scholarly journals Sampling Natural Viral Communities from Soil for Culture-Independent Analyses

2003 ◽  
Vol 69 (11) ◽  
pp. 6628-6633 ◽  
Author(s):  
Kurt E. Williamson ◽  
K. Eric Wommack ◽  
Mark Radosevich
Keyword(s):  
Agricultural Soils ◽  
Plaque Assay ◽  
Potassium Citrate ◽  
Epifluorescence Microscopy ◽  
Virus Like Particles ◽  
Glycine Buffer ◽  
Beef Extract ◽  
Culture Independent ◽  
Phage Strain ◽  
Dry Soil

ABSTRACT An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 × 108 g of dry soil−1), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5×108 g of dry soil−1) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.

scholarly journals Characterization of the Bacterial Community of the Chemically Defended Hawaiian Sacoglossan Elysia rufescens

2013 ◽  
Vol 79 (22) ◽  
pp. 7073-7081 ◽  
Author(s):  
Jeanette Davis ◽  
W. Florian Fricke ◽  
Mark T. Hamann ◽  
Eduardo Esquenazi ◽  
Pieter C. Dorrestein ◽  
...  
Keyword(s):  
Longitudinal Section ◽  
Epifluorescence Microscopy ◽  
Ionization Mass ◽  
Bacterial Assemblage ◽  
Content Type ◽  
Anticancer Properties ◽  
Kahalalide F ◽  
Culture Independent ◽  
Elysia Rufescens

ABSTRACTSacoglossans are characterized by the ability to sequester functional chloroplasts from their algal diet through a process called kleptoplasty, enabling them to photosynthesize. The bacterial diversity associated with sacoglossans is not well understood. In this study, we coupled traditional cultivation-based methods with 454 pyrosequencing to examine the bacterial communities of the chemically defended Hawaiian sacoglossanElysia rufescensand its secreted mucus.E. rufescenscontains a defense molecule, kahalalide F, that is possibly of bacterial origin and is of interest because of its antifungal and anticancer properties. Our results showed that there is a diverse bacterial assemblage associated withE. rufescensand its mucus, with secreted mucus harboring higher bacterial richness than entire-E. rufescenssamples. The most-abundant bacterial groups affiliated withE. rufescensand its mucus areMycoplasmaspp. andVibriospp., respectively. Our analyses revealed that theVibriospp. that were highly represented in the cultivable assemblage were also abundant in the culture-independent community. Epifluorescence microscopy and matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS) were utilized to detect the chemical defense molecule kahalalide F on a longitudinal section of the sacoglossan.

Occurrence of Bacteriophages Infecting Bacteroides fragilis and Other Viruses in Polluted Marine Sediments

1989 ◽  
Vol 21 (3) ◽  
pp. 15-19 ◽  
Author(s):  
J. Jofre ◽  
M. Blasi ◽  
A. Bosch ◽  
F. Lucena
Keyword(s):  
Marine Sediments ◽  
Sodium Nitrate ◽  
Coastal Area ◽  
Bacteroides Fragilis ◽  
Alkaline Ph ◽  
Glycine Buffer ◽  
Beef Extract

Numbers of B.fragilis bacteriophages in comparison to coliphages, enteroviruses and rotaviruses were evaluated by different methods in sediments of a coastal area near Barcelona which receives substantial amounts of pollution of domestic origin. Phages infecting B.fragilis should be eluted from sediments prior to their enumeration, in the same way as solid-associated animal viruses have to.Phages infecting B.fragilis were better eluted by glycine buffer at alkaline pH than by a caotropic agent (beef extract-sodium nitrate). Such differences between glycine buffer and sodium nitrate were not evident when enteroviruses and rotaviruses were eluted from sediments. This suggests that elution with glycine buffer is preferable for bacteriophages, while the use of caotropic agents is advisable for animal viruses, because of the simplicity of the methodology. In the studied area, coliphages were the more abundant viruses. Also, B.fragilis phages outnumbered rotaviruses and enteroviruses by a factor of more than ten. The ratios between phages active against B.fragilis and either enteroviruses or rotaviruses in marine sediments were similar to the ratios found in sewage, thus indicating that they have a similar fate.

scholarly journals Development of an Environmental Functional Gene Microarray for Soil Microbial Communities

2010 ◽  
Vol 76 (21) ◽  
pp. 7161-7170 ◽  
Author(s):  
Ken C. McGrath ◽  
Rhiannon Mondav ◽  
Regina Sintrajaya ◽  
Bill Slattery ◽  
Susanne Schmidt ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Environmental Samples ◽  
Agricultural Soils ◽  
Soil Microbial Communities ◽  
Functional Gene ◽  
Gene Microarray ◽  
Independent Manner ◽  
Soil Microbial ◽  
Functional Attributes ◽  
Culture Independent

ABSTRACT Functional attributes of microbial communities are difficult to study, and most current techniques rely on DNA- and rRNA-based profiling of taxa and genes, including microarrays containing sequences of known microorganisms. To quantify gene expression in environmental samples in a culture-independent manner, we constructed an environmental functional gene microarray (E-FGA) consisting of 13,056 mRNA-enriched anonymous microbial clones from diverse microbial communities to profile microbial gene transcripts. A new normalization method using internal spot standards was devised to overcome spotting and hybridization bias, enabling direct comparisons of microarrays. To evaluate potential applications of this metatranscriptomic approach for studying microbes in environmental samples, we tested the E-FGA by profiling the microbial activity of agricultural soils with a low or high flux of N2O. A total of 109 genes displayed expression that differed significantly between soils with low and high N2O emissions. We conclude that mRNA-based approaches such as the one presented here may complement existing techniques for assessing functional attributes of microbial communities.

scholarly journals A rigorous assessment and comparison of enumeration methods for environmental viruses

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Judith Kaletta ◽  
Carolin Pickl ◽  
Christian Griebler ◽  
Andreas Klingl ◽  
Rainer Kurmayer ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Plaque Assay ◽  
Environmental Water ◽  
Epifluorescence Microscopy ◽  
Nanoparticle Tracking Analysis ◽  
Simple Task ◽  
Viral Ecology ◽  
Reliable Quantification ◽  
Global Population ◽  
Viral Isolates

Abstract Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.

scholarly journals Soil fungal and bacterial biomass determined by epifluorescence microscopy and mycorrhizal spore density in different sugarcane managements

2014 ◽  
Vol 44 (4) ◽  
pp. 588-594 ◽  
Author(s):  
Adriana Pereira Aleixo ◽  
Glaciela Kaschuk ◽  
Odair Alberton
Keyword(s):  
Microbial Biomass ◽  
Nutrient Cycling ◽  
Soil Microbial Biomass ◽  
Bacterial Biomass ◽  
Soil Samples ◽  
Epifluorescence Microscopy ◽  
Spore Density ◽  
Native Forest ◽  
Soil Microbial ◽  
Dry Soil

Crop productivity and sustainability have often been related to soil organic matter and soil microbial biomass, especially because of their role in soil nutrient cycling. This study aimed at measuring fungal and bacterial biomass by epifluorescence microscopy and arbuscular mycorrhizal fungal (AMF) spore density in sugarcane (Saccharum officinarum L.) fields under different managements. We collected soil samples of sugarcane fields managed with or without burning, with or without mechanized harvest, with or without application of vinasse and from nearby riparian native forest. The soil samples were collected at 10cm depth and storage at 4°C until analysis. Fungal biomass varied from 25 to 37µg C g-1 dry soil and bacterial from 178 to 263µg C g-1 dry soil. The average fungal/bacterial ratio of fields was 0.14. The AMF spore density varied from 9 to 13 spores g-1 dry soil. The different sugarcane managements did not affect AMF spore density. In general, there were no significant changes of microbial biomass with crop management and riparian forest. However, the sum of fungal and bacterial biomass measured by epifluorescence microscopy (i.e. 208-301µg C g-1 dry soil) was very close to values of total soil microbial biomass observed in other studies with traditional techniques (e.g. fumigation-extraction). Therefore, determination of fungal/bacterial ratios by epifluorescence microscopy, associated with other parameters, appears to be a promising methodology to understand microbial functionality and nutrient cycling under different soil and crop managements.

scholarly journals Abundance and Diversity of Viruses in Six Delaware Soils

2005 ◽  
Vol 71 (6) ◽  
pp. 3119-3125 ◽  
Author(s):  
Kurt E. Williamson ◽  
Mark Radosevich ◽  
K. Eric Wommack
Keyword(s):  
Land Use ◽  
Forest Soils ◽  
Agricultural Soils ◽  
Dry Weight ◽  
Morphological Data ◽  
Epifluorescence Microscopy ◽  
Viral Abundance ◽  
Wide Range ◽  
Virus Communities ◽  
Abundance And Diversity

ABSTRACT The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 × 109 to 4.17 × 109 g−1 dry weight) than in agricultural soils (8.7 × 108 to 1.1 × 109 g−1 dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.

scholarly journals Modifying Plaque assay and Clearance test as tools in determination of phage typing for E. Coli bacterial interspecies

2013 ◽  
Vol 10 (1) ◽  
pp. 161-167
Author(s):  
Baghdad Science Journal
Keyword(s):  
Plaque Assay ◽  
High Specificity ◽  
Inhibition Zone ◽  
Phage Typing ◽  
Sewage Sample ◽  
Bacterial Strains ◽  
E Coli ◽  
Phage Strain ◽  
Clearance Test

Bacteriophage of E. Coli interspecies from sewage samples were isolated , the phage particles were isolated from two different sewage samples . The first sample was collected from sewage sample of Baghdad university and the second sample was isolated from domestic sewage sample , first sample showed phages specialized for three E. Coli interspecies bacteria (first plate ) and two E. Coli interspecies bacteria (second plate ) , meanwhile second sample showed phage specialized for two E. Coli. interspeciesThe study of appearance of E coli phages from first sample showed three types of E. coli phages with different size of inhibition zone ( 1 , 0.7,0.5 )Cm respectively ( first plate ) , meanwhile E. Coli interspecies bacteria showed phages related with two interspecies with size of inhibition zone ( 0.5 ,0.4) Cm respectively ( second plate ), on other hand , the second sample showed also two interspecies E. coli with inhibition zone (1,0.8)Cm . experimental method has been designed which showed the modifying method of phage assay to determine phage typing assay . phage has been tested particles with different bacterial strains ( E. coli , shagilla and Serratia ) from different sources and the control was the host of each bacteriophages by taking the O.D for all the tests and controls , to setup new criteria for phage typing .:and this test is called ( Clearance Test ) The result showed that O.D for Test 1 , 2 , 3, was ( 1.6 , 1.2 . 1.7) for ( E. coli , shagilla and Serratia ) bacterial strains , meanwhile the control tests was ( 0.3 , 0.2, 0.4 ) for strains isolated from first sample (first and second plate ) and second samples with different interspecies respectively . This result can predict high specificity of phage strain and this method can be used to determine interspecies strains .So from this experiment we can identify only Clearance Test by measuring only O.D. of bacterial strain with different phages instead of going through plaque assay .

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