Evaluation of fluorochromes for imaging bacteria in soil
TL;DR: The fate of introduced bacteria and the location of indigenous bacteria in soil can be confirmed using the microscopic techniques described in this paper.
Abstract: The distribution of inoculated Escherichia coli cells and indigenous bacteria in a silt loam soil and a sandy soil was studied using fluorescence microscopy techniques. Ethidium bromide stained inoculated cells against the soil and resin background. Satisfactory results were also obtained with epifluorescence microscopy of samples stained with calcofluor white M2R (CFW) and 4′,6-diamidino-2-phenylindole (DAPI). Indigenous soil bacteria were visualized in soil thin sections, after staining with ethidium bromide, using fluorescence microscopy. The density of these bacteria was estimated to be 107–108 cells/cm3. Inoculated E. coli cells, stained with one of the green fluorochromes (fluorescein isothiocyanate (FITC), 5-(4,6-dichlorotriazinyl)aminofluorescein, or eosin Y), could be clearly distinguished in sandy soil thin sections. Confocal laser scanning microscopy with 3D image reconstruction was also successfully applied to characterize distribution of E. coli introduced to soil. The fate of introduced bacteria and the location of indigenous bacteria in soil can be confirmed using the microscopic techniques described in this paper.
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TL;DR: In this paper, the mechanisms of microbial adhesion to RO membrane are illustrated along with the key factors that influence the microbial attachment process and the common strategies for biofilm monitoring in water flow systems are reviewed with highlighting applications, advantages and disadvantages of each strategy.
179 citations
Université Paris-Saclay1, Cranfield University2, Michigan State University3, University of Santiago de Compostela4, Gembloux Agro-Bio Tech5, Clemson University6, University of Bremen7, University of Dundee8, Qatar University9, Institut de recherche pour le développement10, Technische Universität München11, Paris-Sorbonne University12, University of Vienna13, Martin Luther University of Halle-Wittenberg14
TL;DR: The objective of the present article is to review progress achieved to date in the significant research program that has ensued, namely the quantification and modeling of the physical-, (bio)chemical-, and microbiological properties of soils, and the integration of these different perspectives into a unified theory.
Abstract: Over the last 60 years, soil microbiologists have accumulated a wealth of experimental data showing that the usual bulk, macroscopic parameters used to characterize soils (e.g., granulometry, pH, soil organic matter and biomass contents) provide insufficient information to describe quantitatively the activity of soil microorganisms and some of its outcomes, like the emission of greenhouse gases. Clearly, new, more appropriate macroscopic parameters are needed, which reflect better the spatial heterogeneity of soils at the microscale (i.e., the pore scale). 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. In terms of microbial aspects, whereas 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 relevant experimental data are extremely scarce. For the overall research to move forward, it will be 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.
171 citations
TL;DR: The development of fluorescent in situ hybridization and confocal laser scanning microscopy techniques provides new potential for microbial distribution studies.
Abstract: Direct microscopic observation of microorganisms is an important tool in many microbial studies. Such observations have been reported for Protozoa, fungi, inoculated bacteria, and rhizosphere microorganisms but few studies have focused on indigenous bacteria and their spatial relationship within various microhabitats. Principles and applications of epifluorescence microscopy and confocal laser scanning microscopy for visualization of soil microorganisms in situ are reviewed. Both cationic and anionic dyes (also commonly referred to as fluorochromes if they are fluorescent) have been used based on their ability to bind to specific cellular components of microbial cells. Common fluorochromes used for imaging of microbial cells include acridine orange, ethidium bromide, fluorescein isothiocyanate, 5-(4,6-dichlorotriazinyl) aminofluorescein, 4′,6-diamidino-2-phenylindole, europium chelate, magnesium salt of 8-anilino-1-naphthalene sulfonic acid, and calcofluor white M2R. Combining fluorescence staining techniques with soil thin section technology allows one to obtain images of microorganisms in situ. Soil texture and the procedures used for resin embedding are important factors affecting the quality of stained soil thin sections. Indeed, general limitations of applying fluorescence microscopy to soil ecological studies are the non-specific binding of dyes to the soil matrix and the autofluorescence of some soil components. The development of fluorescent in situ hybridization and confocal laser scanning microscopy techniques provides new potential for microbial distribution studies.
106 citations
Additional excerpts
...4 (Li et al. 2003)....
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TL;DR: In this article, the fate of photosynthesis-derived organic carbon (OC) in intact wheat rhizosphere, combining stable isotope labeling at field scale with high-resolution 3D-imaging.
Abstract: Plant roots are major transmitters of atmospheric carbon into soil. The rhizosphere, the soil volume around living roots influenced by root activities, represents hotspots for organic carbon inputs, microbial activity, and carbon turnover. Rhizosphere processes remain poorly understood and the observation of key mechanisms for carbon transfer and protection in intact rhizosphere microenvironments are challenging. We deciphered the fate of photosynthesis-derived organic carbon (OC) in intact wheat rhizosphere, combining stable isotope labeling at field scale with high-resolution 3D-imaging. We used nano-scale secondary ion mass spectrometry and focus ion beam-scanning electron microscopy to generate insights into rhizosphere processes at nanometer scale. In immature wheat roots, the carbon circulated through the apoplastic pathway, via cell walls, from the stele to the cortex. The carbon was transferred to substantial microbial communuties, mainly represented by bacteria surrounding peripheral root cells. Iron oxides formed bridges between roots and bigger mineral particles, such as quartz, and surrounded bacteria in microaggregates close to the root surface. Some microaggregates were also intimately associated with the fungal hyphae surface. Based on these results, we propose a conceptual model depicting the fate of carbon at biogeochemical interfaces in the rhizosphere, at the forefront of growing roots. We observed complex interplays between vectors (roots, fungi, bacteria), transferring plant-derived OC into root-free soil and stabilizing agents (iron oxides, root and microorganism products), potentially protecting plant-derived OC within microaggregates in the rhizosphere.
80 citations
Cites background from "Evaluation of fluorochromes for ima..."
...Since then, strong efforts have been made to observe undisturbed soil-root-microorganism assemblages at the microscale under controlled laboratory conditions, in some cases using 13C and 15N tracers (Nunan et al., 2001; Li et al., 2003; Castorena et al., 2016; Vidal et al., 2016)....
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...Since then, strong efforts have been made to observe undisturbed soil-root-microorganism assemblages at the microscale under controlled laboratory conditions, in some cases using 13C and 15N tracers (Nunan et al., 2001; Li et al., 2003; Castorena et al., 2016; Vidal et al., 2016)....
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TL;DR: 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 and will lead to a better understanding of how soil structure can affect soil microorganisms and vice versa.
Abstract: 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.
71 citations
References
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Book•
01 Jan 1995
TL;DR: Quality Control and Quality Assurance in Applied Soil Microbiology and Biochemistry in applied soil microbiology and biochemistry and field methods.
Abstract: (Chapter Headings): Introduction. Quality Control and Quality Assurance in Applied Soil Microbiology and Biochemistry. Soil Sampling, Handling, Storage, And Analysis. Enrichment, Isolation and Counting of Soil Microorganisms. Estimation of Microbial Activities. Anaerobic Microbial Activities in Soil. Enzyme Activities. Micorbial Biomass. Community Structure. Field Methods. Bioremediation of Soil. Subject Index.
2,125 citations
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01 Jan 1976
TL;DR: This document describes the manufacturing processes used in the separation of Na6 from Na6 during the recovery of Na2SO4 from Na3SO4 in the determination of Na4(SO4) through Na2O3 levels.
Abstract: Fixation - Washing, Dehydration and Embedding - Sectioning - Staining - Support Films - Cryofixation - Negative Staining - References - Index
1,071 citations
01 Jan 2007
TL;DR: Soil Environment, D.D. Standing and K. Killham Microbial Phylogeny and Diversity in Soil, V.T. Torsvik and L. Ovreas Horizontal Gene Transfer and Microevolution in So soil, K.K. Finlay and M.I. Prosser Soil Microbial Communities and Global Climate Change-Methanotrophic and Methanogenic Communities as Paradigms.
Abstract: The soil environment / Dominic Standing and Ken Killham -- Microbial phylogeny and diversity in soil / Vigdis Torsvik and Lise Ovreas -- Horizontal gene transfer and microevolution in soil / Kaare M. Nielsen, Pal Johnsen, and Jan Dirk van Elsas -- The bacteria and archaea in soil / Jan Dirk van Elsas, Vigdis Torsvik, Anton Hartmann, Lise Ovreas, and Janet K. Jansson -- The fungi in soil / Roger D. Finlay -- Protozoa and other protista in soil / Marianne Clarholm, Michael Bonkowski, and Bryan Griffiths -- Microbial interactions in soil / Jan Dirk van Elsas, Ling Tam, Roger D. Finlay, Ken Killham, and Jack. T. Trevors -- Plant-associated bacteria : lifestyle and molecular interactions / Jan Sorensen and Angela Sessitsch -- Microorganisms cycling soil nutrients and their diversity / Jim I Prosser -- Soil microbial communities and global climate change : methanotrophic and methanogenic communities as paradigms / Ralf Conrad --^
879 citations
TL;DR: Application of the concepts of architectural analysis to mixed- or pure-species biofilms will allow detailed examination of the relationships among biofilm structure, adaptation, and response to stress.
Abstract: Scanning confocal laser microscopy (SCLM) was used to visualize fully hydrated microbial biofilms. The improved rejection of out-of-focus haze and the increased resolution of SCLM made it preferable to conventional phase microscopy for the analysis of living biofilms. The extent of image improvement was dependent on the characteristics of individual biofilms and was most apparent when films were dispersed in three dimensions, when they were thick, and when they contained a high number of cells. SCLM optical sections were amenable to quantitative computer-enhanced microscopy analyses, with minimal interference originating from overlying or underlying cell material. By using SCLM in conjunction with viable negative fluorescence staining techniques, horizontal (xy) and sagittal (xz) sections of intact biofilms of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Vibrio parahaemolyticus were obtained. These optical sections were then analyzed by image-processing techniques to assess the distribution of cellular and noncellular areas within the biofilm matrices. The Pseudomonas biofilms were most cell dense at their attachment surfaces and became increasingly diffuse near their outer regions, whereas the Vibrio biofilms exhibited the opposite trend. Biofilms consisting of different species exhibited distinctive arrangements of the major biofilm structural components (cellular and extracellular materials and space). In general, biofilms were found to be highly hydrated, open structures composed of 73 to 98% extracellular materials and space. The use of xz sectioning revealed more detail of biofilm structure, including the presence of large void spaces within the Vibrio biofilms. In addition, three-dimensional reconstructions of biofilms were constructed and were displayed as stereo pairs. Application of the concepts of architectural analysis to mixed- or pure-species biofilms will allow detailed examination of the relationships among biofilm structure, adaptation, and response to stress.
858 citations