Application of FISH technology for microbiological analysis: current state and prospects

In order to identify and quantify the microorganisms present in a certain ecosystem, it has become necessary to develop molecular methods avoiding cultivation, which allows to characterize only the countable part of the microorganisms in the sample, therefore losing the information related to the microbial component which presents a vitality condition, although it cannot duplicate in culture medium. In this context, one of the most used techniques is fluorescence in situ hybridization (FISH) with ribosomal RNA targeted oligonucleotide probes. Owing to its speed and sensitivity, this technique is considered a powerful tool for phylogenetic, ecological, diagnostic and environmental studies in microbiology. Through the use of species-specific probes, it is possible to identify different microorganisms in complex microbial communities, thus providing a solid support to the understanding of inter-species interaction. The knowledge of the composition and distribution of microorganisms in natural habitats can be interesting for ecological reasons in microbial ecology, and for safety and technological aspects in food microbiology. Methodological aspects, use of different probes and applications of FISH to microbial ecosystems are presented in this review.     

Molecular biology techniques used in wastewater treatment: An overview

Identification of microorganisms by conventional methods requires the isolation of pure cultures followed by laborious characterization experiments. These procedures are therefore inadequate for study of the biodiversity of a natural or engineered ecosystem. A new set of molecular techniques developed during the 1990s revolutionized microbial ecology research. Among these techniques, cloning and the creation of a gene library, denaturant gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization with DNA probes (FISH) stand out. Cloning provides very precise taxonomical information, but is time consuming and requires specialized personnel and so its introduction in wastewater treatment has been slow. DGGE is a rapid and simple method that provides characteristic band patterns for different samples, allowing quick sample profiling, while retaining the possibility of a more thorough genetic analysis by sequencing of particular bands. FISH makes possible to identify microorganisms at any desired taxonomical level, depending on the specificity of the probe used. It is the only quantitative molecular biology technique, although quantification is either complex or tedious and subjective. Combination with a confocal laser-scanning microscope allows the visualization of three-dimensional microbial structures (granules, biofilms). The methods discussed have deepened our understanding of the microbiology of biological wastewater treatment. PCR-based methods (cloning and DGGE) have proved suitable for identifying the microorganisms that form the sludge. Both DGGE and FISH have been extensively employed. FISH is currently being used for elucidation of the composition, quantification and distribution of different bacterial groups in granules and biofilms, as well as their structure and architecture.     

Co-localized or randomly distributed? Pair cross correlation of in vivo grown subgingival biofilm bacteria quantified by digital image analysis

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 μm) with a pronounced peak at 1.5 μm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.     

实时荧光定量PCR及其在微生物生态学中的应用

定量描述微生物群落的组成,在微生物生态学的许多研究领域都是非常重要的。然而由于可培养技术的局限性,定量描述微生物群落成为比较困难的事情。最近包括PCR技术在内的分子生物学技术为人们提供了有力的工具,使对微生物群落的分布、丰度等有了进一步的了解。实时荧光定量PCR技术作为核酸定量检测技术,自从发明以来在微生物生态学研究中逐渐得到了广泛的应用。从微生物生态学角度,综述了实时荧光定量PCR技术的原理、发展、优缺点及其在微生物生态学研究中的应用与研究进展,并探讨了实时荧光定量PCR技术的发展和应用前景。     

FISH技术在微生物生态学中的研究及进展

分子生物学技术在微生物生态学研究中具有灵敏、精确和快速的优势,但不能提供微生物的形态学、数量性状、空间分布等信息。荧光原位杂交技术结合了分子生物学的精确性和显微镜的可视性信息,可以在自然生境中监测和鉴定不同的微生物个体,尤其是对难培养和未被培养的微生物进行检测。荧光原位杂交技术被广泛用于微生物群落结构诊断和评价,现已成为微生物分子生态学研究中的热点技术。对荧光原位杂交技术的发展和在微生物分子生态学中的应用进行了综述,探讨了该技术应用中存在的问题和发展前景。     

CMEIAS Color Segmentation: An Improved Computing Technology to Process Color Images for Quantitative Microbial Ecology Studies at Single-Cell Resolution

Quantitative microscopy and digital image analysis are underutilized in microbial ecology largely because of the laborious task to segment foreground object pixels from background, especially in complex color micrographs of environmental samples. In this paper, we describe an improved computing technology developed to alleviate this limitation. The system’s uniqueness is its ability to edit digital images accurately when presented with the difficult yet commonplace challenge of removing background pixels whose three-dimensional color space overlaps the range that defines foreground objects. Image segmentation is accomplished by utilizing algorithms that address color and spatial relationships of user-selected foreground object pixels. Performance of the color segmentation algorithm evaluated on 26 complex micrographs at single pixel resolution had an overall pixel classification accuracy of 99+%. Several applications illustrate how this improved computing technology can successfully resolve numerous challenges of complex color segmentation in order to produce images from which quantitative information can be accurately extracted, thereby gain new perspectives on the in situ ecology of microorganisms. Examples include improvements in the quantitative analysis of (1) microbial abundance and phylotype diversity of single cells classified by their discriminating color within heterogeneous communities, (2) cell viability, (3) spatial relationships and intensity of bacterial gene expression involved in cellular communication between individual cells within rhizoplane biofilms, and (4) biofilm ecophysiology based on ribotype-differentiated radioactive substrate utilization. The stand-alone executable file plus user manual and tutorial images for this color segmentation computing application are freely available at . This improved computing technology opens new opportunities of imaging applications where discriminating colors really matter most, thereby strengthening quantitative microscopy-based approaches to advance microbial ecology in situ at individual single-cell resolution.     

荧光原位杂交技术及其在微生物生态学中的应用

综述了荧光原位杂交技术 (fluorescence in situ hybridization FISH)在微生物生态学领域的各种应用 ,同时就其发展过程、原理及种类做了介绍     

利用定量杂交方法对几个人工和环境微生物样品的分析研究

利用真细菌和真核生物两个域特异性探针,初步建立起16SrRNA定量杂交的方法,并对几个人工RNA样品（由提取自S.cerevisiae和P.fluorescens纯培养细胞的RNA按一定比例混合而成）和一个提取自垃圾填埋场渗滤液的RNA样品进行了初步的定量分析。结果表明该方法能从域的水平上对这些样品的组成作较精确的分析,从而在微生物生态学领域具有广泛的应用前景。     

FISH methods in phycology: Phototrophic biofilm and phytoplankton applications

Abstract

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

Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes

Traditional cultivation-based methods to quantify microbial abundance are not suitable for analyses of microbial communities in environmental or medical samples, which consist mainly of uncultured microorganisms. Recently, different cultivation-independent quantification approaches have been developed to overcome this problem. Some of these techniques use specific fluorescence markers, for example ribosomal ribonucleic acid targeted oligonucleotide probes, to label the respective target organisms. Subsequently, the detected cells are visualized by fluorescence microscopy and are quantified by direct visual cell counting or by digital image analysis. This article provides an overview of these methods and some of their applications with emphasis on (semi-)automated image analysis solutions.     

现代分子生物学技术在瘤胃微生态系统研究中的应用

瘤胃中栖息着大量的微生物,由于这些微生物组成复杂且有些细菌在体外无法培养,目前对这些微生物的了解仍然很少。现代分子生物学技术的发展为研究瘤胃微生物提供了有效的方法,利用核酸探针、基因序列分析、遗传指纹技术、全细胞杂交和实时定量PCR等技术可以对瘤胃微生物的分类及进化关系、区系结构图、重要酶的表达以及目的微生物的准确定量进行更为深入和透彻的研究。发展和利用这些技术不仅可以研究微生物之间的关系以及微生物与饲料颗粒之间时间与空间的关系,还能直接在细菌自然生长的环境中对其各种特征进行研究。     

The fluorescent dyes TO-PRO-3 and TOTO-3 iodide allow detection of microbial cells in soil samples without interference from background fluorescence

Visualization of microorganisms in soils and sediments using fluorescent dyes is a common method in microbial ecology studies, but is often hampered by strong nonspecific background fluorescence that can mask genuine cellular signals. The cyanine nucleic acid binding dyes TO-PRO-3 and TOTO-3 iodide enabled a clear detection of microbial cells in a mineral soil, while nonspecific background was greatly reduced compared with commonly used dyes. When used as counterstains for fluorescence in situ hybridization (FISH), both cyanine dyes allowed identification of microbial cells despite strong background from nonspecifically bound probes. TO-PRO-3 and TOTO-3 are easy to use and represent superior alternatives for detecting microorganisms in soil environments.     

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.     

分子微生物生态学及其研究进展

分子微生物生态学是分子生物学实验技术应用于微生物生态学研究领域而发展形成的一门交叉学科,在研究微生物生态系统组成结构、功能的分子机理以及微生物与生物和非生物环境之间相互关系等方面显示了巨大的潜力．十几年来分子微生物生态学研究所取得的成就证明：分子生物学研究技术向微生物生态学领域的不断渗透,为微生物生态学研究领域注入了新的活力,尤其在微生物多样性、微生物区系分子组成及变化规律以及微生物系统进化研究方面取得了重大突破．本文根据近年分子微生物生态学的研究进展,就分子微生物生态学概念的提出、发展历程、主要研究领域、主要研究方法以及未来研究热点领域作以简要综述。     

Application of rRNA-targeted oligonucleotide probes in biotechnology

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.     

Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies.

Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.     

Automated enumeration of groups of marine picoplankton after fluorescence in situ hybridization

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

The identification of microorganisms by fluorescence in situ hybridisation

Fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes facilitates the rapid and specific identification of individual microbial cells in their natural environments. Over the past year there have been a number of methodological developments in this area and new applications of FISH in microbial ecology and biotechnology have been reported.     

微生物分子生态学技术在污水处理系统中的应用

微生物分子生态学作为分子生物学与微生物生态学交叉而形成的学科,在污水处理方面广泛应用。本文从分子生态学实验技术角度,综述了目前污水处理系统中微生物群体结构、多样性及其与功能相关性的研究进展,探讨了分子生态学技术的发展与应用前景,并指出研究该体系微生物对于认识微生物系统发育地位具有重要意义。     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.