Bacterial Pathogenesis and Host Interactions

Source: Jung, A. L., et al. Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages. J. Vis. Exp. (2017)This video demonstrates the isolation of outer membrane vesicles (OMVs) from Legionella pneumophila using sequential centrifugation, sterile filtration, and ultracentrifugation. The purified OMVs, enriched in membrane proteins and lipopolysaccharides, are suitable for host-pathogen interaction studies.

Begin with tubes containing the non-replicating persistent, or NRP stage of Mycobacterium tuberculosis, which has a thickened outer layer.Add glass beads to one tube and shake it to mechanically disrupt the mycobacteria’s outer layer, enhancing permeability. Next, transfer the bead-beaten mycobacterial suspension to a fresh tube.Add fluorescently-labeled rifampicin, an anti-tuberculosis antibiotic, to both the untreated and bead-beaten cell suspensions.Incubate with shaking to allow...

Begin with a suspension of avian pathogenic E. coli or APEC in growth media.APEC is a Gram-negative bacterium that infects poultry by entering through the respiratory tract.After breaching the mucosal layer, APEC adhere to epithelial cells, multiply locally, and disrupt the epithelial barrier to enter the bloodstream. The bacteria then spread to multiple organs, causing systemic infection.Add an equal volume of the bacterial suspension to each well of a multi-well plate containing increasing...

Source: Spiesz, E. M., et al., Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter. J. Vis. Exp. (2019)This video demonstrates a method for comparing 3D-printed alginate patterns containing either control or genetically engineered bacterial strains. The test strain produces curli fibers upon induction, which reinforce the alginate hydrogel structure. Following treatment with a calcium-chelating solution, only the test pattern remains intact, highlighting the...

Source: Tay, S. B., et al. Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases. J. Vis. Exp. (2016)The video demonstrates a method to quantify biofilm biomass in a pathogenic bacterium, comparing untreated controls with samples treated with a quorum-quenching enzyme. The enzyme disrupts bacterial signaling during early incubation, reducing cell adhesion. After washing and further incubation, control wells form dense biofilms, while treated wells show...

Source: Schlafer, S, and Frost Kristensen, M. Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy. J. Vis. Exp. (2020)This video demonstrates the cultivation of Streptococcus mutans and Candida albicans cross-kingdom biofilms under oral-like conditions to model bacterial–fungal interactions, enabling the study of microbial synergy, glucan-mediated adhesion, and acidogenic virulence in oral pathogenesis.

Source: Kurz, D. L., et al. Microfluidic Platform to Study Bioclogging in Porous Media. J. Vis. Exp. (2022)The video demonstrates the use of a microfluidic device to study biofilm formation. A bacterial suspension is introduced into the microchannel, followed by incubation to allow biofilm development. Flow is then resumed, and pressure monitoring along with imaging is used to detect biofilm formation and pore blockage over time.

Source: Perinbam, K., and Siryaporn, A. A Rapid Image-based Bacterial Virulence Assay Using Amoeba. J. Vis. Exp. (2018)This video demonstrates a rapid, image-based method to assess bacterial virulence by incubating amoebae with surface-attached or planktonic bacteria, applying a calcein-acetoxymethyl ester (calcein-AM) agarose pad, and imaging fluorescence to detect host cell damage.

Source: Bucher, T., et al. Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp. (2016)This video demonstrates a pellicle formation assay using Bacillus subtilis to evaluate small-molecule inhibitors that disrupt matrix synthesis and biofilm development, providing a model to screen compounds that selectively inhibit surface-associated growth without affecting free-floating cells.

Source: Bucher, T., et al. Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp. (2016).This video demonstrates how biofilm matrix integrity influences bacterial resistance to ethanol by comparing colony-forming units in untreated and inhibitor-disrupted biofilms. Reduced CFUs in inhibitor-treated samples demonstrate that the extracellular matrix is essential for protecting biofilm-embedded cells from antimicrobial stress.

Source: Beaudoin, T., et al. Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model. J. Vis. Exp. (2016)This video demonstrates the use of a chambered coverglass to study the effect of cystic fibrosis patient-derived sputum host factors on bacterial biofilm development.

Begin with a bacterial suspension in nutrient media.Add the suspension to a multi-well plate and incubate.The nutrients in the media facilitate bacterial growth and attachment.The adhered bacteria secrete extracellular polymeric substances consisting of polysaccharides, proteins, and DNA to form a layer of biofilm.Wash multiple times with buffer to remove unbound bacteria.Invert the plate to ensure the removal of residual liquid from the well and allow the biofilm to dry.Treat with a fixative...

Source: Hu, Y., et al. Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems. J. Vis. Exp. (2019)This video demonstrates the use of thin-layer chromatography (TLC) to assess the degradation of exopolysaccharides (EPS) by human fecal microbiota. EPS fragments in fermentation supernatants are separated based on size and polarity using TLC, then visualized as colored bands following carbohydrate-specific staining.

Source: Schumacher, D., and Sogaard-Andersen, L. Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus. J. Vis. Exp. (2018)This video demonstrates the use of live-cell imaging to track fluorescently tagged chromosome origins during bacterial cell division. It captures chromosome segregation dynamics using time-lapse phase-contrast and fluorescence microscopy.

Source: Tokunou, Y. et. al., Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1. J. Vis. Exp. (2018)This video demonstrates the assembly and operation of an electrochemical reactor model designed to simulate biofilm formation on electrode surfaces. Controlled electrochemical stimulation promotes microbial adhesion, leading to the gradual development of a bacterial monolayer biofilm.

Source: Naji, A., et al. Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans. J. Vis. Exp. (2019)This video demonstrates the use of green fluorescent protein-tagged SKN-1 in C. elegans to monitor oxidative stress by tracking green fluorescence. Worms exposed to S. gordonii show strong nuclear fluorescence, confirming pathogen-induced stress, while those fed E. coli display diffuse green fluorescence in the cytoplasm, indicating minimal SKN-1 activation.

Source: Nielsen, T. B., et al., Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia. J. Vis. Exp. (2018)This video demonstrates the procedure of delivering bacteria through the oropharynx into the lungs of an anesthetized mouse to induce lung infection and trigger an immune response.

Source: Zhao, K., et al. An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm. J. Vis. Exp. (2018)The video demonstrates a photodynamic therapy protocol to assess antibacterial effects on Gram-positive bacterial biofilms. Biofilms are divided into four groups: two receive a photosensitizer precursor, and two receive a buffer. All are incubated in the dark, followed by red light exposure to one precursor-treated group and...

Source: Escudero, J. A., et.al. Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance. J. Vis. Exp. (2018)This video demonstrates how escalating cefazidime exposure is used to compare the survival of three E. coli strains with different copies of the ampicillin resistance gene. It shows that only the strain with a multicopy plasmid survives at higher drug levels, highlighting how increased gene copy number accelerates resistance evolution.

Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp. (2018)This video demonstrates the phage-mediated transfer of donor DNA carrying an excisable antibiotic resistance cassette into Escherichia coli. The integrated cassette replaces the target gene via homologous recombination, resulting in a stable gene deletion.

Source: Claes, J., et al, In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions. J. Vis. Exp. (2015).This video demonstrates a method for studying host-pathogen interactions using a micro-parallel plate flow chamber seeded with human primary endothelial cells. Following endothelial cell activation by an ionophore and perfusion with fluorescently labeled bacteria, the cells release von Willebrand factor (VWF), which binds bacterial adhesins under shear...

Source: Holt-Torres, P. S. et al. Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro. J. Vis. Exp. (2023)This video demonstrates the use of the focused-contact exposure method to evaluate the antibacterial activity of nanostructured surfaces. It outlines the steps involved in establishing direct contact between bacteria and the nanostructured surface, incubation, bacterial collection, and colony counting to assess viability.

Source: Dubois, V., et al. Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes. J. Vis. Exp. (2018)This video demonstrates the procedure of co-culturing amoebae with mutant bacterial strains to evaluate their intracellular survival and identify virulence markers through differences in colony counts.

Source: Nagre, N., et al. Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice. J. Vis. Exp. (2019).This video demonstrates the assessment of complement receptor-mediated phagocytosis using sheep red blood cells opsonized with IgM and complement, followed by their uptake by mouse macrophages. The extent of phagocytosis is quantified by measuring a blue chromogenic product formed through hemoglobin-catalyzed oxidation of diaminofluorene.

Source: Zhang, S., et al. Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model. J. Vis. Exp. (2018).This video demonstrates the detection of target gene expression in a bacteria-infected mouse brain. RNA is extracted, treated with a DNA-degrading enzyme, and reverse-transcribed into cDNA. The cDNA is amplified through thermal cycling with gene-specific primers, while a DNA-binding dye binds to the...

Source: Radulovic, K., et al. Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach. J. Vis. Exp. (2018)This video demonstrates a step-by-step procedure for injecting lipopolysaccharide (LPS) into mice intraperitoneally to induce systemic inflammation.

Take pig kidney cell cultures, with one culture pre-treated with an inhibitor that increases lysosomal pH, impairing autophagic-lysosomal degradation.Add pathogenic bacteria and centrifuge to enhance host-pathogen contact.Incubate to allow bacterial invasion and internalization into endocytic vesicles.The internalized bacteria consume host nutrients to replicate, triggering metabolic stress.This stress activates autophagy, where an autophagic membrane expands around the vesicle, forming an...

Take an adherent culture of murine macrophages.Treat the test well with a buffer containing Wnt5A, a signaling molecule, and the control with buffer only.Infect with a bacterial pathogen and incubate.In the test cells, Wnt5A activates a signaling cascade that enhances cytoskeletal reorganization to boost bacterial uptake into phagosomes, whereas control cells lacking this signaling internalize fewer bacteria.Wash to remove non-internalized bacteria, then incubate with a medium containing...

Source: In, J. G.,et al. Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium. J. Vis. Exp. (2019).This video demonstrates the procedure for culturing colon-derived epithelial cells on a collagen-coated Transwell system to generate a mucus-producing monolayer that models the intestinal barrier and enables host–pathogen interaction studies.

Source: Luukinen, H., et al. Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish. J. Vis. Exp. (2018).

Source: Sigal, N., et al. RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells. J. Vis. Exp. (2016)This video demonstrates RNA extraction from Listeria monocytogenes isolated from infected murine macrophages. The protocol preserves RNA, lyses cells using phenol–chloroform and heat, and separates RNA by centrifugation. Salt and ethanol, combined with cold incubation, promote RNA precipitation. The resulting RNA captures host-induced gene expression, revealing...

Source: Vonaesch, P. et. al., Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells. J. Vis. Exp. (2017)This video demonstrates the infection of epithelial cells with the pathogenic bacterium Shigella flexneri and the resulting host cellular response. Bacterial effector proteins disrupt host translation, triggering the formation of membrane-less organelles called stress granules, which reflect the cell's adaptive response to infection.

Start with an immune cell culture infected with pathogenic bacteria that form intracellular inclusion bodies.The bacterial infection impairs the antigen presentation pathway in the immune cells.Take an uninfected culture as a control.Next, add a model protein antigen and incubate.Immune cells internalize, process, and present the model antigen-derived peptides on MHC-I molecules.After incubation, wash the cells to remove any unprocessed antigen.Fix the cells, then add a quenching agent to...

Source: Chetrit, D., et al. Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System. J. Vis. Exp. (2020).This video demonstrates the preparation and imaging of fluorescently labeled pathogenic bacteria. Agarose pads are first prepared and positioned within an adhesive frame. A suspension of genetically modified bacteria expressing a fluorescent protein is applied to the pad to immobilize the cells. The...

Source: Winston, J. A., et al. Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291. J. Vis. Exp. (2016)This video demonstrates the procedure for inoculating antibiotic-pretreated mice with Clostridium difficile spores to establish a murine model of infection for studying pathogenesis and host responses.

Source: Winston, J. A. et al. Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291. J. Vis. Exp. (2016)This video demonstrates the quantification of Clostridium difficile in fecal samples from an infected mouse model using selective culture techniques. It outlines the procedures for sample collection, anaerobic processing, serial dilution, and colony enumeration to assess bacterial colonization and host-pathogen interactions.

Source: Berens, C. et al. Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling. J. Vis. Exp. (2015)This video demonstrates the use of immunoblotting to assess apoptosis inhibition by the bacterial effector protein CaeB in human cells. It outlines the steps for detecting apoptotic markers following doxycycline-induced expression of pro-apoptotic proteins in host cells expressing the effector.

Source: Yuan, Z., et al. Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury. J. Vis. Exp. (2018)This video demonstrates an in vivo mouse model for inducing and analyzing acute lung injury (ALI) using intraperitoneal administration of bacterial lipopolysaccharide. The inflammatory cascade triggered by alveolar macrophages and neutrophils leads to epithelial and endothelial barrier disruption, allowing fluid and immune cells to accumulate in the alveoli. Alveolar...

Source: Li, T., et al. Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells. J. Vis. Exp. (2020).This video demonstrates how cigarette smoke extract weakens antimicrobial defense in lung epithelial cells by reducing AMP production, leading to increased bacterial invasion. The procedure measures intracellular bacterial load to assess the impact of smoke exposure on host-pathogen interactions.

Source: Ahn, J. J., et al., Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses. J. Vis. Exp. (2016)This video demonstrates the preparation of mouse splenocyte cultures from Listeria monocytogenes-infected mice for downstream cytokine analysis. It includes ex vivo bacteria-derived antigen stimulation and sample processing to study T cell responses.

Source: Barbero, A. M., et al. Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1. J. Vis. Exp. (2025)This video demonstrates the procedure for labeling host immune receptors that bind bacterial surface antigens, enabling their detection through flow cytometry.

Source: Zhang, C. et al. Development and Assessment of Intracellular Infection Models for Staphylococcus aureus. J. Vis. Exp. (2025)This video demonstrates the quantification of intracellular survival of a methicillin-resistant bacterial pathogen in mouse peritoneal macrophages. It outlines the steps involved in infection, extracellular bacterial clearance, host cell lysis, and colony enumeration to assess bacterial survival within host macrophages.

Source: Gutiérrez, S., et al., Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages. J. Vis. Exp. (2017)This video demonstrates the magnetic isolation of phagosomes containing biotin–streptavidin bead–coated bacteria from infected macrophages. The protocol includes buffer treatments, cell disruption, magnetic separation, and washing to yield purified phagosomes for analysis.

Source: Mills, B., et al., Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy. J. Vis. Exp. (2017)This video demonstrates real-time imaging of fluorescently labeled Staphylococcus aureus in suspension and lung tissue using fibered confocal fluorescence microscopy.

Source: Ng, K. M., Tropini, C. Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging. J. Vis. Exp. (2021).This video demonstrates the procedure of fluorescence in situ hybridization (FISH) to stain gut microbiota within mouse intestinal tissue by hybridizing fluorescent probes to bacterial rRNA (ribosomal ribonucleic acid) and counterstaining the host structures for microscopic analysis.

Source: Siva-Jothy, J. A., et al., Oral Bacterial Infection and Shedding in Drosophila melanogaster. J. Vis. Exp. (2018)This video demonstrates the procedure for monitoring bacterial shedding in Drosophila by transferring an infected fly across multiple food-containing tubes, recovering shed bacteria, and quantifying viable colonies through serial dilution and plating.

Source: de Vries, C. R., et al., A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection. J. Vis. Exp. (2020).This video demonstrates a bioluminescent imaging technique to monitor bacterial infection progression in a live mouse model. Pathogenic bacteria engineered with the luxABCDE reporter system emit light at the infection site, allowing visualization of metabolically active bacteria. By imaging the mouse over time, researchers can non-invasively track infection...

Source: Shi, J., et al. Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens. J. Vis. Exp. (2020)This video demonstrates a co-infiltration assay for identifying pathogen effector proteins that suppress RNA silencing in Nicotiana benthamiana 16c. A fluorescent reporter and a candidate effector gene are delivered into plant leaves via Agrobacterium. Normally, RNA silencing degrades reporter transcripts, reducing fluorescence. Suppressor effectors...

Source: Niu, L., et al. Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy. J. Vis. Exp. (2019)This video demonstrates the use of intravital confocal microscopy to visualize macrophage lytic cell death in zebrafish embryos following Mycobacterium marinum infection.

Source: Seman, B. G., et.al. Neonatal Imaging Model of Gram-Negative Bacterial Sepsis. J. Vis. Exp. (2020)This video demonstrates the isolation of neutrophils and inflammatory monocytes from neonatal mouse spleens and their application in a luminescent bacterial killing assay. It covers mechanical dissociation, red blood cell lysis, magnetic bead–based cell enrichment, bacterial incubation, and antibiotic protection. Timed luminescence readings indicate initial bacterial internalization...

Source: Choi, Y. S., et al. In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA. J. Vis. Exp. (2015).This video demonstrates the in situ detection of bacteria in mouse gingival tissue sections using digoxin-labeled DNA probes targeting bacterial DNA. The section is incubated with the probe. Alkaline phosphatase-conjugated antibodies specific to digoxin are added. Endogenous phosphatases are inactivated, and a colored precipitate is...

Source: Valentine, M. E., et al., Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection. J. Vis. Exp. (2020)This video demonstrates the use of a bioluminescence-based imaging approach to monitor bacterial infection in a live mouse model. Sequential bioluminescent and X-ray imaging enables real-time visualization and anatomical localization of infection spread throughout the host.

Source: Luyts, N., et al. Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging. J. Vis. Exp. (2021).This video demonstrates the induction and tracking of urinary tract infection in a mouse model using bioluminescent E. coli injected into the bladder, where bacterial adhesion, invasion, and intracellular replication occur. The emitted light from the bioluminescent operon serves as a real-time marker of bacterial load during infection...

Source: O'Brien, V. P., et. al., Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice. J. Vis. Exp. (2020)This video demonstrates the use of scanning electron microscopy to visualize bacterial colonization and host–pathogen interactions in the bladder epithelium of a pre-infected mouse model.

Source: Bernut, A., et al., Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos. J. Vis. Exp. (2015).This video demonstrates a protocol for isolating and quantifying Mycobacterium abscessus from an infected zebrafish embryo. The procedure involves embryo euthanasia, tissue lysis, and homogenization to release the bacteria, followed by centrifugation and resuspension in a surfactant buffer. Finally, the bacterial suspension is serially diluted, plated...

Source: Bernut, A., et al. Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos. J. Vis. Exp. (2015)This video demonstrates a protocol that uses transgenic zebrafish embryos expressing fluorescent macrophages and infected with fluorescent Mycobacterium abscessus rough strain to visualize host-pathogen interactions. It reveals how extracellular cording enables immune evasion, promoting bacterial survival and host cell damage.

Begin with a contaminated water sample likely to harbor fecal coliforms, including E. coli.Perform a serial dilution in distilled water to reduce turbidity and microbial load.Mix the desired diluted sample with the nutrient medium and load it into a microfluidic chip.Use vacuum filtration to capture bacteria on a membrane within the serpentine channel of the chip.Add nutrient medium to the upper chamber and incubate to enhance bacterial recovery.Replace the medium with a solution of E.

Source: Makowski, Ł., et al. Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy. J. Vis. Exp. (2020)This video demonstrates time-lapse fluorescence imaging of Bdellovibrio bacteriovorus preying on immobilized E. coli beneath an agarose pad, enabling real-time visualization of intracellular replication and progeny release for studying bacterial predation and host-pathogen interactions.

Source: Tay, S. B., et.al. Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases. J. Vis. Exp. (2016)This video demonstrates the process of inhibiting biofilm formation by introducing an enzyme that disrupts bacterial cell signaling. The enzyme prevents the bacteria from producing extracellular polymeric substances, thereby reducing biofilm development on a glass-bottomed microdish. Finally, the biofilm structure is visualized using fluorescent lectins and...

Source: Collins, A. J., et al., Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity. J. Vis. Exp. (2021)This video demonstrates the use of Polymerase Chain Reaction (PCR) to detect Saccharibacteria in a binary culture with Actinomyces. The protocol outlines DNA amplification and gel electrophoresis to confirm a stable host-parasite interaction.

Source: Matthysse, A. G. Adherence of Bacteria to Plant Surfaces Measured in the Laboratory. J. Vis. Exp. (2018).This video demonstrates the evaluation of bacterial attachment to plant roots using phase-contrast microscopy. Root samples from seedlings incubated with bacteria are first mounted to check for free bacteria. The samples are then washed to remove loosely attached bacteria and remounted using a suitable coverslip to prevent damage. Finally, the number of bacteria attached to the root...

Source: Matthysse, A. G. Adherence of Bacteria to Plant Surfaces Measured in the Laboratory. J. Vis. Exp., (2018)This video demonstrates a protocol for measuring root colonization of tomato seedlings by wild-type and cellulose-deficient pathogenic bacteria. Viable counts of tightly adhering bacteria show greater adhesion and microcolony formation by the wild-type strain, highlighting cellulose’s role in stable root colonization.

Source: Brown, A. O., and Orihuela, C. J. Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling. J. Vis. Exp. (2015)The video demonstrates the immunostaining of chemically fixed mouse cardiac tissue sections to visualize pathogenic bacteria localized within infection-induced microlesions. The process includes tissue washing, membrane permeabilization, blocking of nonspecific sites, antibody labeling, nuclear counterstaining, and confocal...

Source: Specker, J. T., et al. Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection. J. Vis. Exp. (2022)This video demonstrates the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to visualize amino acid distribution in intestinal tissues from mice infected with Clostridioides difficile, alone or in combination with Enterococcus faecalis. The procedure involves tissue...

Source: Bucher, T., et. al., Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp.(2016)This video demonstrates the preparation of mature bacterial biofilm colonies for high-resolution imaging. It covers fixation, dehydration, mounting, and metal coating steps to preserve and visualize biofilm morphology.

Source: Beaudoin, T., et al. Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model. J. Vis. Exp. (2016)This video demonstrates an in vitro method to assess how patient-derived cystic fibrosis sputum influences bacterial biofilm formation. Using chamber slides, fluorescent staining, and confocal microscopy, it reveals increased thickness, altered architecture, and higher viable biomass in biofilms grown with sputum, highlighting the role of host components...

Take tubes with a medium containing a nanoparticle composite, engineered from a combination of transition metals and carbon.Introduce antibiotic-resistant Gram-positive and Gram-negative pathogenic bacteria into separate tubes as test samples.Include untreated bacteria as a negative control and antibiotic-treated bacteria as a positive control.Incubate the samples with shaking.In Gram-negative bacteria, the porous outer membrane and thin peptidoglycan layer facilitate nanoparticle entry,...

Source: Bratton, B. P., et al., Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization. J. Vis. Exp. (2019)This video demonstrates the method for acquiring and analyzing three-dimensional fluorescence images of bacterial cells to study protein organization at the single-cell level.

Source: Le, T. A. N., et al., Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans. J. Vis. Exp. (2019)This video demonstrates a fluorescence-based assay to quantify intestinal permeability in C. elegans exposed to Pseudomonas aeruginosa. Compared to controls fed Escherichia coli, pathogen-treated worms show increased fluorophore leakage into the body cavity, indicating epithelial barrier damage.

Source: O'Brien, V.P. et al. Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice. J. Vis. Exp. (2020)This video demonstrates the preparation of a Gardnerella vaginalis inoculum for establishing a mouse model of recurrent urinary tract infection.

Source: Lenhard, A. et al. A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness. J. Vis. Exp. (2022)This video demonstrates a mouse co-infection model in which the influenza virus triggers the transition of Streptococcus pneumoniae from a colonizer to a pathogen. It outlines the steps involved in bacterial colonization, viral infection, and disease progression.

Source: Lenhard, A., et al. A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness. J. Vis. Exp. (2022)This video demonstrates the processing of lungs from a mouse co-infected with a pathogenic bacterium and a virus. Mincing, homogenizing, and plating the lung tissue allows quantification of bacterial load, indicating bacterial spread to the lungs and disease severity.

Source: Cross, T., et al., An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates. J. Vis. Exp.(2015)This video demonstrates the preparation of a bacterial culture for bacteriophage production. It covers the steps of inoculating a soil bacterium into a baffled flask, promoting uniform growth through aeration and shaking, and monitoring cell density using optical density measurements. Once the culture reaches the optimal growth phase,...

Source: Conover, M. S. et. al., Establishment and Characterization of UTI and CAUTI in a Mouse Model. J. Vis. Exp. (2015)This video demonstrates the detection of intracellular bacterial communities (IBCs) in mouse bladder tissue infected with β-galactosidase-expressing Escherichia coli. Following tissue fixation, permeabilization, and staining with X-gal, IBCs appear as blue puncta under a dissecting microscope, confirming bacterial invasion during urinary tract infection.

Source: Wang, Y., et al. In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging. J. Vis. Exp. (2017)This video demonstrates how to establish a burn infection in a mouse model by applying a heated brass block followed by pathogenic bacterial inoculum. The bacteria colonize the exposed extracellular matrix and form a biofilm, enabling in vivo studies of host-pathogen interactions.

Source: Wang, Y., et al. In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging. J. Vis. Exp. (2017)This video demonstrates the procedure for imaging burn wound infections in mice inoculated with Acinetobacter baumannii carrying a bioluminescent operon. It demonstrates how luciferase-catalyzed substrate oxidation produces light proportional to bacterial load, enabling real-time monitoring of...

Source: Barbero, A. M., et.al. Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1. J. Vis. Exp. ( (2025)This video demonstrates a fluorescence-based assay to study the interaction between a macrophage receptor and Mycobacterium tuberculosis antigens. Fluorescently labeled antigens on a coverslip are incubated with macrophage protein extracts, cross-linked, antibody-labeled, and visualized by overlapping fluorescence signals.

Source: Li, J., et al. Development of a Larval Zebrafish Infection Model for Clostridioides difficile. J. Vis. Exp. (2020).This video demonstrates the microgavage procedure, where an anesthetized zebrafish larva is immobilized in agarose and orally injected with a pathogenic bacterium using a tracer-guided needle. The non-invasive delivery mimics natural infection, enabling analysis of bacterial replication, toxin release, and intestinal epithelial damage.

Source: Rufián, J. S., et.al. Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue. J. Vis. Exp. (2022)This video demonstrates a vacuum infiltration method to introduce a bacterial pathogen into Arabidopsis plant leaves. The approach uses a prepared bacterial suspension and vacuum pulses to enable uniform entry through stomata, resulting in controlled leaf infection.

Source: Casanova, A., et al. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells. J. Vis. Exp. (2016)This video demonstrates high-throughput imaging of Brucella abortus infection in epithelial cells with siRNA-mediated STING knockdown to investigate the role of cytosolic DNA sensing in intracellular bacterial replication and host innate immune response.

Source: Miquel Guennoc, C., et al. New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy. J. Vis. Exp. (2017)This video demonstrates the preparation of a fungal culture on treated cellophane membranes to support colony formation and radial growth. It then shows the cultivation and preparation of soil bacterial cells for interaction assays. Finally, it illustrates how bacterial cells adhere to the fungal colony...

Source: Harris, S. L., et al. Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System. J. Vis. Exp. (2019)This video demonstrates a protocol for monitoring the colonization of root-associated bacteria on Arabidopsis thaliana seedlings grown in a soilless system. Following colonization, bacteria are dislodged by sonication, then diluted, plated, and incubated to form colonies that reflect bacterial attachment and viability.

Source: Calatayud Arroyo, M., et al. Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface. J. Vis. Exp. (2018)The video demonstrates a method to evaluate the viability of anaerobic oral bacteria using fluorescent dyes and flow cytometry. It begins with culturing bacteria in oxygen-free broth, followed by resuspending a single colony from a blood agar plate. After incubation and dilution, the culture is stained with membrane-permeable and...

Source: Jackson, L., et al. Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients. J. Vis. Exp. (2020)This video demonstrates the in situ visualization of Pseudomonas aeruginosa within chemically cleared cystic fibrosis sputum using fluorescent labeling and confocal microscopy to reveal bacterial localization and biofilm structure.

Source: Hunt, A. M. A., et al. Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model. J. Vis. Exp.(2017).This video demonstrates the procedure for assessing infection clearance and healing progression in a wounded diabetic mouse using bioluminescent and microscopic images.

Source: Liu, X., et al. Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem. J. Vis. Exp. (2015).This video demonstrates a method to assess the bacterial load in Arabidopsis thaliana leaves infected with Pseudomonas syringae. Leaf discs from infected leaves are homogenized, serially diluted, and plated onto nutrient-rich agar to promote bacterial colony formation. Colony counts are then used to...

Source: Ahn, J. J., et al. Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses. J. Vis. Exp. (2016)The video demonstrates bacterial load measurement in spleen and liver tissues isolated from mice infected with an intracellular bacterial pathogen. Bacteria, accumulated within tissue-resident phagocytes during systemic infection, are released using detergent-based lysis and mechanical disruption. Serial dilutions of the homogenates are plated on...

Source: Siva-Jothy, J. A., et al. Oral Bacterial Infection and Shedding in Drosophila melanogaster. J. Vis. Exp. (2018)The video demonstrates the oral infection of Drosophila melanogaster with pathogenic bacteria. It includes the preparation of infection vials and the transfer of starved flies to enable bacterial ingestion. The procedure captures gut colonization, followed by surface sterilization and homogenization of flies. Finally, it shows quantification of internal bacterial load through...

Source: Mueller, A., et al. An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei. J. Vis. Exp. (2014)This video demonstrates the aerosol delivery of Mycobacterium tuberculosis (Mtb) to mice using a nebulizer-based chamber system to induce pulmonary infection, providing a controlled and reproducible model for studying airborne tuberculosis transmission and host-pathogen interactions in vivo.

Source: de Vries, C. R., et.al. A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection. J. Vis. Exp. (2020)This video demonstrates the delayed inoculation of luminescent Pseudomonas aeruginosa into a mouse bearing two full-thickness dorsal skin wounds covered by a transparent film dressing. This facilitates biofilm formation and the establishment of chronic infection. Regular bioluminescence monitoring enables quantification of bacterial burden and tracking of infection...

Source: Liu, X., et al. A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes. J. Vis. Exp. (2020)This video demonstrates the depletion of native gut microbiota in mosquitoes using an antibiotic-containing sucrose solution. This is followed by controlled recolonization with a bacterial suspension delivered through a membrane feeding system to establish a defined bacterial population in the mosquito gut.

Source: Seman, B. G., et al. A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis. J. Vis. Exp. (2020)The video demonstrates a neonatal mouse model for studying systemic infection using encapsulated bioluminescent bacteria. Neonatal pups are injected with high- or low-dose bacterial suspensions and returned to their mother for postnatal care. The encapsulated bacteria evade early immune detection, spread systemically, and are visualized over time using luminescence imaging. This model...

Source: Shi, J., et al., Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens. J. Vis. Exp. (2020)This video demonstrates the procedure for preparing an Agrobacterium tumefaciens culture by inducing virulence gene expression with acetosyringone, followed by antibiotic removal through washing and resuspension, to generate a transformation-ready suspension for gene delivery into plant tissues.

Source: Behera, R.K. et. al., A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues. J. Vis. Exp. (2019)This video demonstrates the preparation of hemolytic Staphylococcus aureus cultures to standardize bacterial growth and achieve metabolic synchronization for host-pathogen interaction studies.

Source: Shi, J., et al., Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens. J. Vis. Exp. (2020)This video demonstrates the procedure for preparing an Agrobacterium tumefaciens culture by inducing virulence gene expression with acetosyringone, followed by antibiotic removal through washing and resuspension, to generate a transformation-ready suspension for gene delivery into plant tissues.

Source: Chen, Y., et al. Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model. J. Vis. Exp. (2018)This video demonstrates a survival assay using C. elegans to model Aeromonas pathogenesis, where oral ingestion of the bacteria leads to intestinal infection and mortality, enabling quantitative assessment of bacterial virulence in a live host system.

Begin with immunodeficient mice bearing subcutaneous xenograft tumors derived from human colorectal cancer cells.Orally administer Pediococcus pentosaceus, a lactic acid bacterium safe for consumption and engineered to secrete an anticancer protein, to the treated mouse.Administer saline to the control mouse.Being acid-tolerant, the bacteria survive the acidic environment of the stomach and reach the intestine.There, they colonize the intestinal lumen and secrete the anticancer protein.The...

Begin with mice bearing solid tumors implanted subcutaneously in the thigh.Inject the mice via the tail vein with a non-virulent, engineered strain of Salmonella typhimurium.The bacteria circulate in the bloodstream and selectively accumulate in the hypoxic, immunosuppressed tumor microenvironment.They carry a plasmid encoding a therapeutic fusion peptide composed of a cell-penetrating peptide and the mitochondrial target domain of a pro-apoptotic protein, along with phage-derived lysis...

Source: Chen, Y., et al. Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model. J. Vis. Exp. (2018).This video demonstrates the C. elegans Liquid Toxicity Assay with Aeromonas, a method used to evaluate bacterial virulence by measuring worm survival in a nutrient-limited liquid environment. The assay reveals strain-specific pathogenicity based on the extent of toxin-induced damage and mortality in C. elegans.

Source: Nag, D., et al. Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model. J. Vis. Exp. (2018)This video demonstrates a protocol to quantify the intestinal bacterial load in adult zebrafish infected with an antibiotic-resistant strain of Vibrio cholerae. Following euthanasia and dissection, the intestinal tract is isolated, homogenized with glass beads, and processed to release the bacteria. The resulting homogenate is plated onto a selective medium, and...

Source: Nag, D., et.al. Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model. J. Vis. Exp. (2018)This video demonstrates the inoculation of adult zebrafish with a pathogenic Vibrio cholerae strain to study intestinal colonization and disease outcomes. It shows how bacterial toxins disrupt intestinal integrity, leading to fluid leakage, mucus secretion, and watery diarrhea before the fish are prepared for further analysis.

Source: Patras, K. A., Doran, K. S. A Murine Model of Group B Streptococcus Vaginal Colonization. J. Vis. Exp. (2016)This video demonstrates the preparation and vaginal inoculation of a hormone-treated female mouse with Group B Streptococcus to model infection. It explains how to culture and prepare the bacteria, administer the inoculum into the vaginal lumen, and ensure successful infection.

Source: Patras, K. A. & Doran, K. S. A Murine Model of Group B Streptococcus Vaginal Colonization. J. Vis. Exp. (2016)This video demonstrates the quantification of vaginal Group B Streptococcus (GBS) load in a mouse model using swabbing and chromogenic culture. It outlines the steps involved in sample collection, plating, and colony counting to quantify GBS load in the mouse vaginal lumen.

Source: Conover, M. S., et al., J. Establishment and Characterization of UTI and CAUTI in a Mouse Model. J. Vis. Exp. (2015)This video demonstrates the procedure for assembling and implanting a transurethral catheter in a female mouse, followed by bacterial inoculation to establish catheter-associated urinary tract infection and investigate biofilm-mediated host-pathogen interactions.

Source: Flores, E., et al. Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae. J. Vis. Exp. (2019).This video demonstrates the method of establishing a foodborne bacterial intestinal infection model in zebrafish larvae by feeding them paramecia infected with fluorescent E. coli.

Source: Wang, Y., et al. In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging. J. Vis. Exp. (2017)This video demonstrates the procedure for treating burn wound infections caused by bioluminescent, drug-resistant bacteria in mice using blue light therapy, followed by bioluminescence imaging to monitor treatment efficacy.

Source: Ditkowski, B., et al. An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues. J. Vis. Exp. (2019).This video demonstrates the method of using a parallel-plate flow chamber to perfuse fluorescently labeled bacteria over vascular graft tissues, enabling controlled analysis of bacterial adhesion and early pathogenesis under shear flow conditions.

Source: Beernink, B. M. et al. Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones. J. Vis. Exp. (2021)This video demonstrates the Agrobacterium-mediated delivery of recombinant viral clones into maize seedlings for gene silencing. It outlines the process of Agrobacterium infection, systemic viral spread, and RNA interference-driven phenotypic changes.

Source: Gupta, K., et al. Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis. J. Vis. Exp. (2017).This video demonstrates the intrarenal injection of E. coli into the renal pelvis of an anesthetized rat to induce a localized kidney infection, modeling acute pyelonephritis. It highlights key surgical steps and aseptic techniques necessary for establishing a reproducible in vivo infection model for downstream analysis.

Source: Behera, R. K., et al. A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues. J. Vis. Exp. (2019)This video demonstrates a method for preparing infected mouse kidney tissue, staining DNA, and detecting bacterial gene expression using dual fluorescent reporters. Red marks all bacteria, and green highlights infection-responsive cells.

Source: Brzozowski, R. S. et al. Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria. J. Vis. Exp. (2019)This video demonstrates live-cell fluorescence microscopy to visualize real-time morphological changes in bacteria. It outlines the steps involved in imaging, time-lapse acquisition, and deconvolution to obtain high-resolution images in real time.

Source: McMullen, P. D., Freitag, N. E. Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes. J. Vis. Exp. (2015)This video demonstrates the injection of Listeria monocytogenes into the tail vein of a mouse to establish systemic infection. It shows bacterial circulation, organ colonization, and the quantification of bacterial load in the heart by homogenizing tissue, performing serial dilutions, and plating.

Source: Zhan, Y., et al. Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus. J. Vis. Exp. (2018).This video demonstrates the procedure for photobleaching pigments in cyanobacterial cells using xenon light under cold conditions to improve the fluorescence imaging of growth-regulating proteins.

Source: Zhang, M. et. al., Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium. J. Vis. Exp. (2022)This video demonstrates the application of stimulated Raman scattering microscopy to assess susceptibility to antibiotics by tracking deuterium-labeled metabolic activity. By monitoring carbon–deuterium signals, the method enables rapid detection of resistant and susceptible bacterial populations.

Source: Hiroyasu, A., D et. al., Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis. J. Vis. Exp. (2018)This video demonstrates the procedure for establishing a bacterial infection in Drosophila larvae by placing them into a bacterial droplet and pricking them with a tungsten needle. The larvae are then incubated on a grooved, nutrient-rich agar plate to study host-pathogen interactions.

Source: Hiroyasu, A., et al. Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis. J. Vis. Exp. (2018)This video demonstrates high-resolution confocal imaging of Drosophila melanogaster hemocytes infected with Coxiella burnetii to visualize pathogen localization in 3D, enabling detailed analysis of intracellular infection dynamics and host-pathogen interactions.

Source: Rufián, J. S., et al. Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue. J. Vis. Exp. (2022).This video demonstrates a method for analyzing gene expression in plant-extracted pathogenic bacteria at the single-cell level using a fluorescently tagged transcription factor. Confocal imaging reveals heterogeneity in virulence gene expression, highlighting functional diversity within a clonal bacterial population.

Source: Montefusco-Pereira, C. V. et al. P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives. J. Vis. Exp.(2020)This video demonstrates a co-culture model of bronchial epithelial cells and macrophage-like cells at the air-liquid interface to study host-pathogen interactions. Pathogenic bacterial infection compromises epithelial membrane integrity, triggering macrophage-like cell activation and...

Source: Nguyen, J. Q., et. al. Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis. J. Vis. Exp. (2017)This video demonstrates the procedure of intratracheal inoculation in an anesthetized rat, involving catheter placement, bacterial delivery into the trachea, and subsequent lung infection establishment for studying host-pathogen interactions.

Source: Claes, J., et al, In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions. J. Vis. Exp. (2015).This video demonstrates an in vivo method for analyzing shear-dependent bacterial adhesion in a live mouse model. Following surgical preparation and catheterization, the mesenteric vasculature is exposed for real-time imaging under a fluorescence microscope. Perfusion with an ionophore and fluorescently labeled bacteria reveals how shear stress triggers...

Source: Poore, K., et al. Epithelial Cell Infection Analyses with Shigella. J. Vis. Exp. (2024)This video demonstrates an in vitro assay to assess Shigella invasion and intracellular replication in human colonic epithelial monolayers. It highlights steps for infection, antibiotic selection, host cell lysis, and recovery of intracellular bacteria for downstream analysis.

Source: Zhang, C., et al., Development and Assessment of Intracellular Infection Models for Staphylococcus aureus. J. Vis. Exp. (2025)The video demonstrates the isolation of kidneys containing Staphylococcus aureus-infected macrophages, followed by tissue homogenization to release intracellular bacteria. It quantifies bacterial persistence within macrophages in control and antibiotic-treated groups by counting colony-forming units.

Source: Berthold, A., et al. Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia. J. Vis. Exp.(2022)This video demonstrates the cultivation of pathogenic spirochetes from infected skin tissue to model dermal infection. It highlights their localization in the dermis, interaction with the extracellular matrix, and survival during selective culture conditions.

Source: Morgan, E., et al. Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria. J. Vis. Exp. (2021)This video demonstrates a protocol for isolating intestinal bacteria of interest from wild Caenorhabditis worms. The worms are first starved and washed to eliminate residual feeding bacteria from their surface and intestinal lumen while preserving the target microbes. After surface cleaning and multiple crawling steps, the worms are dissected to extract...

Source: Petersen, C. et. al., Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans. J. Vis. Exp. (2022)This video demonstrates the isolation and cultivation of bacterial species associated with Caenorhabditis elegans. A bead-based lysis of C. elegans, followed by selective culturing, enables the isolation of diverse microbial strains for further analysis.

Source: Liu, X., et al. A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes. J. Vis. Exp. (2020)This video demonstrates the dissection of the mosquito midgut to isolate gut bacteria. The midgut is extracted from the digestive tract, homogenized to release the bacteria, and plated on nutrient agar to culture bacterial colonies.

Take a control mouse with a normal gut microbiota and an experimental mouse with depleted gut microbiota. The depletion compromises the integrity of the gut barrier.This enables bacteria to translocate through the blood vessel into the liver tissue.Inject both mice with the hepatitis B virus or HBV plasmid to induce an HBV infection in the liver tissue.In the control mouse, the T cell detects an infected liver cell through the interaction of its receptors with viral antigens, and produces...

Begin with a bacterial biosensor culture suspended in a medium.These engineered bacteria produce a transcription factor that, upon binding to a specific fecal metabolite, activates a promoter associated with a fluorescent reporter gene.To this culture, add a filtered human fecal sample containing metabolites, such as lactate, that are involved in host-microbiome interactions.Dilute this mixture with a fresh medium to maintain an optimal reaction volume.Incubate without shaking to mimic the...

Begin with a rat model with sutures tied around its upper molar teeth to support bacterial colonization.Repeatedly smear the sutures with a gum disease-causing oral pathogenic bacterium using a gel-like solution.The bacteria colonize the gum around the sutures.Colonization triggers inflammation and damages the tissues.The bacteria release toxins that diffuse through damaged tissues and enter the bloodstream.These circulating toxins reach the gut, cause inflammation, disrupt the epithelial...

Source: Yang, E., et al. Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis. J. Vis. Exp. (2019)This video demonstrates the manual isolation of single epithelial cells containing intracellular bacterial communities from infected mouse bladder using mouth micropipetting, enabling downstream single-cell analysis of viable host-associated E. coli populations in urinary tract infection...

Source: Chen, Y. et. al., Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model. J. Vis. Exp. (2018)This video demonstrates a muscle necrosis assay in C. elegans (Caenorhabditis elegans) using pathogenic Aeromonas strains. By feeding green fluorescent protein (GFP)-expressing worms on Aeromonas-seeded plates, the assay models bacterial infection and reveals tissue damage through fluorescence imaging. The progressive loss of muscle structure and GFP...

Source: Hu, Y. et.al., Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems. J. Vis. Exp. (2019)This video demonstrates the analysis of short-chain fatty acid production by human gut microbiota in response to exopolysaccharides. This highlights microbial fermentation products separation and quantification using gas chromatography. The results reveal that exopolysaccharides modulate microbial metabolites that influence host-pathogen...

Source: Lo, B. C., et al. Chronic Salmonella Infection Induced Intestinal Fibrosis. J. Vis. Exp. (2019)The video demonstrates the quantification of bacterial load in a mouse model of chronic infection with streptomycin-resistant Salmonella typhimurium. Following euthanasia and sterile dissection, cecal and splenic tissues are harvested, homogenized with steel beads to release bacteria, and serially diluted. Dilutions are plated on selective agar, and visible colonies are counted to estimate...

Source: Figueroa, D. et. al. Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization. J. Vis. Exp. (2018).This video demonstrates the preparation of spheroplasts from rod-shaped, gram-negative bacteria. Bacteria are grown in the presence of an antibiotic to form elongated filaments. These are washed with a hypertonic solution and treated with a lysis buffer to remove the outer cell wall. Hypotonic treatment forms spheroplasts,...

Source: Sigal, N., et al. RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells. J. Vis. Exp. (2016).This video demonstrates the isolation of internalized pathogenic bacteria from infected bone marrow-derived macrophages (BMDMs). Bacteria are allowed to attach to host cells, and unattached bacteria are washed away. After internalization, an antibiotic is added to eliminate non-internalized bacteria. The host cells are lysed, centrifuged, and the supernatant is...

Source: Temkin, M. I. et al. High Throughput Co-culture Assays for the Investigation of Microbial Interactions. J. Vis. Exp. (2019)This video demonstrates an in vitro assay to study microbial interactions between Corynebacterium propinquum and Staphylococcus in a co-culture.

Source: Madenspacher, J. H. and Fessler, M. B. A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice. J. Vis. Exp. (2016)This video demonstrates the process of quantifying bacterial load in the spleen of an infected mouse to assess the severity of infection. The homogenized spleen is serially diluted and plated to determine total bacterial burden. The resulting data provide insights into host-pathogen interactions within...

Take cultures of human urinary bladder epithelial cells in a multiwell plate.Incubate the control culture with wild-type Enterobacter cloacae, a pathogenic bacterium with pili, hair-like structures on the bacterial surface.The FimH subunit of the pili binds to the host cell receptor, anchoring the wild-type bacteria to the host cell.Incubate the experimental culture with a mutant Enterobacter cloacae lacking FimH.Without FimH, the mutant bacteria fail to adhere to the host...

Take suspensions of an antibiotic-resistant strain of a pathogenic bacterium.Treat one suspension with a short, membrane-disrupting antibiotic compound and the other with both the short compound and a longer, membrane-intercalating compound.Incubate both suspensions with agitation. These compounds mimic the lipid bilayer structure, enabling their spontaneous insertion into bacterial membranes.The short compound spans only part of the membrane’s thickness, creating a structural mismatch. This...

Source: Brown, A. O., et al. Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling. J. Vis. Exp. (2015).This video demonstrates the microscopic visualization of bacterial microlesions in mouse heart tissue. The heart of a mouse infected with Streptococcus pneumoniae is harvested, washed, and fixed before being processed to obtain coronal sections. These sections are stained with hematoxylin and eosin and examined under a microscope to visualize...

Source: Pava-Ripoll, et al. Detection of Foodborne Bacterial Pathogens from Individual Filth Flies. J. Vis. Exp. (2015)This video demonstrates a PCR-based method that amplifies target bacterial DNA from enriched fly samples using pathogen-specific tablets, enabling real-time fluorescence detection and melting curve analysis to confirm the presence of foodborne pathogens in individual insects.

Source: Calatayud Arroyo, M., et al. Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface. J. Vis. Exp. (2018).This video demonstrates an in vitro gut host–microbe model to study bacterial viability. A co-culture of Caco-2 and HT29-MTX cells is incubated with bacteria pre-treated under simulated digestive conditions. Epithelial barrier integrity is assessed using transepithelial electrical resistance, and fractions from the medium, solubilized...

Source: Chen, H., et al. Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples. J. Vis. Exp. (2018)This video demonstrates the extraction of total genomic DNA from citrus leaf tissue infected with a pathogenic bacterium. The protocol enables reliable DNA recovery for downstream PCR-based detection of the phloem-limited pathogen.

Source: Pett, N., et al. T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo. J. Vis. Exp. (2024).This video demonstrates the in vivo interaction between T4 bacteriophage and E. coli in the murine intestine, and the fecal spot plating method to quantify their coexistence by measuring bacterial colonies and phage plaques.

Source: Alshehri, M. et. al., Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro. J. Vis. Exp. (2025)This video demonstrates a co-culture model using multi-species oral biofilms and human oral epithelium. It highlights epithelial immune activation and enables the downstream analysis of host-pathogen interaction.