The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications.
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (β-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.
23 Related JoVE Articles!
Design and Construction of an Urban Runoff Research Facility
Institutions: Texas A&M University, The Scotts Miracle-Gro Company.
As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2
facility was constructed which consists of 24 individual 33.6 m2
field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4
, and Ca2+
had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.
Environmental Sciences, Issue 90, urban runoff, landscapes, home lawns, turfgrass, St. Augustinegrass, carbon, nitrogen, phosphorus, sodium
Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity
Institutions: Case Western Reserve University.
Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.
Environmental Sciences, Issue 85, Coexistence, community assembly, environmental drivers, plant-soil feedback, soil heterogeneity, soil microbial communities, soil patch
Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading
Institutions: Grand Valley State University.
Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ
measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.
Environmental Sciences, Issue 85, Limnology, internal loading, eutrophication, nutrient flux, sediment coring, phosphorus, lakes
A Protocol for Conducting Rainfall Simulation to Study Soil Runoff
Institutions: University of Maryland Eastern Shore, USDA - Agricultural Research Service, University of Maryland Eastern Shore.
Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24 hr after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.
Environmental Sciences, Issue 86, Agriculture, Water Pollution, Water Quality, Technology, Industry, and Agriculture, Rainfall Simulator, Artificial Rainfall, Runoff, Packed Soil Boxes, Nonpoint Source, Urea
Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells
Institutions: Monash University.
One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations. These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes. This limits their use in in vitro
and in vivo
applications, such as in vitro
modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered. However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient. This protocol describes targeting of the Pituitary homeobox 3
) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3
-specific DNA donor vector. Following the generation of the PITX3
reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro
Parkinson’s disease modeling or cell replacement therapy.
Molecular Biology, Issue 90, Electroporation, human embryonic stem cell, genome editing, reporter cell line, midbrain dopaminergic neurons
Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation
Institutions: North Carolina State University, North Carolina State University.
Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.
Environmental Sciences, Issue 89,
Lysimetry, porewater, soil, chemical leaching, pesticides, turfgrass, waste
Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)
Institutions: University of Arizona.
Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.
Environmental Sciences, Issue 89, Entomology, Nematology, Steinernema, Heterorhabditis, nematodes, soil sampling, insect-bait, modified White-trap
Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions
Institutions: University of California, Los Angeles (UCLA), University of California, Los Angeles (UCLA).
Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1
. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2
. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3
. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology—provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications—for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.
Stem Cell Biology, Issue 93, Human induced pluripotent stem cells, STEMCCA, factor-free, GMP, xeno-free, quantitative PCR
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface
Institutions: Colorado School of Mines.
Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study evaporation under various surface boundary conditions to improve our current understanding and characterization of this multiphase phenomenon as well as to validate numerical heat and mass transfer theories that couple Navier-Stokes flow in the atmosphere and Darcian flow in the porous media. Experimental data were collected using a unique soil tank apparatus interfaced with a small climate controlled wind tunnel. The experimental apparatus was instrumented with a suite of state of the art sensor technologies for the continuous and autonomous collection of soil moisture, soil thermal properties, soil and air temperature, relative humidity, and wind speed. This experimental apparatus can be used to generate data under well controlled boundary conditions, allowing for better control and gathering of accurate data at scales of interest not feasible in the field. Induced airflow at several distinct wind speeds over the soil surface resulted in unique behavior of heat and mass transfer during the different evaporative stages.
Environmental Sciences, Issue 100, Bare-soil evaporation, Land-atmosphere interactions, Heat and mass flux, Porous media, Wind tunnel, Soil thermal properties, Multiphase flow
Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Institutions: The Recombinant Antibody Network, University of Toronto, University of California, San Francisco at Mission Bay, The University of Chicago.
The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.
Immunology, Issue 95, Bacteria, Viruses, Amino Acids, Peptides, and Proteins, Nucleic Acids, Nucleotides, and Nucleosides, Life Sciences (General), phage display, synthetic antibodies, high throughput, antibody selection, scalable methodology
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Institutions: Colorado State University, Oak Ridge National Laboratory, University of Colorado.
Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e.
C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e.
colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ
soil type and temperature can influence enzyme kinetics.
Environmental Sciences, Issue 81, Ecological and Environmental Phenomena, Environment, Biochemistry, Environmental Microbiology, Soil Microbiology, Ecology, Eukaryota, Archaea, Bacteria, Soil extracellular enzyme activities (EEAs), fluorometric enzyme assays, substrate degradation, 4-methylumbelliferone (MUB), 7-amino-4-methylcoumarin (MUC), enzyme temperature kinetics, soil
Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells
Institutions: University of Florida, Shiraz University of Medical Sciences, Inc..
The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies < 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide
neural stem cells is calculated respectively by counting the number of colonies that are < 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated.
Neuroscience, Issue 49, Stem Cells, Neural Colony Forming Cell Assay, Progenitor Cells, enumeration
Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example
Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1
. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2
. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1
.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1
. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria
spp. Several species of African grasses of the genus Brachiaria
are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4
.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5
Plant Biology, Issue 52, host plant resistance, antibiosis, antixenosis, tolerance, Brachiaria, spittlebugs
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil
Institutions: University of Wisconsin, Madison, University of Wisconsin, Madison, University of Florida .
Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2
. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4
. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7
. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8
. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using δ13
C or ion fragments of these biomarkers in biogeochemical studies9, 10
Molecular Biology, Issue 63, Glucosamine, muramic acid, microbial residue, aldononitrile acetate derivatization, isotope incorporation, ion structure, electron ionization, GC, MS
Transmitting Plant Viruses Using Whiteflies
Institutions: University of Florida .
Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci
, a complex of morphologically indistinquishable species5
, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus
) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16
). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17
). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13
, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14
, new cultural approaches1,4,19
, or the selection and development of resistant cultivars7,8,18
, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7
. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.
Plant Biology, Issue 81, Virology, Molecular Biology, Botany, Pathology, Infection, Plant viruses, Bemisia tabaci, Whiteflies, whitefly, insect transmission, Begomovirus, Carlavirus, Crinivirus, Ipomovirus, host pathogen interaction, virus, insect, plant
Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
Institutions: Yale University, Virginia Tech, The Hebrew University of Jerusalem.
The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2
. Plant litter comprises the majority of detritus3
, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5
. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6
. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9
because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6
. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7
. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10
, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6
. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira
), a dominant grasshopper herbivore (Melanoplus femurrubrum
),and a variety of grass and forb plants9
Environmental Sciences, Issue 73, Microbiology, Plant Biology, Entomology, Organisms, Investigative Techniques, Biological Phenomena, Chemical Phenomena, Metabolic Phenomena, Microbiological Phenomena, Earth Resources and Remote Sensing, Life Sciences (General), Litter Decomposition, Ecological Stoichiometry, Physiological Stress and Ecosystem Function, Predation Risk, Soil Respiration, Carbon Sequestration, Soil Science, respiration, spider, grasshoper, model system
Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression
Institutions: UCL Cancer Institute, Friedrich Miescher Institute for Biomedical Research .
A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli
tetracycline resistance operon1
, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3
. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1
). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4
, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11
. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al
modified the shRNA expression vector pSUPER5
by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14
and apoptosis signaling15,16
. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2
). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.
Genetics, Issue 73, Medicine, Biomedical Engineering, Bioengineering, Cellular Biology, Molecular Biology, Anatomy, Physiology, Mammals, Proteins, Cell Biology, tissue culture, stable manipulation of cell lines, tetracycline regulated expression, cDNA, DNA, shRNA, vectors, tetracycline, promoter, expression, genes, clones, cell culture
Purification of Transcripts and Metabolites from Drosophila Heads
Institutions: University of Florida , University of Florida , University of Florida , University of Florida .
For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila
as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila
are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.
Genetics, Issue 73, Biochemistry, Molecular Biology, Neurobiology, Neuroscience, Bioengineering, Cellular Biology, Anatomy, Neurodegenerative Diseases, Biological Assay, Drosophila, fruit fly, head separation, purification, mRNA, RNA, cDNA, DNA, transcripts, metabolites, replicates, SCA3, neurodegeneration, NMR, gene expression, animal model
Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture
Institutions: Western Washington University, Washington State University Northwestern Research and Extension Center, Texas Tech University.
Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ
degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g.
fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.
Microbiology, Issue 75, Plant Biology, Environmental Sciences, Agricultural Sciences, Soil Science, Molecular Biology, Cellular Biology, Genetics, Mycology, Fungi, Bacteria, Microorganisms, Biodegradable plastic, biodegradable mulch, compostable plastic, compostable mulch, plastic degradation, composting, breakdown, soil, 18S ribosomal DNA, isolation, culture
Using Coculture to Detect Chemically Mediated Interspecies Interactions
Institutions: University of North Carolina at Chapel Hill .
In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e.
biofilm formation, sporulation, virulence factor production, etc
.) Screening is performed under growth conditions where this phenotype is not
expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis
; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.
Microbiology, Issue 80, High-Throughput Screening Assays, Genes, Reporter, Microbial Interactions, Soil Microbiology, Coculture, microbial interactions, screen, fluorescent transcriptional reporters, Bacillus subtilis
An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates
Institutions: University of the Sciences, Arcadia University, Immaculata University, Neumann University, Cabrini College.
Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter
hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter
phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp.
KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.
Environmental Sciences, Issue 98, Phage enrichment, Arthrobacter phages, phage purification, plaque isolation, soil phages, Acintobacteriaceae,