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Evaluation of tyramide solutions for an improved detection and enumeration of single microbial cells in soil by CARD-FISH
Abstract
Several tyramide solutions were evaluated for the application of fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) in soil. Fluorescently labeled tyramide solutions were synthesized and compared to commercially available tyramides for the detection and quantification of single microbial cells. Among the tyramide solutions tested, a succinimidyl ester of fluorescein diluted with dimethylformamide containing iodophenolboronic acid (SFX-DMF-IPBA) gave the best results, yielding highly reproducible cell numbers and detection rates of archaea and bacteria along with negligible non-specific signals. The addition of organic and inorganic compounds to the amplification reagents had a positive impact on the detection of prokaryotic cells. The applicability of SFX-DMF-IPBA for CARD-FISH in soil was further evaluated in soils of different texture. Cell numbers and detection rates of bacteria and archaea remained on a high level independent of the clay or organic matter content. Based on the results obtained in this study, the choice of the tyramide solution used for CARD-FISH has a significant influence on the detection and quantification of single microbial cells in soil. Therefore, we suggest the application of the presented tyramide signal amplification procedure including the tyramide solution SFX-DMF-IPBA for comprehensive CARD-FISH studies investigating the abundance and spatial distribution of soil microorganisms.
... The roots were then washed in 19 PBS twice (5 min at RT), cut into sections of c. 20 mm length, and stored in 19 PBS/EtOH (1 : 1) at À20°C. The fixation of rhizosphere soil washed off from these roots was performed according to Schmidt et al. (2012b). ...
... Details on HRP-labeled oligonucleotide probes and hybridization conditions are given in Table 1. For CARD, the hybridized root sections were transferred to a 0.5-mL reaction tube and incubated in 450 lL amplification buffer [100 mg mL À1 dextran sulfate, 1% (v/v) blocking reagent, 2 M NaCl, 19 PBS] containing 0.0015% (v/v) H 2 O 2 and 3 lL mL À1 of fluorescein-labeled tyramide solution (Schmidt et al., 2012b) for 20 min at 37°C while rotating. Afterward, the root sections were washed in 19 TXP (5 min) and H 2 O MQ (5 min) at RT and subsequently prepared for sequential hybridization or fluorescence microscopy. ...
... The understanding of rhizosphere processes including activity and spatial distribution patterns of microbial cells has received considerable attention in recent years (Sørensen et al., 2009;Spohn et al., 2013). With the application of the presented CARD-FISH protocol for root samples in combination with the protocol for soil samples (Schmidt et al., 2012b), questions regarding the visualization, localization, and quantification of single cells in soilroot interfaces in situ can be routinely addressed in almost any soil-plant system of interest. As such, an adapted version of our protocol has been applied recently to prove the actual presence of microbial populations on roots of Arabidopsis thaliana identified as main rhizoplane colonizers by pyrosequencing (Bulgarelli et al., 2012;Lundberg et al., 2012). ...
Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was applied to detect microbial cells on the rhizoplane of wetland rice (Oryza sativa L.). Fluorescent signals of high intensity and specificity allowed for a reliable quantification of selected microbial phyla. Absolute cell numbers of archaea and bacteria were observed to be highest at flowering stage of rice plant development (P<0.05) showing values of 1.32 and 6.26 ×10(4) cells per mm(2) rhizoplane, respectively. Highest colonization densities shifted from the root tip towards more mature regions with increasing plant age. Significant differences between cell numbers observed within a short distance (0-15 mm) indicated irregular distribution patterns of microbiota. Root tips, elongation zones, and openings at the base of lateral roots represented preferential areas for microbial colonization, which were often covered with iron coatings and densely colonized with potential iron-oxidizing Betaproteobacteria (59% of bacteria). Methanogenic archaea were abundant on the rhizoplane (up to 0.96 ×10(3) cells mm(-2) rhizoplane) and the decline of their relative abundance with plant age was also found in the associated rhizosphere soil. Cell numbers of methanotrophic bacteria significantly increased at flowering (6.38 ×10(3) cells mm(-2) rhizoplane; P<0.05), indicating their stimulation by root-derived substrates which was less pronounced in the rhizosphere soil. This article is protected by copyright. All rights reserved.
... 25 The SIMBIOS Centre, University of Abertay Dundee, UK, and the Institute of Soil Science, University of Bremen, Germany, respectively. the thin sections, as this technique promises to be a more sensitive method than standard FISH or the use of fluorescent stains that bind to DNA (deoxyribonucleic acid) to detect the presence of microorganisms [5,19,81]. Figure 6. PET film samplers can be used to visualize the microbiological complexity of soils. ...
... The ability to produce A-L interface biofilms in static microcosms is common amongst environmental pseudomonads, including water, soil and plant-associated, plant and mushroom pathogens, and psychrotrophic meat-spoiling pseudomonads [57,66,74] (see also the review of cellulose expression in Pf. SBW25 and other environmental pseudomonads by Spiers et al. [68]), and amongst pathogenic, environmental and produce-related Enterobacteriaceae [64,81]. The formation of biofilms by bacteria is a key strategy in the colonisation of natural environments, though they are only one of a range of assemblages which range from isolated surface-attached bacteria, microcolonies, and multi-layered, multi-species and differentiated biofilms, as well as flocs and slime (for recent biofilm reviews see [3,18,25,48,56,78]). ...
Investigation on transformation of organic pollutants in microcosm is an important way of understanding their fate and behavior in the environment. The common methods used in microcosm experiment include (1) determination of concentration change of organic pollutants during the whole process by gas chromatography (GC) or high performance liquid chromatography (HPLC); (2) identification of degradation products by gas chromatography-mass spectrometry (GC-MS) or high performance liquid chromatography-mass spectrometry (HPLC-MS). Over the past decades, a new and innovative method of compound-specific isotope analysis (CSIA) using gas chromatography coupled isotope ratio mass spectrometry via a combustion interface (GC-C-IRMS) has been established as a powerful tool for assessing transformation of organic pollutants based on the fractionation of stable isotopes during degradation process. Two stable isotopes, 13C and 2H, have been extensively exploited to evaluate the transformation processes of contaminants. The enrichment of δ13C and δ2H offer a direct evidence of transformation, which does not prominently influenced by physical processes such as volatilization and sorption. Moreover, isotopic fractionation effect is closely associated with the reaction mechanism and the type of bond being broken. In this chapter, we review studies on degradation pathway of organic pollutants in microcosm by mean of CSIA. Especially with diethyl phthalate (DEP) as an example, we discuss how the CSIA technique combined with GC-MS analysis was used for studying the degradation pathway of organic pollutants in microcosm.
... The utilization of catalyzed reporter depositionfluorescence in situ hybridization (CARD-FISH), a sophisticated method for the identification and enumeration of single microbial cells in environmental samples (Amann and Fuchs, 2008), may help to fill this knowledge gap. In paddy soils, CARD-FISH has been reported to significantly improve the detection of archaea and bacteria compared to traditional FISH and to allow for a reliable acquisition of quantitative data of single prokaryotic cells (Eickhorst and Tippkötter, 2008;Schmidt et al., 2012). ...
... The preparation of soil samples on filter membranes for CARD-FISH was performed according to Schmidt et al. (2012). For in situ hybridizations with catalyzed reporter deposition, 5-7 filter sections were incubated with 400 L hybridization buffer (0.9 M NaCl, 20 mM Tris, 10% (v/v) Blocking Reagent (Roche, Germany), 1% (w/v) SDS, 10% (w/v) Dextran Sulfate (Sigma-Aldrich, Germany), a probe dependent concentration of formamide (%FA (v/v), Table 1; Fluka, Germany), and (70% -%FA) H 2 O MQ ) and 1.5 L probe working solution containing 50 ng L −1 horseradish peroxidase (HRP)-labeled oligonucleotide probes (Biomers, Ulm, Germany) for 120 min at 35 • C while rotating in a hybridization oven. ...
The puddled layer of paddy soils represents a highly dynamic environment regarding the spatio-temporal variability of biogeochemical conditions. To study these effects on the abundance and community structure of microbial populations, a rhizotron experiment was conducted throughout an entire growing season of wetland rice. Soil samples were taken from selected areas of the puddled layer (bulk soil, oxidized layer, rhizosphere) at main plant developmental stages such as (i) the initial stage, (ii) tillering, (iii) panicle initiation, (iv) flowering, and (v) maturity. Cell numbers of archaea, bacteria, and selected phyla were assessed by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The structure and diversity of microbial communities was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) along with sequencing of selected bands. Following submergence of the paddy soil, shifts of bacterial community structure were observed in the oxidized layer and the rhizosphere. Members of the β-Proteobacteria became predominant in the rhizosphere at tillering stage and were affiliated with aerobic, iron-oxidizing bacteria of the genus Sideroxydans. Seasonal effects were mainly visible in the rhizosphere, as several phylogenetic subgroups including methanotrophic bacteria showed increased cell numbers at flowering stage. Cell numbers of methanogenic archaea were also highest at flowering stage (bulk soil, rhizosphere) and members of the Methanocellales were identified as predominant archaeal populations in areas of oxic and anoxic conditions. In contrast to bacteria, the communities of archaea in the puddled layer of the studied paddy soil were less influenced by spatio-temporal variations of biogeochemical conditions.
... In the two decades since, remarkable progress has been achieved in the quantitative description of the geometry of the pore space and the architecture of soils (e.g., Pierret et al., 2002;Vogel et al., 2010Vogel et al., , 2021Beckers et al., 2014;Smet et al., 2018;Chirol et al., 2021;Baveye et al., 2022), the heterogeneous microscale distribution of inorganic or organic chemical species (e.g., Schumacher et al., 2005;Solomon et al., 2005;Kinyangi et al., 2006;Jacobson et al., 2007;Strawn and Baker, 2008;Mueller et al., 2012;Spohn et al., 2013;Pedersen et al., 2015;Yamaguchi et al., 2021;Kravchenko et al., 2022), the microscale spatial distribution of microorganisms (Nunan et al., 2002(Nunan et al., , 2003Otten et al., 2004;Eickhorst and Tippkötter, 2008;Young et al., 2008;Schmidt et al., 2012;Fraser et al., 2016;Juyal et al., 2018Juyal et al., , 2019Juyal et al., , 2021, as well as that of plant roots and the dynamics of the associated rhizosphere (e.g., Roose et al., 2016;Zarebanadkouki et al., 2018;Schnepf et al., 2022). In recent years, efforts have been made to perform these different complementary measurements on the same soil samples (Hapca et al., 2015;Schlüter et al., 2019;Bandara et al., 2021;Kravchenko et al., 2022), as well as on 2-dimensional micromodels simulating the architecture of soils (e.g., Deng et al., 2015;Soufan et al., 2018;Aleklett et al., 2018;Pucetaite et al., 2021). ...
Over the last few years, several researchers working on the development of “biogeochemical” or “ecosystem-scale” models of soil carbon dynamics have reported struggling with a number of difficult challenges. At the same time, work in this area has focused exclusively on microbial activity described at a macro-ecological level, and has entirely bypassed the abundant literature produced in the last two decades on the study of soil processes at the microscale. Juxtaposition of these different observations suggests that a radical shift of perspective is in order. In this general context, the present article carries out an in-depth analysis of several of the key limitations of current ecosystem-scale models and recommends a number of steps to shift the perspective to one that is argued to have a better chance of success in the relatively short time we have to address several pressing soil-related environmental problems. These steps, in particular, require the development of large-spatial-scale models of soil carbon dynamics to be far more interdisciplinary than it has been till now, and to adopt a “bottom-up” approach, building on what the research at the microscale reveals about soil processes. Nevertheless, because it may assist in upscaling efforts, it is argued that some room should be preserved for work to continue on the search for empirical models applicable at large spatial scales.
... CARD-FISH was performed according to [84]. Briefly, after the filters thawed, they were dehydrated in a series of EtOH and embedded in 0.1% low melting point agarose. ...
Hot spring biofilms are stable, highly complex microbial structures. They form at dynamic redox and light gradients and are composed of microorganisms adapted to the extreme temperatures and fluctuating geochemical conditions of geothermal environments. In Croatia, a large number of poorly investigated geothermal springs host biofilm communities. Here, we investigated the microbial community composition of biofilms collected over several seasons at 12 geothermal springs and wells. We found biofilm microbial communities to be temporally stable and highly dominated by Cyanobacteria in all but one high-temperature sampling site (Bizovac well). Of the physiochemical parameters recorded, temperature had the strongest influence on biofilm microbial community composition. Besides Cyanobacteria, the biofilms were mainly inhabited by Chloroflexota, Gammaproteobacteria, and Bacteroidota. In a series of incubations with Cyanobacteria-dominated biofilms from Tuhelj spring and Chloroflexota- and Pseudomonadota-dominated biofilms from Bizovac well, we stimulated either chemoorganotrophic or chemolithotrophic community members, to determine the fraction of microorganisms dependent on organic carbon (in situ predominantly produced via photosynthesis) versus energy derived from geochemical redox gradients (here simulated by addition of thiosulfate). We found surprisingly similar levels of activity in response to all substrates in these two distinct biofilm communities, and observed microbial community composition and hot spring geochemistry to be poor predictors of microbial activity in the study systems.
... CARD-FISH was performed according to [79]. Brie y, after the lters thawed, they were dehydrated in a series of EtOH and embedded in 0.1% low melting point agarose. ...
Hot spring biofilms are stable, highly complex microbial structures. They form at dynamic redox and light gradients and are composed of microorganisms adapted to the extreme temperatures and fluctuating geochemical conditions of geothermal environments. In Croatia, a large number of poorly investigated geothermal springs hosts biofilm communities. Here, we investigated the microbial community composition of biofilms collected over several seasons at 12 geothermal springs and wells. We found biofilm microbial communities to be temporally stable and highly dominated by Cyanobacteria in all but one high-temperature sampling site (Bizovac well). Of the physiochemical parameters recorded, temperature was found to have the strongest influence on biofilm microbial community composition. Besides Cyanobacteria , the biofilms were mainly inhabited by Chloroflexi , Gammaproteobacteria and Bacteroides . In a series of incubations with Cyanobacteria -dominated biofilms from Tuhelj spring, and Chloroflexi - and Proteobacteria -dominated biofilms from Bizovac well, we stimulated either chemoorganotrophic or chemolithotrophic community members, to determine the fraction of microorganisms in biofilms dependent on organic carbon ( in situ predominantly produced via photosynthesis) versus energy derived from geochemical redox gradients (here simulated by addition of thiosulfate). We found a surprisingly similar level of activity in response to all substrates in these two distinct biofilm communities and observed microbial community composition and hot spring geochemistry to be poor predictors of microbial activity in the studies systems.
... When dealing with a very bright background of fluorescing soil constituents, FISH probing can be combined with tyramide signal amplification. This technique is called catalysed reporter deposition (CARD)-FISH, and is very useful for obtaining higher signal intensities and reduced background interference [156][157][158]. The combination of CARD-FISH with confocal laser scanning microscopes (CLSM) allows quantification, localization, and visualization of microbial cells with even more depth-resolution. ...
Due to their small size, microorganisms directly experience only a tiny portion of the environmental heterogeneity manifested in the soil. The microscale variations in soil properties constrain the distribution of fungi and bacteria, and the extent to which they can interact with each other, thereby directly influencing their behavior and ecological roles. Thus, to obtain a realistic understanding of bacterial–fungal interactions, the spatiotemporal complexity of their microenvironments must be accounted for. The objective of this review is to further raise awareness of this important aspect and to discuss an overview of possible methodologies, some of easier applicability than others, that can be implemented in the experimental design in this field of research. The experimental design can be rationalized in three different scales, namely reconstructing the physicochemical complexity of the soil matrix, identifying and locating fungi and bacteria to depict their physical interactions, and, lastly, analyzing their molecular environment to describe their activity. In the long term, only relevant experimental data at the cell-to-cell level can provide the base for any solid theory or model that may serve for accurate functional prediction at the ecosystem level. The way to this level of application is still long, but we should all start small.
... Tyramide amplification was modified from Schmidt et al. (2012). Tyramide and amplification buffer were home-made. ...
Les déchets sulfurés issus de l’extraction des minerais métalliques génèrent des drainages miniers acides (DMA), contenant des éléments toxiques tels que l’arsenic (As), l’antimoine (Sb) et le thallium (Tl). Des procédés de remédiation utilisant des communautés microbiennes ont été développés afin d’éliminer ces polluants des DMA, mais les processus biologiques en jeu dans ces traitements doivent encore être maîtrisés pour garantir leur efficacité. Les verrous scientifiques, objets de la thèse, résident dans (1) la méconnaissance des transformations microbiennes, directe et indirectes, de Sb et Tl, et, (2) pour l’As, la faible compréhension des relations qui existent entre la dynamique des communautés microbiennes, leur potentiel fonctionnel, la physico-chimie des eaux et l’efficacité des traitements appliqués aux DMA. Une approche pluridisciplinaire, principalement basée sur des outils d’écologie microbienne et de physico-chimie, a permis de caractériser la diversité des communautés microbiennes capables de transformer directement ou indirectement As et Sb, à différentes échelles expérimentales : réacteur batch, dispositif à flux continu de laboratoire et pilote de terrain. Un consortium microbien capable de tolérer jusqu’à 100 mM d’antimonite et de l’oxyder en conditions acides (pH < 4), équivalentes à celles des DMA, a été obtenu à partir d’un sol contaminé au Sb. Un bioréacteur colonne de laboratoire, alimenté en continu avec un DMA réel et inoculé avec un consortium bactérien sulfato-réducteur issu de ce DMA, a permis l’élimination de la quasi-totalité de l’As, du Sb et du Tl présents dans l’eau. Enfin, la dynamique des communautés bactériennes au sein d’un dispositif aérobie de traitement d’un DMA riche en As installé sur site a été décrite. Ces communautés sont dominées par des bactéries Fe-oxydantes, et les modifications spatiales et temporelles de leur structure apparaissent associées aux variations de la physico-chimie de l’eau (concentration en oxygène dissous, température, pH, potentiel rédox, concentrations en sulfate, arsenic et fer(II)). Les connaissances acquises au cours de cette thèse pourront servir de base à la conception d’éco-technologies passives et peu coûteuses applicables à la gestion et la remédiation d’anciens sites miniers.
... After these novel techniques became available, an initial stage in the research has consisted of identifying and resolving the problems associated with their use to elucidate microbial processes in heterogeneous soils and sediments. Significant progress has been achieved in this respect, for example in the development of objective (operator-independent), local segmentation techniques adapted for X-ray µCT images [e.g., (Schlüter et al., 2010;Hapca et al., 2013;Houston et al., 2013a,b)], in terms of improvements of hybridization (FISH) technologies to locate bacterial and archaeal cells in soil thin sections (Eickhorst and Tippkötter, 2008;Schmidt et al., 2012), or in the in elaboration of statistical tools to interpolate 2-D measurements to produce 3-D data (Hapca et al., 2011(Hapca et al., , 2015. ...
Editorial: Elucidating Microbial Processes in Soils and Sediments: Microscale Measurements and Modeling
... Gold-FISH has been applied on a variety of environmental samples to date. Enumerations of single microbial cells in soil and sediment samples showed comparable results to those obtained with established CARD-FISH protocols (12,47). Fluorescent signals were only slightly weaker compared to CARD-FISH (Fig. 3a), which was confirmed for the visualization of single microbial cells of Rhizobium leguminosarum on the rhizoplane of inoculated rice roots as well. ...
The advent of new sophisticated spectroscopic and tomographic techniques arise interest in the study of environmental conditions within microbial habitats on a submicroscopic level. These methods are based on electromagnetic radiation and result in either elemental characterization or structure visualization. Both aspects are relevant for the investigation of microbe-habitat interactions why a correlative detection of microbial cells would be useful. Fluorescence in situ hybridization is an ideal technique to identify and localize microorganisms but requires a cell-detection via fluorescence microscopy which has a limited optical and elemental resolution. Therefore the utilization of nanogold as marker for in situ hybridization approaches is of great potential. Gold labels can be visualized with one of the aforementioned techniques on resolutions beyond light microscopy and allow the identification and localization of single microbial cells in their habitat in situ. The basic principal and potential of this method is described in this chapter giving an overview on the development steps of gold-targeted cell detection as well. Selected results exemplarily show applications in environmental microbiology both via fluorescence microscopy and electron microscopy including elemental mapping.
... Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) coupled with CLSM can be used to significantly enhance the signal intensities of hybridized cells, although is rather expensive and requires enzymatic pretreatment. Currently, it has been applied to improve the retrieval of information from biofilms without the destruction of the biofilm structure (Amann et al. 1998;Lupini et al. 2011), and it has been also found the usefulness of CARD-FISH to heterotrophic bacteria in marine samples and soil (Pernthaler et al. 2002;Schmidt et al. 2012). ...
Quantitative image analysis techniques have gained an undeniable role in several fields of research during the last decade. In the field of biological wastewater treatment (WWT) processes, several computer applications have been developed for monitoring microbial entities, either as individual cells or in different types of aggregates. New descriptors have been defined that are more reliable, objective, and useful than the subjective and time-consuming parameters classically used to monitor biological WWT processes. Examples of this application include the objective prediction of filamentous bulking, known to be one of the most problematic phenomena occurring in activated sludge technology. It also demonstrated its usefulness in classifying protozoa and metazoa populations. In high-rate anaerobic processes, based on granular sludge, aggregation times and fragmentation phenomena could be detected during critical events, e.g., toxic and organic overloads. Currently, the major efforts and needs are in the development of quantitative image analysis techniques focusing on its application coupled with stained samples, either by classical or fluorescent-based techniques. The use of quantitative morphological parameters in process control and online applications is also being investigated. This work reviews the major advances of quantitative image analysis applied to biological WWT processes.
... Although the embedding process can result in sample shrinkage, they are able to retrospectively identify the position of sliced sections in X-ray CT images of the original core, similar to the mapping of buried PET film samplers. Thilo Eickhorst is also developing CARD-FISH (catalyzed reporter deposition-FISH) for use with the thin sections, as this technique promises to be a more sensitive method than standard FISH or the use of fluorescent stains that bind to DNA (deoxyribonucleic acid) to detect the presence of microorganisms [5,19,81]. Figure 6. PET film samplers can be used to visualize the microbiological complexity of soils. ...
The fast pace of scientific research often means that contemporary investigators are unaware of critical developments resulting from the work of previous generations of scientists. We were superficially aware of Sergei Winogradsky (1856 – 1953) through his columns, currently in teaching to illustrate aspects of microbial succession and community function, but were unaware of his importance as the founder of microbial ecology and the first to use microcosms to study bacterial physiology. Here we take the opportunity to remind fellow microbiologists of his work, presenting in homage columns constructed using water and sediments from the Dnipro (Dnieper) collected close to his place of birth. We then provide a review of our own research using microcosms to investigate aspects of plant-bacterial interactions, bacterial evolution, biofilm-formation, and soil colonization, as well as recent advances in developing plastic and transparent soils to investigate root development and fungal hyphae invasion in soil pore networks, to show that more than one hundred years after the invention of the microcosm, they are still being used profitably in microbial research.
A PDF of this is available on request to AS.
Au cours des dernières années, plusieurs chercheurs travaillant au développement de modèles « biogéochimiques » ou « à l'échelle de l'écosystème » de la dynamique du carbone dans les sols ont rapporté être confrontés avec un certain nombre de défis difficiles. Parallèlement, les travaux dans ce domaine se sont concentrés exclusivement sur l'activité microbienne décrite au niveau macro-écologique et ont complètement ignoré l'abondante littérature produite au cours des deux dernières décennies sur l'étude des processus du sol à l'échelle microscopique. Juxtaposition de ces différentes observations suggère qu'un changement radical de perspective s'impose. Dans ce contexte général, le présent article procède à une analyse approfondie de plusieurs des principales limites des modèles actuels à l'échelle de l'écosystème et recommande un certain nombre d'étapes pour déplacer l'attention vers une perspective qui est censée avoir de meilleures chances de succès dans le laps de temps relativement court dont nous disposons, nous devons résoudre plusieurs problèmes environnementaux urgents liés aux sols. Ces étapes nécessitent en particulier que le développement de modèles à grande échelle de la dynamique du carbone dans les sols soit beaucoup plus interdisciplinaire qu'il ne l'a été jusqu'à présent, et qu'il adopte une approche « ascendante », en s'appuyant sur ce que la recherche à l'échelle microscopique révèle sur les processus du sol. Néanmoins, parce que cela peut contribuer à intensifier les efforts, il faudrait préserver un peu d'espace à la marge pour poursuivre les travaux sur la recherche de modèles empiriques applicables à de grandes échelles spatiales. 1. Contexte Au cours des quatre dernières décennies, un nombre considérable de recherches ont été consacrées au développement et à l'utilisation de modèles informatiques « biogéochimiques » ou « à l'échelle de l'écosystème » de la dynamique du carbone dans le sol. Parmi d'autres applications pratiques, ils ont largement servi à prédire la séquestration à long terme du carbone du sol résultant de diverses activités agricoles ou des pratiques de géo-ingénierie destinées à atténuer le changement climatique (voir, par exemple, Riggers et al., 2021; Berthelin et al., 2022; Bruni et al., 2022; Baveye et al., 2023). Initialement, ces modèles supposaient que la matière organique pourrait être répartie en pools distincts avec des temps de résidence distincts, et que sa cinétique de minéralisation pourrait être décrite par de simples réactions de premier ordre, sans tenir compte explicitement de la présence ou activité des micro-organismes. Ces a Ceci est la traduction en français de l'article de 2023 intitulé " Ecosystem-scale modelling of soil carbon dynamics: Time for a radical shift of perspective? ", Soil Biology and Biochemistry 184, 109112.
Cambridge Core - Geochemistry and Environmental Chemistry - Analytical Geomicrobiology - edited by Janice P. L. Kenney
Over the last 60 years, soil microbiologists have accumulated a wealth of experimental data showing that the bulk, macroscopic parameters (e.g., granulometry, pH, soil organic matter, and biomass contents) commonly used to characterize soils provide insufficient information to describe quantitatively the activity of soil microorganisms and some of its outcomes, like the emission of greenhouse gasses. Clearly, new, more appropriate macroscopic parameters are needed, which reflect better the spatial heterogeneity of soils at the microscale (i.e., the pore scale) that is commensurate with the habitat of many microorganisms. For a long time, spectroscopic and microscopic tools were lacking to quantify processes at that scale, but major technological advances over the last 15 years have made suitable equipment available to researchers. In this context, the objective of the present article is to review progress achieved to date in the significant research program that has ensued. This program can be rationalized as a sequence of steps, namely the quantification and modeling of the physical-, (bio)chemical-, and microbiological properties of soils, the integration of these different perspectives into a unified theory, its upscaling to the macroscopic scale, and, eventually, the development of new approaches to measure macroscopic soil characteristics. At this stage, significant progress has been achieved on the physical front, and to a lesser extent on the (bio)chemical one as well, both in terms of experiments and modeling. With regard to the microbial aspects, although a lot of work has been devoted to the modeling of bacterial and fungal activity in soils at the pore scale, the appropriateness of model assumptions cannot be readily assessed because of the scarcity of relevant experimental data. For significant progress to be made, it is crucial to make sure that research on the microbial components of soil systems does not keep lagging behind the work on the physical and (bio)chemical characteristics. Concerning the subsequent steps in the program, very little integration of the various disciplinary perspectives has occurred so far, and, as a result, researchers have not yet been able to tackle the scaling up to the macroscopic level. Many challenges, some of them daunting, remain on the path ahead. Fortunately, a number of these challenges may be resolved by brand new measuring equipment that will become commercially available in the very near future.
Introduction
In contrast to conventional approaches molecular microbiology leads to a deeper understanding of the biodiversity of soil microorganisms. Nevertheless, there is a lack of knowledge regarding the spatial distribution of microbiota in the complex soil matrix and the interaction between the soil structure and microorganisms. DNA analytical methods such as fluorescence in situ hybridization (FISH) are being utilized to improve the characterization of microbial biocoenosis.Micropedological procedures which preserve the soil structure by embedding it with resin, in combination with FISH, allow the localization and identification of soil microorganism diversity in relation to the specific properties of their microhabitats. In this study, FISH was used prior to resin embedding in undisturbed soil samples of four different soils.The polished sections provided visualization of the bound probes as well as the undisturbed soil matrix via fluorescence microscopy. Furthermore, cell counts of active bacteria, locating of hot spots and their relationship to microsites rich in nutrients and water, such as humus or clay minerals, are now easy to perform. This will lead to a better understanding of how soil structure can affect soil microorganisms and vice versa.Derived from the use of 16S rRNA targeted oligonucleotide probes, EUB338 and NON338, the cell counts of FISH-detected bacteria were in the same order of magnitude in the undisturbed and the suspended soil samples. Counterstaining with DAPI showed varying detection rates caused by differing activities of the soil microorganisms.
The root-zone of wetland rice was monitored in a paddy soil throughout a vegetation period with the aid of a rhizotron experiment.
For this purpose (a) digital images of the root-zone were taken daily, and (b) the redox potential was measured in situ every
day. The images were processed by image analysis in order to display areas of oxidation and reduction in the soil. Therefore,
thresholds were set to simplify the localization and quantification of discrete areas which were colourized due to the redox
potential. Both, images and measured redox potentials, provide the basis for the visualization of the root and redox dynamics
in the root-zone. The anaerobic root-zone of flooded paddy soils is significantly influenced by the aerenchymal transport
of oxygen to rice roots. The release of oxygen into the rhizosphere, which causes different patterns of oxidized and reduced
areas in the course of the vegetation period, also affects microbial communities such as methane producing archaea or methane
oxidizing bacteria. The visualization of redox dynamics may, therefore, be useful to localize potential hotspots for the microorganisms
in the root-zone of paddy soils. The reduced and oxidized conditions changed spatiotemporally. Oxidized areas were mostly
found in the surrounding of active roots and in a distinct layer next to the soil surface. Reduced areas shifted from beneath
the oxidized surface layer into sparsely-rooted soil. The ratio of the analyzed oxidized and reduced areas was oscillating
with increasing intensity throughout the monitored vegetation period.
KeywordsRhizotron–Paddy soil–Rhizosphere–Root-zone–Redox potential–Image analysis
rRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material. Introduced Pseudomonas aeruginosa cells were directly fixed in soils and applied to slides after separation of large soil minerals only. Remaining soil minerals (clay minerals) and organic material (up to 8%) did not significantly interfere with signal expression after hybridization. Background signals were mainly caused by autofluorescence of organic material. Non-specific binding of labelled oligonucleotides to soil particles was not observed. In situ detection of introduced cells of Pseudomonas cepacia in a sandy loam spiked with a mixture of selected soil micro-organisms was possible after hybridization with a specific probe. Analysis of natural bacterial populations in soil, however, was not possible by in situ hybridization without activation of these micro-organisms by adding nutrients. Growing cells, e.g. Streptomyces scabies hyphae growing in amended soil, were easily detected.
Enhanced biological phosphate removal in an anaerobic-aerobic activated sludge system has generally been ascribed to members of the genus Acinetobacter. A genus-specific 16S rRNA-targeted oligonucleotide probe was developed to investigate the role of Acinetobacter spp. in situ. Nonisotopic dot blot hybridization to 66 reference strains, including the seven described Acinetobacter spp., demonstrated the expected probe specificity. Fluorescent derivatives were used for in situ monitoring of Acinetobacter spp. in the anaerobic and aerobic compartments of a sewage treatment plant with enhanced biological phosphate removal. Microbial community structures were further analyzed with oligonucleotide probes specific for the alpha, beta, or gamma subclasses of the class Proteobacteria, for the Cytophaga-Flavobacterium cluster, for gram-positive bacteria with a high G + C DNA content, and for all bacteria. Total cell counts were determined by 4',6-diamidino-2-phenylindole staining. In both the anaerobic and the aerobic basins, the activated sludge samples were dominated by members of the class Proteobacteria belonging to the beta subclass and by gram-positive bacteria with a high G + C DNA content. Acinetobacter spp. constituted less than 10% of all bacteria. For both basins, the microbial community structures determined with molecular techniques were compared with the compositions of the heterotrophic saprophytic microbiota determined with agar plating techniques. Isolates on nutrient-rich medium were classified by whole-cell hybridization with rRNA-targeted probes and fatty acid analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
Microprofiles of O2 and NO3- were measured in nitrifying biofilms from the trickling filter of an aquaculture water recirculation system. By use of a newly developed biosensor for NO3-, it was possible to avoid conventional interference from other ions. Nitrification was restricted to a narrow zone of 50 microns on the very top of the film. In the same biofilms, the vertical distributions of members of the lithoautotrophic ammonia-oxidizing genus Nitrosomonas and of the nitrite-oxidizing genus Nitrobacter were investigated by applying fluorescence in situ hybridization of whole fixed cells with 16S rRNA-targeted oligonucleotide probes in combination with confocal laser-scanning microscopy. Ammonia oxidizers formed a dense layer of cell clusters in the upper part of the biofilm, whereas the nitrite oxidizers showed less-dense aggregates in close vicinity to the Nitrosomonas clusters. Both species were not restricted to the oxic zone of the biofilm but were also detected in substantially lower numbers in the anoxic layers and even occasionally at the bottom of the biofilm.
The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.
A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0-8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.
In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.
A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 micrometer). A nonsense probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the probes and for binding by the rhodamine part of the probe, control experiments in which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 x 10(8) active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 x 10(8) active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 micrometer), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4', 6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic probes and for automatic counting of soil bacteria.
We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections > or =70% of total cell counts, e.g., coastal surface waters during spring and summer.
We present a publicly available software tool (http://www.unm.edu/∼compbio/software/GenomeHistory) that identifies all pairs of duplicate genes in a genome and then determines the degree of synonymous and non‐synonymous
divergence between each duplicate pair. Using this tool, we analyze the relations between (i) gene function and the propensity
of a gene to duplicate and (ii) the number of genes in a gene family and the family’s rate of sequence evolution. We do so
for the complete genomes of four eukaryotes (fission and budding yeast, fruit fly and nematode) and one prokaryote (Escherichia coli). For some classes of genes we observe a strong relationship between gene function and a gene’s propensity to undergo duplication.
Most notably, ribosomal genes and transcription factors appear less likely to undergo gene duplication than other genes. In
both fission and budding yeast, we see a strong positive correlation between the selective constraint on a gene and the size
of the gene family of which this gene is a member. In contrast, a weakly negative such correlation is seen in multicellular
eukaryotes.
An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency
of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy
transfer for a large number of combinations of commonly used fluorophores and quenchers. We have also measured the quenching
effect of nucleotides on the fluorescence of each fluorophore. Quenching efficiencies were measured for both the resonance
energy transfer and the static modes of quenching. We found that, in addition to their photochemical characteristics, the
tendency of the fluorophore and the quencher to bind to each other has a strong influence on quenching efficiency. The availability
of these measurements should facilitate the design of oligonucleotide probes that contain interactive fluorophores and quenchers,
including competitive hybridization probes, adjacent probes, TaqMan probes and molecular beacons.
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.
Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification
methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization
(FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed
as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies
that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic
microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community
composition.
The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.
A hierarchical set of five 16S rRNA-targeted oligonucleotide DNA probes for phylogenetically defined groups of autotrophic ammonia- and nitrite-oxidizing bacteria was developed for environmental and determinative studies. Hybridization conditions were established for each probe by using temperature dissociation profiles of target and closely related nontarget organisms to document specificity. Environmental application was demonstrated by quantitative slot blot hybridization and whole-cell hybridization of nitrifying activated sludge and biofilm samples. Results obtained with both techniques suggested the occurrence of novel populations of ammonia oxidizers. In situ hybridization experiments revealed that Nitrobacter and Nitrosomonas species occurred in clusters and frequently were in contact with each other within sludge flocs.
The enumeration of methanotrophic bacteria in the cover soil of an aged municipal landfill was carried out using (1) fluorescent in situ hybridization (FISH) with horseradish peroxidase-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition-FISH (CARD-FISH), and (2) most probable number (MPN) method. The number of methanotrophs was determined in cover soil samples collected during April-November 2003 from a point with low CH(4) emission. The number of types I and II methanotrophs obtained by CARD-FISH varied from 15 +/- 2 to 56 +/- 7 x 10(8) cells g(-1) absolute dry mass (adm) of soil and methanotrophs of type I dominated over type II. The average number of methanotrophs throughout the cover soil profile was highest during May-September when the cover soil temperature was above 13 degrees C. Methanotrophs accounted for about 50% of the total bacterial population in the deepest cover soil layer owing to higher availability of substrate (CH(4)). A lower number of methanotrophs (7 x 10(2) to 17 x 10(5) cells g(-1) adm of soil) was determined by the MPN method compared to the CARD-FISH counts, thus confirming previous results that the MPN method is limited to the estimation of the culturable species that can be grown under the incubation conditions used. The number of culturable methanotrophs correlated with the methane-oxidizing activity measured in laboratory assays. In comparison to the incubation-based measurements, the number of methanotrophs determined by CARD-FISH better reflected the actual characteristics of the environment, such as release and uptake of CH(4), temperature, and moisture, and availability of substrates.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
A hierarchical set of five 16S rRNA-targeted oligonucleotide DNA probes for phylogenetically defined groups of autotrophic ammonia- and nitrite-oxidizing bacteria was developed for environmental and determinative studies. Hybridization conditions were established for each probe by using temperature dissociation profiles of target and closely related nontarget organisms to document specificity. Environmental application was demonstrated by quantitative slot blot hybridization and whole-cell hybridization of nitrifying activated sludge and biofilm samples. Results obtained with both techniques suggested the occurrence of novel populations of ammonia oxidizers. In situ hybridization experiments revealed that Nitrobacter and Nitrosomonas species occurred in clusters and frequently were in contact with each other within sludge flocs.
The chapter discusses the fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, and describes probe designing and testing. Fluorescence FISH with rRNA-targeted probes is a staining technique that allows phylogenetic identification of bacteria in mixed assemblages without prior cultivation by means of epifluorescence and confocal laser scanning microscopy, or by flow cytometry. FISH with oligonucleotide probes is for the purpose of bacterial identification that is to analyze bacterial community structure, and to follow the spatial and temporal dynamics of individual microbial populations in their habitat. Numerous aspects and applications of this method are discussed. FISH is successfully applied in freshwater, coastal, and offshore marine planktonic habitats, and in coastal sediments. It is shown that the fraction of bacteria detectable by FISH corresponds well with the abundance of active cells as determined by microautoradiography in coastal marine bacterioplankton.
Adsorption or binding of DNA by montmorillonite or kaolinite, homoionic to Ca2+, was not affected by base composition, blunt or cohesive ends. Fitting data to both Freundlich and Langmuir adsorption isotherms showed that the amount of lower molecular mass DNA adsorbed and bound by both clay minerals was higher than that of the higher molecular mass DNA. The relevance of phosphate groups for the adsorption of DNA by clay minerals was investigated by adding sodium metaphosphate before and after the addition of DNA to clay minerals: DNA was partially not adsorbed even at low concentrations of sodium metaphosphate. The fact that the observed DNA was partially desorbed by washing with double-distilled H2O indicated that bonds with different degrees of strength were formed between DNA molecules and clay minerals. The higher molecular mass DNA could interact with a larger number of binding sites on the external surface of clay mineral than the lower molecular mass DNA. The number of external surface binding sites was higher on kaolinite than on montmorillonite.
We developed a 16S rRNA-targeted oligonucleotide probe (S-P-GPos-1200-a-A-13) for the Gram-positive bacteria, confirmed its specificity by database searches and hybridization studies, and investigated the effects of humic acids on membrane hybridizations with this probe. S-P-GPos-1200-a-A-13 was used to estimate the abundance of Gram-positive populations in the bovine rumen and Lake Michigan sediments. This probe should be useful for studies of the environmental distribution of Gram-positive bacteria and the detection of uncultured, phylogenetically Gram-positive bacteria with variable or negative Gram staining reactions, and could serve for Gram staining in some diagnostic settings.
Adsorption and desorption of salmon sperm DNA on four different colloidal fractions from Brown Soil and clay minerals were studied. The adsorption isotherms of DNA on the examined soil colloids and minerals conformed to the Langmuir equation. The amount of DNA adsorbed followed the order: montmorillonite≫fine inorganic clay>fine organic clay>kaolinite>coarse inorganic clay>coarse organic clay. A marked decrease in the adsorption of DNA on organic clays and montmorillonite was observed with the increase of pH from 2.0 to 5.0. Negligible DNA was adsorbed by organic clays above pH 5.0. As for inorganic clays and kaolinite, a slow decrease in DNA adsorption was found with increasing pH from 2.0 to 9.0. The results implied that electrostatic interactions played a more important role in DNA adsorption on organic clays and montmorillonite. Magnesium ion was more efficient than sodium ion in promoting DNA adsorption on soil colloids and minerals. DNA molecules on soil colloids and minerals were desorbed by sequential washing with 10 mM Tris, 100 mM NaCl and 100 mM phosphate at pH 7.0. A percentage of 53.7–64.4% of adsorbed DNA on organic clays and montmorillonite was released, while only 10.7–15.2% of DNA on inorganic clays and kaolinite was desorbed by Tris and NaCl. The percent desorption of DNA from inorganic clays, organic clays, montmorillonite and kaolinite by phosphate was 39.7–42.2, 23.6–28.8, 29.7 and 11.4%, respectively. Data from this work indicated that fine clays dominate the amount of DNA adsorption and coarse clays play a more important role in the binding affinity of DNA in soil. Organic matter may not favor DNA adsorption in permanent-charge soil. The information obtained is of fundamental significance for the understanding of the ultimate fate of extracellular DNA in soil.
In recent years, methods of molecular microbiology have been used for the investigation of soil microbial diversity. Fluorescence in situ hybridization (FISH) represents a method which allows a specific staining and enumeration of soil microorganisms by using fluorescent-labelled oligonucleotide probes. However, the detection of FISH-stained cells is often affected by strong autofluorescence of the background, especially in samples of the top soils.In this study a more efficient FISH-approach coupled with catalyzed reporter deposition (CARD) was adapted to soils. Due to tyramide signal amplification (TSA) the fluorescence intensity has been considerably increased at the target binding site of a probe.Six different soils were investigated to evaluate the effect of sample preparation and pre-treatments, TSA, and the procedure of detection. The results show that both cell permeabilization and TSA are two important factors which improve in situ hybridization of soil microorganisms. Soils with higher clay contents have shown better results when prepared on polycarbonate filters rather than on glass slides.Using specific fluorescence filter systems and dye combinations the detection of hybridized cells was extensively increased compared with the application of monolabelled oligonucleotide probes in regular FISH-analysis. As a result, CARD-FISH-stained cells were suitable for automated counting using digital image analysis. Nevertheless, the counterstain with DAPI had to be analyzed manually as it was strongly affected by autofluorescence.
A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100mM arsenate and 10mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.
The bacterial community composition of the active layer (0-45 cm) of a permafrost-affected tundra soil was analysed by fluorescence in situ hybridisation (FISH). Arctic tundra soils contain large amounts of organic carbon, accumulated in thick soil layers and are known as a major sink of atmospheric CO(2). These soils are totally frozen throughout the year and only a thin active layer is unfrozen and shows biological activity during the short summer. To improve the understanding of how the carbon fluxes in the active layer are controlled, detailed analysis of composition, functionality and interaction of soil microorganisms was done. The FISH analyses of the active layer showed large variations in absolute cell numbers and in the composition of the active microbial community between the different horizons, which is caused by the different environmental conditions (e.g., soil temperature, amount of organic matter, aeration) in this vertically structured ecosystem. Universal protein stain 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) showed an exponential decrease of total cell counts from the top to the bottom of the active layer (2.3 x 10(9)-1.2 x 10(8) cells per gram dry soil). Using FISH, up to 59% of the DTAF-detected cells could be detected in the surface horizon, and up to 84% of these FISH-detected cells could be affiliated to a known phylogenetic group. The amount of FISH-detectable cells decreased with increasing depth and so did the diversity of ascertained phylogenetic groups.
In situ identification of prokaryotic cells in subsurface sediments is hampered by the low cellular rRNA contents of the target organisms. Fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) has the potential to overcome this limitation, and was therefore optimized for a 40 cm deep sediment core sampled from a tidal sandy flat of the German Wadden Sea. Treatment with methanol and H(2)O(2) inactivated endogenous peroxidases and effectively reduced the background signal. Percentage of DAPI stained cells detected with the probe combination EUB(I-III), targeting nearly all the Bacteria, were comparable for CARD-FISH with a horseradish peroxidase (HRP)-labeled probe and FISH with a fluorescently monolabeled probe in the 2-3 cm depth interval (92% and 82%, respectively), but significantly higher with the HRP-labeled probe at 35-40 cm, the deepest layer sampled (63% with HRP vs. 26% with monolabeled probe). With CARD-FISH Alphaproteobacteria and the Desulfobulbaceae group of sulfate-reducing bacteria were detected only in the upper layers. In contrast, Desulfosarcinales, the Bacteroidetes group, Planctomycetes, Betaproteobacteria, and Gammaproteobacteria were found at all depths. Archaea were detectable with ARCH915-HRP after achromopeptidase treatment. Surprisingly, aggregates of Bacteria and Archaea were found, below 12 cm depth, that strongly resemble consortia involved in anoxic oxidation of methane that have previously been found in sediments near methane hydrate deposits. With the optimized CARD-FISH protocol, microbial populations could also be detected in deeper sediment horizons. Furthermore, the intensity of the CARD-FISH signals improved detection of rare organisms such as Archaea.
A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.
Fluorescent-dye-conjugated oligonucleotides were used to classify 14 Fibrobacter strains by fluorescence microscopy. On the basis of partial 16S rRNA sequences of six Fibrobacter strains, four hybridization probes were designed to discriminate between the species Fibrobacter succinogenes and Fibrobacter intestinalis and to identify F. succinogenes subsp. succinogenes. After in situ hybridization to whole cells of the six sequenced strains, epifluorescence microscopy confirmed probe specificity. The four probes were then used to make presumptive species and subspecies assignments of eight additional Fibrobacter strains not previously characterized by comparative sequencing. These assignments were confirmed by comparative sequencing of the 16S rRNA target regions from the additional organisms. Single-mismatch discrimination between certain probe and nontarget sequences was demonstrated, and fluorescent intensity was shown to be enhanced by hybridization to multiple probes of the same specificity. The direct detection of F. intestinalis in mouse cecum samples demonstrated the application of this technique to the characterization of complex natural samples.
In a previous publication (Bobrow et al., J. Immunol. Methods (1989) 279-285), we described a novel signal amplification method, catalyzed reporter deposition (CARD), and its application to microplate immunoassays. The method utilizes the analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter onto the surface of a solid-phase immunoassay system. In this paper, we describe the utilization of CARD amplification for nonradiometric membrane assays where detection is facilitated by the formation of an insoluble chromogenic product. In the examples described, deposition of reporter is accomplished in two steps: (i) a horseradish peroxidase (HRP) ADRE catalyzes the deposition of either a biotin or fluorescein labeled phenol, and (ii) incubation with either enzyme labeled streptavidin or anti-fluorescein, respectively, results in the deposition of additional enzyme. Using this method, we have improved detection limits from 8- to greater than 200-fold depending on the amplification format and the chromogen used.
Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.
Rapid phylogenetic identification of single microbial cells was achieved with a new staining method. Formaldehyde-fixed, intact
cells were hybridized with fluorescently labeled oligodeoxynucleotides complementary to 16S ribosomal RNA (rRNA) and viewed
by fluorescence microscopy. Because of the abundance of rRNA in cells, the binding of the fluorescent probes to individual
cells is readily visualized. Phylogenetic identification is achieved by the use of oligonucleotides (length 17 to 34 nucleotides)
that are complementary to phylogenetic group-specific 16S rRNA sequences. Appropriate probes can be composed of oligonucleotide
sequences that distinguish between the primary kingdoms (eukaryotes, eubacteria, archaebacteria) and between closely related
organisms. The simultaneous use of multiple probes, labeled with different fluorescent dyes, allows the identification of
different cell types in the same microscopic field. Quantitative microfluorimetry shows that the amount of an rRNA-specific
probe that binds to Escherichia coli varies with the ribosome content and therefore reflects growth rate.
A novel signal amplification method, catalyzed reporter deposition (CARD), and its application to immunoassays is described. The method involves utilizing an analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter on the surface in a solid-phase immunoassay. In the examples described, deposition of reporter is facilitated by using a horseradish peroxidase (HRP) ADRE to catalyze the deposition of biotin labeled phenols. The deposited biotins are then reacted with streptavidin-labeled enzyme, thereby resulting in deposition of enzyme. Using the ADRE to catalyze the deposition of additional enzyme results in an amplification of the signal of the ADRE alone and improves the detection limit of the assay. The method is highly sensitive, simple, flexible, and easy to implement.
A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of nonspecific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA-targeted oligonucleotide probes to well-growing microbial cells. Two probes of different specificities--one labeled with fluorescein, the other with tetramethylrhodamine--could be applied simultaneously for dual color analysis.
In situ hybridization with rRNA-targeted oligonucleotide probes has become a widely applied tool for direct analysis of microbial population structures of complex natural and engineered systems. In such studies probe EUB338 (AMANN et al., 1990) is routinely used to quantify members of the domain Bacteria with a sufficiently high cellular ribosome content. Recent reevaluations of probe EUB338 coverage based on all publicly available 16S rRNA sequences, however, indicated that important bacterial phyla, most notably the Planctomycetales and Verrucomicrobia, are missed by this probe. We therefore designed and evaluated two supplementary versions (EUB338-II and EUB338-III) of probe EUB338 for in situ detection of most of those phyla not detected with probe EUB338. In situ dissociation curves with target and non-target organisms were recorded under increasing stringency to optimize hybridization conditions. For that purpose a digital image software routine was developed. In situ hybridization of a complex biofilm community with the three EUB338 probes demonstrated the presence of significant numbers of probe EUB338-II and EUB338-III target organisms. The application of EUB338, EUB338-II and EUB338-III should allow a more accurate quantification of members of the domain Bacteria in future molecular ecological studies.
The Molecular Biology Database Collection is an online resource listing key databases of value to the biological community.
This Collection is intended to bring fellow scientists' attention to high-quality databases that are available throughout
the world, rather than just be a lengthy listing of all available databases. As such, this up-to-date listing is intended
to serve as the jumping-off point from which to find specialized databases that may be of use in advancing biological research.
The databases included in this Collection provide new value to the underlying data by virtue of curation, new data connections
or other innovative approaches. Short, searchable summaries and updates for each of the databases included in this Collection
are available through the Nucleic Acids Research Web site at http://nar.oupjournals.org.
Fluorescence in situ hybridisation with rRNA-targeted nucleic acid probes can be used to directly identify microorganisms within complex samples in a few hours and therefore has widespread application in environmental and medical microbiology. The past year has seen significant methodological improvements in fluorescence in situ hybridisation, as well as in the combination of this method with other techniques for inferring functional traits of microorganisms within their environment.
The anaerobic digestion rate for flocculated sludge has been considered to be lower than that of original sludge, particularly in the later stages of digestion; attributed this relatively slower rate to the increased mass transfer resistance for reactants through the large flocs after flocculation. This study confirmed that methane production was retarded by flocculation. The structure of the floc was identified with fluorescence in situ hybridization (FISH) and a confocal laser scanning microscope (CLSM) technique. To verify the mass transfer resistance induced by flocculation, microsensors were applied to assess the response of oxygen concentration distribution inside the flocs that are subjected to sudden changes in ambient oxygen levels. Response time for the electrode at a floc's center was five times greater than the response time in original sludge flocs. Although the effective diffusivity of oxygen in the floc increased by 2.3 times after flocculation, the increased size of the flocculated floc was the major contributor to the total mass transfer resistance to oxygen.
Pesticide and nitrate contamination of soil and groundwater from agriculture is an environmental and public health concern worldwide. Simazine, 6-chloro-N2,N4-diethyl-1,3,5-triazine-2,4-diamine, is a triazine herbicide used in agriculture for selective weed control with several types of crops and it is frequently applied to soils receiving N-fertilizers. Degradation experiments were performed in the laboratory to assess whether the biodegradation of simazine in soil may be influenced by the presence of urea. Simazine degradation rates under different experimental conditions (presence/absence of urea, microbiologically active/sterilized soil) were assessed together with the formation, degradation and transformation of its main metabolites in soil. Simazine degradation was affected by the presence of urea, in terms both of a smaller half-life (t(1/2)) and of a higher amount of desethyl-simazine formed. The soil bacterial community was also studied. Microbial abundances were determined by epifluorescence direct counting. Moreover in situ hybridization with rRNA-targeted fluorescent oligonucleotide probes was used to analyze the bacterial community structure. Fluorescent in situ hybridization (FISH) was used to detect specific groups of bacteria such as the alpha,beta,gamma-subdivisions of Proteobacteria, Gram-positive bacteria with a high G + C DNA content, Planctomycetes, Betaproteobacterial ammonia-oxidizing bacteria and nitrifying bacteria. The presence of the herbicide and/or urea affected the bacterial community structure, showing that FISH is a valuable tool for determining the response of bacterial populations to different environmental conditions.
A CARD-FISH protocol was developed and applied to analyse surface-associated bacteria on the marine algae Ulva lactuca, Delisea pulchra, Corallina officinalis, Amphiroa anceps, Porphyra sp. and Sargassum linearifolium. The combination of Alexa(546)-labelled tyramide as the reporter molecule with SYBR Green II counterstain allowed for superior detection of the hybridised probe fluorescence against plant tissue from which pigment autofluorescence has been reduced.
Native bacteria, Pseudomonas and filamentous bacteria were quantified and localized on wheat roots grown in the field using fluorescence in situ hybridization (FISH). Seminal roots were sampled through the season from unploughed soil in a conservation farming system. Such soils are spatially heterogeneous, and many roots grow slowly through hard soil with cracks and pores containing dead roots remnant from previous crops. Root and rhizosphere morphology, and contact with soil particles were preserved, and autofluorescence was avoided by observing sections in the far-red with Cy5 and Cy5.5 fluorochromes. Spatial analyses showed that bacteria were embedded in a stable matrix (biofilm) within 11 microm of the root surface (range 2-30 microm) and were clustered on 40% of roots. Half the clusters co-located with axial grooves between epidermal cells, soil particles, cap cells or root hairs; the other half were not associated with visible features. Across all wheat roots, although variable, bacteria averaged 15.4 x 10(5) cells per mm(3) rhizosphere, and of these, Pseudomonas and filaments comprised 10% and 4%, respectively, with minor effects of sample time, and no effect of plant age. Root caps were most heavily colonized by bacteria along roots, and elongation zones least heavily colonized. Pseudomonas varied little with root development and were 17% of bacteria on the elongation zone. Filamentous bacteria were not found on the elongation zone. The most significant factor to rhizosphere populations along a wheat root, however, was contact with dead root remnants, where Pseudomonas were reduced but filaments increased to 57% of bacteria (P < 0.001). This corresponded with analyses of root remnants showing they were heavily colonized by bacteria, with 48% filaments (P < 0.001) and 1.4%Pseudomonas (P = 0.014). Efforts to manage rhizosphere bacteria for sustainable agricultural systems should continue to focus on root cap and mucilage chemistry, and remnant roots as sources of beneficial bacteria.
This chapter presents a protocol for the phylogenetic identification of microorganisms in environmental samples (water and sediments) by means of fluorescence in situ hybridization (FISH) with ribosomal RNA-targeted oligonucleotide probes and signal amplification (catalyzed reporter deposition [CARD]). The FISH probes are labeled with the enzyme, horseradish peroxidase (HRP). A subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities of target cells than after FISH with probes directly labeled with fluorochromes. Sample preparation and cell permeabilization strategies for various microbial cell wall types are discussed. The custom labeling of tyramides with different fluorochromes is described. A sequential multicolor CARD-FISH protocol is outlined for the simultaneous detection of different phylogenetic groups.
The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled with fluorescent dyes or the enzyme horseradish peroxidase, they can be used to identify single microbial cells directly by fluorescence in situ hybridization. In this Review, we provide an update on the recent methodological improvements that have allowed more reliable quantification of microbial populations in situ in complex environmental samples, with a particular focus on the usefulness of group-specific probes in this era of ever-growing rRNA databases.
Modified protocols of fluorescence in situ hybridization (FISH) and catalyze reporter deposition fluorescence in situ hybridization (CARD-FISH) were developed in order to detect bacteria in situ in calcified stromatolite biofilms. Smooth, well-preserved thin sections of calcified biofilms (approximately 5 microm thin, vertical sectioning of approximately 1 cm deep) were obtained by cryo-sectioning using the adhesive tape-stabilization technique. A modified hybridization buffer was applied during hybridization to prevent calcite dissolution as well as false binding of oligonucleotide probes to the charged mineral surfaces. Particularly, bright and specific CARD-FISH signals allowed the detection of bacteria in intensively calcified biofilms even at low magnification, which is suitable for investigating millimeter- to centimeter-scale vertical distribution patterns of bacteria.
- H Schmidt
H. Schmidt et al. / Journal of Microbiological Methods 91 (2012) 399–405
Enhanced catalyzed reporter deposition
- M N Bobrow
- K E Adler
- K A Roth
Bobrow, M.N., Adler, K.E., Roth, K.A., 2003. Enhanced catalyzed reporter deposition. U.S.
Pat. US 6,593,100 B2.