natural Samples Treated with Chloramphenicol

Despite the numerous advantages of fluorescent in situ hybridization for the identification of single prokaryotic cells with 16S rRNA probes, use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluorescently labeled cells constitutes the primary problem for in situ hybridization of natural samples, probably due to low cellular rRNA content. This study represents an attempt to improve detection of marine prokaryotes by increasing cellular rRNA content without changing the species composition. Cells from three California coastal sites were treated with chloramphenicol, an inhibitor of protein synthesis and rRNA degradation, at 100 (mu)g/ml and then were probed with a "universal" 16S rRNA fluorescent probe and viewed by image-intensified video microscopy. Counts of fluorescent cells increased from ca. 75% for untreated samples to ca. 93 to 99% for chloramphenicol-treated samples, compared to counts produced by DAPI (4(prm1),6-diamidino-2-phenylindole) staining, after at least 45 min of exposure to the drug (these percentages include autofluorescent cells, which averaged 6%). This suggests that most cells in these samples were active. We hypothesize that the low fluorescent-cell counts previously reported were probably often due to the fluorescence intensity of labeled cells being below the detection level rather than to high levels of dead cells in marine environments. This method may aid in the characterization of bacterioplankton with fluorescent probes.     

Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 × 10⁷ ± 1.9 × 10⁷ cells ml⁻¹. Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.     

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle.

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Comparison of Fluorescence Microscopy and Solid-Phase Cytometry Methods for Counting Bacteria in Water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10⁶ cells filter⁻¹. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10⁵, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

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°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°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°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%).