Bacteria:
One of the three domains of life (the others being Eukarya and Archaea), also called Eubacteria. They are unicellular prokaryotic microorganisms which generally possess rigid cell walls, multiply by cell division, and exhibit three principal forms: round or coccal, rodlike or bacillary, and spiral or spirochetal. Bacteria can be classified by their response to Oxygen: aerobic, anaerobic, or facultatively anaerobic; by the mode by which they obtain their energy: chemotrophy (via chemical reaction) or Phototrophy (via light reaction); for chemotrophs by their source of chemical energy: Chemolithotrophy (from inorganic compounds) or chemoorganotrophy (from organic compounds); and by their source for Carbon; Nitrogen; etc.; Heterotrophy (from organic sources) or Autotrophy (from Carbon dioxide). They can also be classified by whether or not they stain (based on the structure of their Cell walls) with Crystal violet dye: gram-negative or gram-positive.
JoVE General
Solid Plate-based Dietary Restriction in Caenorhabditis elegans
1Department of Internal Medicine, Division of Geriatric Medicine, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan
Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.
JoVE Immunology and Infection
In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells
This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.
JoVE Immunology and Infection
Invasion of Human Cells by a Bacterial Pathogen
Department of Biology and Biochemistry, University of Bath
A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.
JoVE Immunology and Infection
Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens
1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University
Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.
JoVE Immunology and Infection
Introducing Shear Stress in the Study of Bacterial Adhesion
Blood vessels as a target for infection, Paris center for cardiovascular research, INSERM U970
During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.
JoVE Immunology and Infection
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.
JoVE General
Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment
1School of Earth Sciences, The Ohio State University, 2School of Environment & Natural Resources, The Ohio State University, 3Institute of Geology and Geophysics, Chinese Academy of Sciences
We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.
JoVE General
Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions
1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biomedical Engineering, Texas A&M University
This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.
JoVE Clinical and Translational Medicine
Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
JoVE General
Bacterial Delivery of RNAi Effectors: Transkingdom RNAi
Institute of Pathology, Charité Campus Mitte
For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.
JoVE Bioengineering
Bacterial Immobilization for Imaging by Atomic Force Microscopy
1Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, 2Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 3Department of Surgery, Eastern Virginia Medical School, 4Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory
Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).
JoVE General
Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria
Department of Molecular and Cell Biology, University of Connecticut
This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.
JoVE Immunology and Infection
Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging
Department of Cellular and Molecular Medicine, University of Bristol, UK
Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).
JoVE General
Measuring Caenorhabditis elegans Life Span on Solid Media
1Department of Pathology, University of Washington, 2Molecular and Cellular Biology Program, University of Washington
In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.
JoVE Immunology and Infection
Colonization of Euprymna scolopes Squid by Vibrio fischeri
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University
The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.
JoVE Immunology and Infection
Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging
Biology, University of Texas at Arlington
We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.
JoVE General
A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients
1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biology, Texas A&M University, 3Department of Biomedical Engineering, Texas A&M University
This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.
JoVE Immunology and Infection
Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Department of Molecular Microbiology & Immunology, Oregon Health & Science University
This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.
JoVE General
The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh
The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.
JoVE Immunology and Infection
Bioluminescent Bacterial Imaging In Vivo
Cork Cancer Research Centre, BioSciences Institute, University College Cork
This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.
JoVE Immunology and Infection
Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples
Department of Basic Sciences, Mississippi State University
Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.
JoVE General
Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans
1Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania
Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.
JoVE General
Visualizing Bacteria in Nematodes using Fluorescent Microscopy
Department of Bacteriology, University of Wisconsin-Madison
To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.
JoVE General
Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans
Department of Molecular and Cellular Biology, Harvard University
The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.
JoVE Immunology and Infection
Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky
This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.
JoVE Immunology and Infection
Using Luciferase to Image Bacterial Infections in Mice
Microbial & Molecular Pathogenesis, Texas A&M Health Science Center
Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.
JoVE Bioengineering
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, 2Clinical Research Laboratory, 81st Medical Group, Keesler Air Force Base
Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.
JoVE Immunology and Infection
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Department of Microbiology, Immunology, & Cancer Biology, University of Virginia Health System
We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.
JoVE Immunology and Infection
Quantitative Measurement of the Immune Response and Sleep in Drosophila
Center for Sleep and Circadian Neurobiology, University of Pennsylvania Perelman School of Medicine
To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.
JoVE Immunology and Infection
Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis
Department of Urology, Northwestern University Feinberg School of Medicine
Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.
JoVE General
Aseptic Laboratory Techniques: Plating Methods
Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.
JoVE Clinical and Translational Medicine
Experimental Human Pneumococcal Carriage
1Respiratory Infection Group, Liverpool School of Tropical Medicine, 2Royal Liverpool and Broadgreen, University Hospital Trust, 3Comprehensive Local Research Network, 4NIHR Biomedical Research Centre in Microbial Diseases, Royal Liverpool and Broadgreen University Hospitals NHS Trust, 5Institute of Lung Health, Respiratory Biomedical Unit, University Hospitals of Leicester NHS Trust & University of Leicester, 6Department of Clinical Infection Microbiology & Immunology, Institute of Infection & Global Health, University of Liverpool
Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.
JoVE General
Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism
1Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, 2Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)
Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.
JoVE General
Fabrication of the Thermoplastic Microfluidic Channels
Department of Biomedical Engineering, Boston University
Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.
JoVE Immunology and Infection
TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination
Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL)
A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.
JoVE General
Transformation of Plasmid DNA into E. coli Using the Heat Shock Method
Department of Physiology and Biophysics, University of California, Irvine (UCI)
JoVE General
Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates
1Department of Chemical Physiology, The Scripps Research Institute, 2Department of Molecular and Experimental Medicine, The Scripps Research Institute
In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.
JoVE Immunology and Infection
In vitro Biofilm Formation in an 8-well Chamber Slide
Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital
This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide
JoVE Immunology and Infection
An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci
1Center for Oral Biology, University of Rochester Medical Center, 2State Key Laboratory of Oral Diseases, Sichuan University, 3Department of General Medicine, Glostrup Hospital, Glostrup, Denmark, 4Department of Microbiology and Immunology, University of Rochester Medical Center
Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.
JoVE General
High-throughput Screening and Biosensing with Fluorescent C. elegans Strains
1Department of Biology, University of Florida, 2Mount Desert Island Biological Laboratory
A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.
JoVE Immunology and Infection
Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions
Division of Basic Medical Sciences, Mercer University School of Medicine
This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.
JoVE Immunology and Infection
Purification of Pathogen Vacuoles from Legionella-infected Phagocytes
Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität
This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.
JoVE Bioengineering
Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon
1UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Université de Lyon, 2Département Biosciences, INSA de Lyon, Université de Lyon, 3INSERM U758, Ecole Normale Supérieure de Lyon, Université de Lyon, 4Laboratoire de Génie Civil et Ingénierie Environnementale, INSA de Lyon, Université de Lyon
The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.
JoVE Clinical and Translational Medicine
Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry
1Sanford-Burnham Medical Research Institute, 2Division of Dermatology, University of California, San Diego, 3VA San Diego Healthcare Center, 4Moores Cancer Center, University of California, San Diego
Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.
JoVE General
Engineering Cell-permeable Protein
Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.
JoVE Immunology and Infection
Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells
Laboratory for Epithelial Immunobiology, RIKEN Research Center for Allergy and Immunology
M cells in a specialized follicle-associated epithelium covering Peyer’s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.
JoVE Immunology and Infection
Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence
Department of Bacteriology, University of Wisconsin-Madison
The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.
JoVE Neuroscience
Assaying β-amyloid Toxicity using a Transgenic C. elegans Model
1Institute for Behavioral Genetics, University of Colorado, 2Integrative Physiology, University of Colorado
The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human β-amyloid peptide (Aβ). Induced expression of Aβ in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate Aβ toxicity.
JoVE Immunology and Infection
Growth of Mycobacterium tuberculosis Biofilms
1Department of Infectious Diseases and Microbiology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh
Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.
JoVE Immunology and Infection
A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Institute for Bioscience and Biotechnology Research, University of Maryland
A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.
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