Fungi:
A kingdom of eukaryotic, heterotrophic organisms that live parasitically as saprobes, including Mushrooms; Yeasts; smuts, molds, etc. They reproduce either sexually or asexually, and have life cycles that range from simple to complex. Filamentous fungi, commonly known as molds, refer to those that grow as multicellular colonies.
JoVE General
Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi
Department of Bioproducts and Biosystems Engineering, University of Minnesota
This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.
JoVE General
Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies
Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.
JoVE General
Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture
1Biology Department, Western Washington University, 2Washington State University Northwestern Research and Extension Center, 3Department of Plant and Soil Science, Texas Tech University
Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.
JoVE Immunology and Infection
Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays
Plant Pathology and Microbiology, Texas A&M University
The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.
JoVE General
Sexual Development and Ascospore Discharge in Fusarium graminearum
1Genetics Program, Michigan State University, 2Department of Plant Biology, Michigan State University, 3Human Biology Program, Michigan State University, 4Department of Plant Pathology, Michigan State University
Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.
JoVE Immunology and Infection
Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens
1Department of Laboratory Medicine, University of California, San Francisco, 2Division of Infectious Diseases, University of California, San Francisco
The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.
JoVE General
Competitive Genomic Screens of Barcoded Yeast Libraries
1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, 6Department of Pharmaceutical Sciences, University of Toronto
We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.
JoVE Bioengineering
Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control
1Entomology, International Center for Tropical Agriculture (CIAT), Cali, Colombia, 2Sustainable Perennial Crops Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, USA
This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.
JoVE Immunology and Infection
Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
1Institute for Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, 2Department of Immunoregulation, Helmholtz Center for Infection Research
We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).
JoVE Immunology and Infection
Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens
Department of Microbiology and Immunology, Albert Einstein College of Medicine
C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.
JoVE General
Yeast Colony Embedding Method
School of Biological Sciences, University of Missouri - Kansas City
A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.
JoVE Immunology and Infection
Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology
1Biosciences, University of Exeter, 2BICMS, Queen Mary University of London, 3St. Bartholomew's Hospital and The London NHS Trust
A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.
JoVE General
High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli
1Department Of Chemistry and Chemical Engineering, California Institute of Technology, 2Chemistry and Chemical Engineering, California Institute of Technology
We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.
JoVE Immunology and Infection
PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP
1Department of Orthopaedics, School of Medicine, West Virginia University, 2Department of Orthopaedics, Stem Cell Research Center, University of Pittsburgh, 3WVNano Initiative, 4Mary Babb Randolph Cancer Center
Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.
JoVE General
High-Throughput Measurement and Classification of Organic P in Environmental Samples
School of Engineering, University of Vermont
This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.
JoVE General
Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels
Biological Medical Research Center (BMFZ), University Duesseldorf
This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.
JoVE Immunology and Infection
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University
This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).
JoVE Immunology and Infection
Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles
Institute of Biochemical Engineering, Technische Universität Braunschweig
A method to precisely generate and to comprehensively characterize morphology of filamentous fungus Aspergillus niger is described, which allows the mathematical correlation of morphological appearance and productivity.
JoVE Immunology and Infection
Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae
Department of Molecular and Biomedical Sciences, University of Maine
The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.
JoVE Immunology and Infection
Microtiter Dish Biofilm Formation Assay
Microbiology and Immunology, Dartmouth Medical School
The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.
JoVE General
A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation
1Max Planck Institute of Molecular Plant Physiology, 2University of Kaiserslautern
We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.
JoVE General
Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University
Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).
JoVE General
Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis
Boyce Thompson Institute for Plant Research, Cornell University
Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.
JoVE General
Establishing Primary Adult Fibroblast Cultures From Rodents
Department of Biology, University of Rochester
This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.
JoVE Bioengineering
GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization
1DOE Plant Research Laboratory, Michigan State University, 2DOE Great Lakes Bioenergy Research Center, Michigan State University
GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.
JoVE Chemistry
LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations
1Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, 2Department of Physics and Astronomy, Ohio University
Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.
JoVE General
Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
1School of Forestry and Environmental Studies, Yale University, 2Department of Biological Sciences, Virginia Tech, 3Department of Ecology, Evolution and Behavior, The Hebrew University of Jerusalem
We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.
JoVE Immunology and Infection
Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis
Louisiana State University Health Sciences Center
Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.
JoVE Immunology and Infection
Neutrophil Extracellular Traps: How to Generate and Visualize Them
1Core Facility Microscopy, Max Planck Institute for Infection Biology, 2Cellular Microbiology, Max Planck Institute for Infection Biology
Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.
JoVE Immunology and Infection
RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract
1Department of Pediatrics, University of Texas Southwestern Medical Center, 2Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Pediatrics and Microbiology, University of Texas Southwestern Medical Center
A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.
JoVE Immunology and Infection
Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice
1Research Animal Diagnostic Services, Charles River, 2Research Models and Services, Charles River, 3Department of Comparative Medicine, University of Washington
This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.
JoVE Bioengineering
Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count
Department of Biomedical Engineering, McGill University, Montreal, QC Canada
Nanoparticles such as semiconductor quantum dots (QDs) can be used to create photoactivatable agents for anti-microbial or anti-cancer applications. This technique shows how to water-solubilize cadmium telluride (CdTe) QDs, conjugate them to an antibiotic, and perform a bacterial inhibition assay based upon growth curves and plate count.
JoVE General
Assay for Adhesion and Agar Invasion in S. cerevisiae
Department of Molecular Biology, Princeton University
We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.
JoVE General
OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides
1Energy Biosciences Institute, University of California, Berkeley, 2Department of Plant and Microbial Biology, University of California, Berkeley
A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.
JoVE Immunology and Infection
Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging
1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University
A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.
JoVE Neuroscience
Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila
1Center for Advanced Biotechnology and Medicine, Rutgers University, 2Current Address: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, 3Department of Molecular Biology and Biochemistry, Rutgers University
We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.
JoVE Immunology and Infection
Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay
We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.
JoVE General
Gibberella zeae Ascospore Production and Collection for Microarray Experiments.
1Cereal Disease Laboratory, USDA, 2University of Minnesota/ Agroinnova, University of Torino, 3Cereal Disease Laboratory, University of Minnesota
To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.
JoVE General
Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans
Centre d’Immunologie de Marseille-Luminy, Université de la Méditerranée
We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.
JoVE General
Glycan Profiling of Plant Cell Wall Polymers using Microarrays
1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen
A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.
JoVE General
In-vivo Centrifugation of Drosophila Embryos
Department of Biology, University of Rochester
We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.
JoVE General
Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors
Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
When working in a laboratory, it is imperative to minimize sources of contamination. Aseptic technique refers to procedures that permit transfer of cultures and reagents while avoiding contact with non-sterile surfaces. Serological pipettes and micropipettors are used to measure precise volumes without compromising sterility of solutions used in experiments.
JoVE Immunology and Infection
Live-cell Video Microscopy of Fungal Pathogen Phagocytosis
1Division of Applied Medicine, University of Aberdeen, 2Aberdeen Fungal Group, University of Aberdeen
We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.
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