Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.
21 Related JoVE Articles!
Mechanical Expansion of Steel Tubing as a Solution to Leaky Wellbores
Institutions: Louisiana State University.
Wellbore cement, a procedural component of wellbore completion operations, primarily provides zonal isolation and mechanical support of the metal pipe (casing), and protects metal components from corrosive fluids. These are essential for uncompromised wellbore integrity. Cements can undergo multiple forms of failure, such as debonding at the cement/rock and cement/metal interfaces, fracturing, and defects within the cement matrix. Failures and defects within the cement will ultimately lead to fluid migration, resulting in inter-zonal fluid migration and premature well abandonment. Currently, there are over 1.8 million operating wells worldwide and over one third of these wells have leak related problems defined as Sustained Casing Pressure (SCP)1
The focus of this research was to develop an experimental setup at bench-scale to explore the effect of mechanical manipulation of wellbore casing-cement composite samples as a potential technology for the remediation of gas leaks.
The experimental methodology utilized in this study enabled formation of an impermeable seal at the pipe/cement interface in a simulated wellbore system. Successful nitrogen gas flow-through measurements demonstrated that an existing microannulus was sealed at laboratory experimental conditions and fluid flow prevented by mechanical manipulation of the metal/cement composite sample. Furthermore, this methodology can be applied not only for the remediation of leaky wellbores, but also in plugging and abandonment procedures as well as wellbore completions technology, and potentially preventing negative impacts of wellbores on subsurface and surface environments.
Physics, Issue 93, Leaky wellbores, Wellbore cement, Microannular gas flow, Sustained casing pressure, Expandable casing technology.
In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)
Institutions: University of Arizona, University of Arizona.
Entomopathogenic nematodes (EPN) (Steinernematidae
) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus
bacteria are associated with steinernematids nematodes while Photorhabdus
are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo
and in vitro
techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont–free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock’s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.
Bioengineering, Issue 91, entomology, nematology, microbiology, entomopathogenic, nematodes, bacteria, rearing, in vivo, in vitro
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
High-Sensitivity Nuclear Magnetic Resonance at Giga-Pascal Pressures: A New Tool for Probing Electronic and Chemical Properties of Condensed Matter under Extreme Conditions
Institutions: University of Leipzig.
Nuclear Magnetic Resonance (NMR) is one of the most important techniques for the study of condensed matter systems, their chemical structure, and their electronic properties. The application of high pressure enables one to synthesize new materials, but the response of known materials to high pressure is a very useful tool for studying their electronic structure and developing theories. For example, high-pressure synthesis might be at the origin of life; and understanding the behavior of small molecules under extreme pressure will tell us more about fundamental processes in our universe. It is no wonder that there has always been great interest in having NMR available at high pressures. Unfortunately, the desired pressures are often well into the Giga-Pascal (GPa) range and require special anvil cell devices where only very small, secluded volumes are available. This has restricted the use of NMR almost entirely in the past, and only recently, a new approach to high-sensitivity GPa NMR, which has a resonating micro-coil inside the sample chamber, was put forward. This approach enables us to achieve high sensitivity with experiments that bring the power of NMR to Giga-Pascal pressure condensed matter research. First applications, the detection of a topological electronic transition in ordinary aluminum metal and the closing of the pseudo-gap in high-temperature superconductivity, show the power of such an approach. Meanwhile, the range of achievable pressures was increased tremendously with a new generation of anvil cells (up to 10.1 GPa), that fit standard-bore NMR magnets. This approach might become a new, important tool for the investigation of many condensed matter systems, in chemistry, geochemistry, and in physics, since we can now watch structural changes with the eyes of a very versatile probe.
Physics, Issue 92, NMR, micro-coil, anvil cell, high pressures, condensed matter, radio-frequency
Helminth Collection and Identification from Wildlife
Institutions: Purdue University, Helm West Laboratory.
Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are "roundworms" (nematodes), "thorny-headed worms" (acanthocephalans), "flukes" (trematodes), and "tapeworms" (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 μm sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris' hematoxylin or Semichon's carmine. Helminths are keyed to species by examining different structures (e.g
. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.
Environmental Sciences, Issue 82, Helminths, eukaryotic parasites, worms, nematodes, cestodes, trematodes, acanthocephalans, wildlife
Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp
Institutions: The George Washington University, Clarkson University.
The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in vitro
driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e.
a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.
Bioengineering, Issue 84, oil-flow visualization, vocal fold polyp, three-dimensional flow separation, aerodynamic pressure loadings
Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice
Institutions: Duquesne University.
Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.
Medicine, Issue 86, Bladder pain, electromyogram (EMG), interstitial cystitis/bladder pain syndrome (IC/BPS), urinary bladder distension (UBD), visceromotor response (VMR)
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
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
Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates
Institutions: Cytori Therapeutics Inc, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA.
In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.
Cellular Biology, Issue 79, Adipose Tissue, Stem Cells, Humans, Cell Biology, biology (general), enzymatic digestion, collagenase, cell isolation, Stromal Vascular Fraction (SVF), Adipose-derived Stem Cells, ASCs, lipoaspirate, liposuction
Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control
Institutions: International Center for Tropical Agriculture (CIAT), Cali, Colombia , United States Department of Agriculture, Beltsville, Maryland, USA.
is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana
may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana
endophytically in the common bean (Phaseolus vulgaris
), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana
treatment of 108
conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana
to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana
within a given plant part (i.e.
leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.
Bioengineering, Issue 74, Plant Biology, Microbiology, Infection, Environmental Sciences, Molecular Biology, Mycology, Entomology, Botany, Pathology, Agriculture, Pest Control, Fungi, Entomopathogen, Endophyte, Pest, Pathogen, Phaseolus vulgaris, Beauveria bassiana, Sustainable Agriculture, hemocytometer, inoculation, fungus
Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
Institutions: Massachusetts General Hospital and Harvard Medical School, Massachusetts Institute of Technology.
The nematode C. elegans
has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans
triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans
. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans
Genetics, Issue 73, Biochemistry, Cellular Biology, Molecular Biology, Developmental Biology, Physiology, Anatomy, Caenorhabditis elegans, Obesity, Energy Metabolism, Lipid Metabolism, C. elegans, fluorescent lipid staining, lipids, Nile red, fat, high throughput screening, obesity, gas chromatography, mass spectrometry, GC/MS, animal model
Protein Crystallization for X-ray Crystallography
Institutions: Yale University.
Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.
Molecular Biology, Issue 47, protein crystallization, nucleic acid crystallization, vapor diffusion, X-ray crystallography, precipitant
Electrophysiology of Scorpion Peg Sensilla
Institutions: University of Oklahoma.
We describe a modification of an existing tip-recording technique1,2
for electrophysiologically investigating short, peg-like sensory sensilla3,4
. On the mid-ventral surface of all scorpions are two appendages called pectines, which have dense fields of mechano- and chemosensory peg sensilla5,6
. One method for assessing chemoresponsiveness of these sensilla uses a tungsten electrode for extracellularly recording neural activity within a sensillum as a volatile odorant is introduced to the sensory field5,7
. The limitations of this method include slow data collection and uncontrolled stimulant introduction to, and removal from, the peg field. To overcome these limitations, we developed a new tip-recording technique that uses nonpolar mineral oil as a medium through which to deliver water-based tastants to individual peg sensilla8,9
. We have successfully applied this method to obtain sensillar chemoresponses to citric acid, ethanol, and salt. Here we describe the experimental protocol for such a study9
. We think this new method may be useful for studying the response properties of other arthropod chemosensory systems, including those of insects10, 11
Neuroscience, Issue 50, Electrophysiology, sensory neurobiology, extracellular, tip-recording, mineral oil, Scorpion, Peg Sensilla
Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice
Institutions: Charles River, Charles River, University of Washington.
Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.
Immunology, Issue 55, rat, mouse, endoparasite, ectoparasite, diagnostics, mites, pinworm, helminths, protozoa, health monitoring
High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.
Institutions: University of California, Riverside .
Root-knot nematodes (genus Meloidogyne
) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28°C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.
Immunology, Issue 61, Cowpea, Meloidogyne, root infection, root-knot nematodes, tomato, seedling growth pouches
Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans
Institutions: Harvard University .
The genetically tractable model organism C. elegans
has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans
researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans
. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.
Cellular Biology, Issue 61, Caenorhabditis elegans, extracellular fluid, reverse microinjection, vampiric isolation, pseudocoelom
High Throughput Single-cell and Multiple-cell Micro-encapsulation
Institutions: Vanderbilt University.
Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1
of 83.7% and a double-particle encapsulation efficiency Pk=2
of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed.
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2
The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3
Encapsulation of cells in drops has been utilized to improve detection of protein expression,4
and metabolic activity8
for high throughput screening, and could be used to improve high throughput cytometry.9
Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10
and targeted surface cell coatings.11
Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12
which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13
For a given nozzle geometry, the drop generation frequency f
and drop size can be altered by adjusting oil and aqueous flow rates Qoil
. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk
cells is dictated by Poisson statistics, where Dk
exp(-λ)/(k!) and λ is the average number of cells per drop. The fraction of cells
which end up in the "correctly" encapsulated drops is calculated using Pk
= (k x Dk
)/Σ(k' x Dk'
). The subtle difference between the two metrics is that Dk
relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk
relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low λ) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk
would be high, the majority of drops would be empty (low Dk
), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16
and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18
Here, the Reynolds number Re =uDh/ν
and particle Reynolds number Rep =Re(a/Dh
, where u
is a characteristic flow velocity, Dh
] is the hydraulic diameter, ν
is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep
increase. Note that the high Re and Rep
requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12
However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20
determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12
a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15
In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12
the average number of particles per drop λ was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when λ is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.
Bioengineering, Issue 64, Drop-based microfluidics, inertial microfluidics, ordering, focusing, cell encapsulation, single-cell biology, cell signaling
Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence
Institutions: University of Wisconsin-Madison.
, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta
larvae to a variety of entomopathogenic bacterial species1-5
, as well as the wealth of information available regarding the insect's immune system6-8
, and the pending genome sequence9
make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta
larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta
larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus
species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10
. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11
and oral toxicity assays12.
Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta
immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7
; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13
. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6
. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14
Infection, Issue 70, Microbiology, Immunology, Bacteriology, Entomology, Bacteria, injection, pathogenesis, insect larvae, instar, Manduca sexta, tobacco hornworm, animal model, host pathogen interactions
The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol
Institutions: University of Waterloo.
Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3
. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.
Developmental Biology, Issue 25, Drosophila, embryos, live-imaging, GFP
Proper Care and Cleaning of the Microscope
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.
Basic Protocols, Issue 18, Current Protocols Wiley, Microscopy, Cleaning the Microscope