Salmonella:
A genus of gram-negative, facultatively anaerobic, rod-shaped bacteria that utilizes citrate as a sole carbon source. It is pathogenic for humans, causing enteric fevers, gastroenteritis, and bacteremia. Food poisoning is the most common clinical manifestation. Organisms within this genus are separated on the basis of antigenic characteristics, sugar fermentation patterns, and bacteriophage susceptibility.
JoVE General
Chronic Salmonella Infected Mouse Model
Department of Medicine, University of Rochester
Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.
JoVE Bioengineering
Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry
1Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, 2Amnis Corporation, 3Department of Chemical and Biological Engineering, Iowa State University
In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.
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
An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
Department of Population Health, University of Georgia
We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.
JoVE Immunology and Infection
Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens
1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey
Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.
JoVE General
Catheterization of Intestinal Loops in Ruminants
1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge
We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.
JoVE General
A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants
1Department of Experimental Oncology, European Institute of Oncology, 2Department of Pathology and Laboratory Medicine, European Institute of Oncology, 3U.O. Gastroenterologia 2, IRCCS Ca' Granda, Ospedale Policlinico di Milano
We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.
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 General
An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase
Department of Biochemistry, University of Toronto
The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.
JoVE General
Method for the Isolation of Francisella tularensis Outer Membranes
Department of Microbiology, University of Texas Southwestern Medical Center
A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.
JoVE General
Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays
Department of Biochemistry and Molecular Biology, University of Chicago
We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.
JoVE General
Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography
1School of Biology, Georgia Institute of Technology, 2Department of Molecular Pharmacology, RWTH Aachen University, 3School of Chemistry and Biochemistry, Georgia Institute of Technology
Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 – 90kDa.
JoVE General
Protein Crystallization for X-ray Crystallography
Molecular Biochemistry and Biophysics, Yale University
The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.
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 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 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 Bioengineering
Microfluidic Device for Recreating a Tumor Microenvironment in Vitro
Department of Chemical Engineering, University Of Massachusetts Amherst
We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.
JoVE Immunology and Infection
'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens
1BioSciences Division, Guild Associates, Inc., 2Department of Molecular Genetics and Microbiology, University of Texas at Austin, 3Department of Craniofacial Biology, Medical University of South Carolina
A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.
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
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
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 General
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Centre for Infectious Diseases and Microbiology, University of Sydney
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
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 Bioengineering
Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model
1Department of Microbiology and Immunology, Tulane University Medical School, 2Physician/Scientist Program, Tulane University Medical School, 3Department of Molecular and Cellular Biology, Baylor College of Medicine
Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.
JoVE Immunology and Infection
Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies
1Molecular and Cell Biology Program, Ohio University, 2Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, 3Department of Biomedical Engineering, Russ College of Engineering and Technology, Ohio University
Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.
JoVE General
Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models
Basic Medical Sciences, University of Arizona College of Medicine - Phoenix
A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.
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
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 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.
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