Bacterial Growth and Techniques

Source: Cian, M. B. et. al., Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii. J. Vis. Exp. (2020)This video demonstrates the separation of the inner and outer membrane fractions of Gram-negative bacteria using sucrose gradient ultracentrifugation. The membrane fractions are purified and stored at low temperatures for targeted analysis of membrane-specific components.

Source: Mercier, N., et al. MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)This video demonstrates a step-by-step procedure for harvesting bacterial cells and mechanically lysing them to release intracellular contents.

Source: Mercier, N., et. al., MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation of MS2-tagged small regulatory RNA (sRNA) bound to its bacterial target RNA using affinity purification with a maltose-binding protein (MBP)–MS2 coat protein fusion immobilized on amylose resin.

Source: Feltwell, T., et.al. Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient. J. Vis. Exp. (2019)This video demonstrates the use of a discontinuous density gradient to separate bacterial strains based on capsule amount. By centrifuging a transposon mutant library through the gradient, bacteria with different capsule densities localize to distinct layers. The separated fractions can then be collected for downstream analysis.

Source: Gupta, S., Marcela Rodriguez, G. Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria. J. Vis. Exp. (2019)This video demonstrates the stepwise isolation of extracellular vesicles (EVs) from Mycobacterium tuberculosis cultured under iron-limiting conditions that mimic the host environment. It outlines the use of ultrafiltration, differential centrifugation, and density gradient separation to enrich and identify EV-containing fractions.

Source: Watson, D. C., et.al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation and concentration of extracellular vesicles (EVs) derived from the outer membrane of genetically engineered bacteria. The process involves culturing the bacteria to release EVs, followed by sequential centrifugation and filtration steps to remove cells and debris. Finally, ultrafiltration is used to...

Source: Watson, D. C., et al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)This video demonstrates the use of size exclusion chromatography with porous resin beads to isolate bacterial extracellular vesicles (EVs) from a bacterial culture filtrate. Larger EVs elute early by bypassing the resin pores, while smaller proteins enter the pores and elute later.

Source: Fattahi, N., et al. Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation. J. Vis. Exp. (2021).This video demonstrates the extraction of bacterial colonies from a photodegradable hydrogel using UV light. A specific colony is selected, and a patterned light exposure is designed to initiate localized hydrogel degradation. After adjusting light intensity and duration, the UV light is applied, releasing the colony. The colony is then aspirated through tubing...

Source: Ghadigaonkar, D. and Rath, A. Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes. J. Vis. Exp. (2023)This video demonstrates the isolation and preparation of antibiotic-resistant bacteria from a water sample using selective media. The DNA is extracted from antibiotic-resistant bacteria and amplified to identify the bacterial antibiotic-resistant genes.

Source: Kazi, M. I., et. al., Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing. J. Vis. Exp. (2020)This video demonstrates the selection, quantification, and preservation of Gram-negative recipient bacteria carrying transposon insertions based on kanamycin resistance.

Source: Fernández-Coll, L. and Cashel, M. Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography. J. Vis. Exp. (2019)This video presents a high-throughput protocol for measuring the levels and dynamics of the bacterial stress-response nucleotides ppGpp and pppGpp using thin-layer chromatography (TLC). The method provides a powerful, rapid, and cost-effective approach to quantifying (p)ppGpp in bacterial cultures...

Source: Streather, B. R., et al. Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli. J. Vis. Exp. (2023)This video demonstrates the production and isolation of recombinant proteins tagged with Vesicle Nucleating Peptide (VNp) from bacterial vesicles. An inducer triggers VNp-tagged protein expression, driving vesicle formation and encapsulation, followed by centrifugation and sonication for efficient protein recovery without cell...

Source: Shivaiah, K., et al. Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria. J. Vis. Exp. (2022).This video demonstrates the isolation of plastoglobules from cyanobacteria using sucrose density gradient centrifugation, highlighting their lipid-rich, low-density nature and ensuring their structural preservation for downstream analysis.

Source: Armbruster, K. M., Meredith, T. C. Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry. J. Vis. Exp. (2018)This video demonstrates the enrichment of bacterial lipoproteins using a non-ionic detergent that undergoes phase separation upon warming. Through sequential temperature shifts and centrifugation steps, lipoproteins are selectively partitioned into the detergent phase, while non-lipoprotein contaminants...

Source: Leuthold, M. M., et al. Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography. J. Vis. Exp. (2016).This video demonstrates the purification of recombinant human norovirus P domain using size exclusion chromatography, highlighting the separation of the target protein from higher and lower molecular weight impurities based on differential pore accessibility. The process is monitored by UV absorbance and confirmed through SDS-PAGE analysis of eluted...

Source: Kuo, T., et al. One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode. J. Vis. Exp. (2016)The video demonstrates a negative chromatography technique to purify a virulence-associated protein from a bacterial lysate. By using a positively charged resin in a buffered solution, host-cell proteins are selectively retained, while the near-neutral target protein remains unbound and is collected in the...

Source: Murata, T., et al. Purification of a High Molecular Mass Protein in Streptococcus mutans. J. Vis. Exp. (2019)This video demonstrates a step-by-step procedure for the purification of a polyhistidine-tagged protein secreted from Streptococcus mutans.

Source: Venuprasad, K., et al. Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation. J. Vis. Exp. (2016)This video demonstrates the collection and processing of bronchoalveolar lavage fluid from a euthanized mouse infected with non-typeable Haemophilus influenzae for downstream analysis.

Source: Ginies, C., et al. Identification of Fatty Acids in Bacillus cereus. J. Vis. Exp. (2016)This video demonstrates the process of preparing fatty acid methyl esters (FAMEs) from bacterial samples by treating the biomass with an alkaline solution, followed by phase separation and solvent evaporation for downstream analysis.

Source: Shivaiah, K., et al. Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria. J. Vis. Exp. (2022)The video demonstrates the isolation of plastoglobules from cyanobacteria. It begins with a culture of cyanobacteria and proceeds through cell disruption using mechanical force. The release of photosynthetic pigments indicates successful lysis. A sucrose step gradient is then used to separate cellular components by density, allowing low-density plastoglobules to form a...

Begin with cultures of Gram-negative Yersinia species and non-Yersinia Enterobacteriaceae.Using sterile needle inoculators, dot the cultures onto both traditional Cefsulodin-Irgasan-Novobiocin or CIN agar and modified CIN agar.Incubate the plates to promote bacterial growth.Both traditional and modified CIN agars support the growth of Yersinia and non-Yersinia Enterobacteriaceae that ferment mannitol to produce acidic byproducts.Acid production lowers the pH and triggers a color change,...

Begin with a wetland soil-derived enrichment culture containing bacterial communities.The culture exhibits Feammox activity, ammonium oxidation coupled to ferric iron reduction, indicating the enrichment of a bacterium capable of this metabolic process.Serially dilute the culture using media.Streak the diluted samples onto test agar plates supplemented with ferric iron and ammonium.Use control plates that lack either ferric iron or ammonium, or contain ferric iron with organic carbon instead of...

Source: Becker, B., et al. A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast. J. Vis. Exp. (2017).This video demonstrates the purification of a polyhistidine-tagged recombinant protein using nickel-based affinity chromatography. It outlines key steps, including bacterial lysis by sonication, clarification and filtration of the lysate, and selective binding and elution of the target protein using imidazole.

Source: Wunderlin, T., et al. Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells. J. Vis. Exp. (2016)This video demonstrates a method to isolate endospores from an environmental sample containing both vegetative cells and various bacteria. The procedure involves filtration, mechanical disruption, and selective chemical treatment to weaken and lyse vegetative cells while preserving endospores. The final step isolates the resilient endospores through...

Source: Gulla, S., et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis. J. Vis. Exp. (2019).This protocol extracts genomic DNA from Yersinia ruckeri by suspending isolated colonies in ultrapure water and applying heat to lyse the cells. Centrifugation separates the DNA-containing supernatant from cell debris, yielding template DNA suitable for PCR-based genotyping and epidemiological analysis.

Source: Gulla, S., et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis. J. Vis. Exp. (2019).This protocol extracts genomic DNA from Yersinia ruckeri by suspending isolated colonies in ultrapure water and applying heat to lyse the cells. Centrifugation separates the DNA-containing supernatant from cell debris, yielding template DNA suitable for PCR-based genotyping and epidemiological analysis.

Source: Stones, D. H., et al. Biomimetic Materials to Characterize Bacteria-host Interactions. J. Vis. Exp. (2015)This video demonstrates the use of bacteriomimetic beads to quantify bacterial adhesion to host epithelial cells under competitive conditions. It outlines the steps involved in treating the cells, lysing them, and plating serial dilutions to measure colony-forming units.

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 pre-treatment of macrophages with Legionella pneumophila outer membrane vesicles (OMVs) to examine their effect on bacterial replication. By altering host immune signaling, OMVs make macrophages more permissive to infection. Higher colony counts in treated cells, compared to untreated ones, indicate...

Source: Jagau, H. et al. Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow. J. Vis. Exp. (2019)This video demonstrates the real-time visualization of pneumococcal attachment to Von Willebrand Factor (VWF) strings on endothelial cells under constant media flow conditions. It outlines the steps for stimulating VWF release, labeling the strings, and quantifying bacterial binding.

Source: Morrill, S. R., et al. Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria. J. Vis. Exp. (2022)This video demonstrates a protocol for modeling vaginal colonization in mice using antibiotic-resistant anaerobic bacteria. It begins with hormonal treatment to increase epithelial susceptibility, followed by vaginal inoculation and co-inoculation under anaerobic conditions. The procedure concludes with post-inoculation sampling and culture to evaluate bacterial colonization...

Source: Li, J. et al. Development of a Larval Zebrafish Infection Model for Clostridioides difficile. J. Vis. Exp. (2020)This video demonstrates the microinjection-based delivery of fluorescently labeled Clostridioides difficile into the intestinal lumen of an anesthetized zebrafish larva. It outlines the steps involved in larval preparation, bacterial injection, and fluorescence microscopy–based confirmation of bacterial localization within the intestine.

Source: Cruz-Adalia, A., et al. T Cells Capture Bacteria by Transinfection from Dendritic Cells. J. Vis. Exp. (2016).This video demonstrates bacterial transinfection through antigen-specific T cell–dendritic cell interactions. Bacteria-infected dendritic cells presenting ovalbumin peptide are incubated with T cells, either directly or separated by a membrane. Direct contact allows immunological synapse formation, enabling bacterial transfer, while the membrane prevents interaction. Gentamicin...

Source: Balczon, R., et al. Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa. J. Vis. Exp. (2018)This video demonstrates the procedure for generating cytotoxic supernatant from pulmonary endothelial cells following infection with Pseudomonas aeruginosa. It outlines how bacterial effector proteins disrupt host cell structure and induce the release of cytotoxic amyloid oligomers into the supernatant.

Source: Lee, H. et. al., Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection. J. Vis. Exp. (2018)This video demonstrates a method for monitoring bacterial infection and effector protein delivery in plant cells using split superfolder green fluorescent protein (sfGFP) complementation combined with confocal microscopy. The approach enables precise visualization of effector translocation into the host cell cytosol.

Source: Islam, A., et al., Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates. J. Vis. Exp. (2023)This video demonstrates the procedure for assessing transcytosis of neonatal Escherichia coli across intestinal epithelial monolayers using transwell inserts, electrical resistance measurement, and bacterial quantification.

Source: Anderson, Q. L., et al. A High-throughput, High-content, Liquid-based C. elegans Pathosystem. J. Vis. Exp. (2018)The video demonstrates a liquid-based assay where genetically modified C. elegans larvae are exposed to pathogenic bacteria in a microplate. Bacterial toxins secreted into the medium induce host stress and death. A fluorescent dye stains dead worms, allowing imaging-based quantification of mortality to assess genetic susceptibility or resistance.

Source: Yarringto, K. D., et al. Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions. J. Vis. Exp. (2020)This video demonstrates the preparation of a coculture system using motile and non-motile bacteria under an agarose pad for live-cell imaging. It shows how motile bacteria detect chemical attractants and invade non-motile colonies, revealing bacterial interactions.

Source: Sorokulova, I., et al. Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain. J. Vis. Exp. (2016)This video demonstrates rats pre-treated with beneficial bacteria or phosphate-buffered saline (PBS) and exposed to heat stress to evaluate gut barrier integrity. Fewer gut-derived bacterial colonies from the liver of treated rats, in contrast to numerous colonies in control rats, indicate an intact epithelial barrier and minimal bacterial translocation.

Source: Yi, Y., et al. Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates. J. Vis. Exp. (2018)This video demonstrates the preparation and treatment of Bacillus mycoides cultures to study plant-microbe interactions. It outlines the steps from streaking a stock culture and incubating it in liquid medium to exposing the bacteria to plant root exudates under agitation. The process culminates in harvesting and flash-freezing the bacterial cells for...

Source: Venuprasad, K., et al. Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation. J. Vis. Exp. (2016)This video demonstrates the intratracheal delivery of a lung-pathogenic bacterial inoculum into an anesthetized mouse to model lower respiratory tract infection.

Source: Taylor, M. N., et.al. Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions. J. Vis. Exp. (2022)This video demonstrates the exposure of germ-free, age-matched Caenorhabditis elegans worms to a bacterial strain for intestinal colonization. It explains the selective removal of non-adhered and external bacteria, leaving only viable bacteria adhered to the gut epithelium for quantification.

Source: Taylor, M. N. et al. Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions. J. Vis. Exp. (2022)This video demonstrates the quantification of intestinal bacterial load variability in individual Caenorhabditis elegans through mechanical disruption. It outlines the steps for sample preparation, mechanical disruption, serial dilution, and colony counting to assess differences in intestinal bacterial load among individual worms.

Endophytic bacteria colonize plant tissues and promote plant growth.To study their impact on tomato plant growth under salt stress, start with sterile seedlings showing developing leaves and roots.Immerse the roots of the experimental seedling in a suspension of endophytic bacteria; leave the control uninoculated.Transfer both groups into sterilized soil to prevent external microbial interference and incubate in a growth chamber.From the soil, the bacteria enter through transient root openings,...

Take an inoculum mixture of phosphate-solubilizing bacteria or PSB and purple non-sulfur bacteria or PNSB.Spray the mixture onto the soil mixed with fertilizer and cow dung manure.Sow the seed and cover it with the soil.Allow the plant to grow.The bacteria localize near the root in a zone called the rhizosphere.PSB converts insoluble soil phosphates into soluble forms.Meanwhile, PNSB fixes nitrogen by converting atmospheric nitrogen gas into ammonia.The plant absorbs the soluble phosphates and...

Begin with rice seedlings pre-germinated under dark, aseptic conditions to ensure uniform sprouting.Transfer these seedlings to a flask containing a nutrient-rich, semi-solid medium pre-inoculated with a test bacterium.As the plant grows, the roots release chemotactic compounds into the surrounding medium.These compounds stimulate bacterial proliferation, leading to adhesion, multiplication, and colony formation on the root surface.As the roots develop, the bacteria enter the plant through...

Source:Ocean E. Clarke1, Romy A. Dop1, Tom Hasell1, Joanne L. Fothergill1, Daniel R. Neill21University of Liverpool, 2 University of DundeeThis video demonstrates the use of a 3D-printed, silicone-coated device that mimics the inner surface of urinary catheters to simulate catheter-associated urinary tract infection.

Source: Arroyo, M. C., 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 the viability assessment of a synthetic oral microbial community during simulated oral, gastric, and intestinal digestion. Bacterial survival is quantified after each phase, revealing a progressive decline due to digestive stress.

Source: Okamoto, A., et al. Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site. J. Vis. Exp. (2018)This video demonstrates the use of an on-site electrochemical system to enrich anode-respiring bacteria (ARB) from natural reservoirs. A redox gradient is established by placing the anode in oxygen-free deep water and the cathode in oxygen-rich surface water. ARB form biofilms and transfer electrons to the anode, which flows to the cathode, where oxygen is reduced,...

Source: Ahmadi, S., et al. An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota. J. Vis. Exp. (2019).This video demonstrates a method for modeling gut microbial fermentation by incubating fecal samples with dietary fermentable fiber under anaerobic conditions to study metabolite production by gut microbiota.

Source: Biller, S. J., et al. Isolation and Characterization of Cyanobacterial Extracellular Vesicles. J. Vis. Exp. (2022)This video demonstrates the preparation of cyanobacterial extracellular vesicles for transmission electron microscopy by negative staining, enabling high-resolution visualization of vesicle morphology, size, and purity for structural and comparative analyses.

Source: Zhang, Y., et al. Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria. J. Vis. Exp. (2015)This video demonstrates the use of fluorescent nanoparticles to visualize endo-lysosomal remodeling in human epithelial cells infected by intracellular bacteria. Following bacterial invasion, the pathogens manipulate the host cytoskeleton and endo-lysosomal system, forming vacuoles with tubular projections that alter vesicle transport.

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 bioluminescence to visualize and compare the in vivo behavior of virulent and attenuated bacterial strains in mice. It outlines the preparation of bioluminescent bacterial suspensions and their intraperitoneal injection into mice. The virulent strain exhibits...

Source: Guo, S., et al. Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates. J. Vis. Exp. (2022).This video demonstrates the use of a double-layer gradient swarm plate to study bacterial swarming behavior under varying inhibitor concentrations. A plate with a gradient-forming inhibitor in the bottom layer is inoculated with bacteria and incubated. Images are captured at regular intervals to analyze the effect of inhibitor concentration on bacterial swarming.

Source: Zhang, X., et al. A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection. J. Vis. Exp. (2019)This video demonstrates fluorescence imaging of zebrafish embryos injected with Staphylococcus aureus, enabling quantification of infection progression and bacterial distribution to assess the impact of biomaterial microspheres on host-pathogen interactions in vivo.

Source: Schlimpert, S. et al. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device. J. Vis. Exp. (2016)This video demonstrates fluorescence time-lapse imaging of the complete life cycle of the bacteria Streptomyces venezuelae using a microfluidic device to visualize germination, hyphal growth, and sporulation in real time.

Source: Bru, J., et.al. Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response. J. Vis. Exp. (2020)This video demonstrates how to visualize and measure swarm avoidance behavior in Pseudomonas aeruginosa using a swarming agar plate assay. It shows the setup for time-lapse imaging under controlled conditions and explains how antibiotic-induced stress signals redirect bacterial movement, which is quantified through image analysis.

Source: Heering, J., et al., Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells. J. Vis. Exp. (2017).This video demonstrates the preparation of swarmer cells from a Vibrio parahaemolyticus colony for fluorescence imaging. The bacteria are genetically engineered to express a fluorophore-tagged protein that localizes unipolarly in swimmer cells and bipolarly in elongated swarmer cells, allowing for their visual differentiation. An...

Source: Bennett, J. A. et al. Visual and Microscopic Evaluation of Streptomyces Developmental Mutants. J. Vis. Exp. (2018)This video demonstrates the use of phase-contrast microscopy to examine phenotypic differences between wild-type and mutant Streptomyces coelicolor. It outlines the steps involved in preparing and imaging aerial filaments to detect developmental defects in spore formation.

Source: Chu, W., and Zhuang, X. Visualizing Bacterial Motility Based on a Color Reaction. J. Vis. Exp. (2022)This video demonstrates a redox dye-based semisolid agar assay for assessing bacterial motility by visually distinguishing motile and non-motile strains.

Source: Pioli, R., et al., Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly. J. Vis. Exp. (2021)This video demonstrates the method of capturing single fluorescently tagged Escherichia coli (E. coli) cells in microfluidic traps using a carbon-free suspension and controlled flow, followed by nutrient addition to trigger growth and fluorescence imaging of microcolonies.

Source: Wen, J., Pasman, et. al., Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores. J. Vis. Exp. (2019)This video demonstrates the use of structured illumination microscopy to acquire three-dimensional fluorescence images that reveal the organization of germination proteins within the bacterial spore.

Source: Oms, T., et al. Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli. J. Vis. Exp. (2023)This video presents a protocol for monitoring individual bacterial cells expressing a fluorescently labeled DNA-binding protein within a microfluidic system. It illustrates how antibiotics inhibit most cells, while a subpopulation of persister cells survives the treatment and regrows following antibiotic removal.

Source: Murata, K. et. al., Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography. J. Vis. Exp. (2018)This video demonstrates the synchronous cultivation of cyanobacteria under light-dark cycles to study circadian-regulated DNA compaction. Fluorescence microscopy reveals condensed DNA at defined time points, confirming cell cycle alignment across the population.

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 use of live-cell fluorescence imaging to quantify the polarity and dynamics of secretion system components in Legionella pneumophila, enabling analysis of spatial recruitment and cytosolic redistribution critical for understanding bacterial...

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)The video describes how to prepare Legionella pneumophila samples for cryo-electron tomography by combining bacterial culture, gold nanoparticle marking, and vitrification techniques. The process preserves native cellular structures and enables high-resolution imaging of membrane-associated secretion...

Source: Weber, N., et al., Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna. J. Vis. Exp. (2022)This video demonstrates the procedure to assess gliding motility in Phormidium lacuna by homogenizing filament cultures to create a uniform suspension, preparing a Petri plate assay setup over a microscope slide, and visualizing filament movement.

Source: Sorokulova, I., et al. Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain. J. Vis. Exp. (2016)This video presents histological and blood microscopy analyses used to evaluate the protective effects of Bacillus subtilis in rats exposed to heat stress.

Source: Chaimovitz, E. A., et al. Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy. J. Vis. Exp. (2021)This video demonstrates real-time visualization of biological noise in E. coli by tracking fluctuations in GFP fluorescence during bacterial growth and division using time-lapse microscopy.

Source: Fattahi, N., et al. Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation. J. Vis. Exp. (2021)This video demonstrates the use of photodegradable hydrogels to isolate bacterial colonies within silicon microwell arrays. Bacterial cells are first loaded into microwells, encapsulated in a crosslinked hydrogel, and cultured into distinct colonies. Upon exposure to ultraviolet light, targeted degradation of the hydrogel enables selective extraction of individual...

Source: Feltwell, T., et.al. Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient. J. Vis. Exp. (2019)This video demonstrates the preparation and characterization of bacterial strains with varying capsule production. Pathogenic strains carrying different mutations in capsule-related genes are cultured, grown in liquid media, and subjected to centrifugation to distinguish capsule phenotypes based on pellet density. Buffer treatment is used to wash and stabilize the cells...

Source: Le Nagard, L., et al. Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1. J. Vis. Exp. (2018).This video demonstrates how to culture Magnetospirillum bacteria under microaerobic conditions using both liquid and semi-solid media. It highlights the formation of a visible bacterial band at the oxic-anoxic interface and its upward migration as oxygen is depleted. The video also shows how to assess magnetotactic behavior by observing directional...

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 generation of double-crossover recombinants in Pseudomonas aeruginosa using a plasmid-based gene deletion strategy. The process involves selecting bacteria that harbor a non-replicative plasmid integrated into the genome via a single crossover and then inducing a second...

Source: Schlimpert, S., et al. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device. J. Vis. Exp. (2016)This video demonstrates the cultivation and isolation of spores from Streptomyces venezuelae. Spores, dormant cells that help bacteria survive stress, are produced during nutrient limitation and collected through centrifugation. The method ensures a pure, cold-stored spore suspension for downstream applications.

Source: Heering, J., et al. Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells. J. Vis. Exp. (2017)This video demonstrates the induction of swarmer cell differentiation in Vibrio parahaemolyticus using a specially prepared agar medium. By manipulating environmental cues such as iron depletion, calcium supplementation, and surface contact, the bacteria are stimulated to transition from their swimmer form into elongated swarmer cells...

Source: Li, J., et. al., Development of a Larval Zebrafish Infection Model for Clostridioides difficile. J. Vis. Exp. (2020)This video demonstrates the isolation and culturing of Clostridioides difficile bacteria from the intestine of larval zebrafish. The infected intestine is dissected, homogenized, and incubated in selective anaerobic media containing antibiotics. These conditions suppress competing microbes and promote the growth of viable cells.

Source: Ayivi, R. et al., The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective. J. Vis. Exp. (2022)This video demonstrates the method for recovering cold-stressed lactic acid bacteria from a frozen glycerol stock, restoring bacterial viability through anaerobic incubation, and isolating active colonies for culture expansion.

Source: Kuo, C., et al. Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System. J. Vis. Exp. (2018)This video demonstrates the preparation of a luciferase-expressing enterohemorrhagic Escherichia coli suspension from frozen stock for use as an oral inoculum in animal models. This enables real-time, bioluminescent tracking of intestinal colonization during in vivo infection studies.

Source: Nathoo, N., et al. A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling. J. Vis. Exp. (2017).This video demonstrates a hydroponic co-cultivation system for studying plant–bacteria interactions under sterile conditions. Plant seedlings are transferred into a sterile, acidic growth medium, incubated, and inspected for contamination before inoculation with Agrobacterium. The activated bacteria attach to plant...

Source: Figueroa, D. M., et al. Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization. J. Vis. Exp. (2018)This video demonstrates a step-by-step procedure for generating protoplasts from the Gram-positive bacterium Bacillus megaterium.

Source: Eggers, C. H. Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi. J. Vis. Exp. (2022)This video demonstrates the preparation of a genetically modified Borrelia burgdorferi culture engineered with dormant bacteriophage DNA carrying an antibiotic resistance gene. Following antibiotic selection and incubation, a chemical inducer triggers DNA damage, leading to excision, replication, and assembly of new virions containing the resistance gene. After a...

Source: Morales-Soto, N. et. al., Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays. J. Vis. Exp. (2015)This video demonstrates a swarming assay using bacteria that express green fluorescent protein to observe surface motility on soft agar. Time-lapse imaging captures coordinated expansion and tendril formation, highlighting bacterial surface motility.

Source: Anwar, Z., et al. Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples. J. Vis. Exp. (2019).In this video, sulfite-reducing bacteria are cultured in a sulfite-containing agar medium, where they grow anaerobically and reduce sulfite to hydrogen sulfide, forming black precipitates with iron ions. Black colonies are isolated from the agar depth using aseptic techniques and transferred to fresh medium for continued anaerobic incubation.

Source: Morita, M., et al. Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles. J. Vis. Exp. (2019).This video demonstrates a method for culturing and observing individual bacterial cells encapsulated inside giant vesicles anchored to a supported lipid bilayer. The system enables stable, long-term imaging of single-cell growth dynamics in a confined, physiologically relevant microenvironment.

Source: Tomás, J. M., et al. Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility. J. Vis. Exp. (2022).This video demonstrates the initiator-dependent amplification of a suicide plasmid in E. coli. The plasmid, which carries an antibiotic resistance gene, replicates only in the presence of an initiator protein encoded by the host. After electroporation and incubation in recovery medium, the mixture is plated on antibiotic-containing agar.

Source: Cafini, F., et.al. Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus. J. Vis. Exp. (2017)This video demonstrates competence-induced DNA uptake in Staphylococcus aureus through natural transformation. A chloramphenicol-sensitive strain engineered to constitutively express a sigma factor is cultured under nutrient-limiting conditions to enhance competence, then incubated with donor DNA carrying a resistance gene. Successful gene transfer is confirmed by the...

Source: Anderson, Q. L., et al., A High-throughput, High-content, Liquid-based C. elegans Pathosystem. J. Vis. Exp. (2018)This video demonstrates the preparation of RNA interference (RNAi) bacteria carrying plasmids to inhibit target gene expression. It outlines RNAi bacterial selection, growth, and induction steps to produce dsRNA-expressing plates for downstream assays.

Source: Brodl, E., et al. In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria. J. Vis. Exp. 1 (2018)This video demonstrates the cultivation of marine bioluminescent bacteria to observe quorum-sensing–mediated activation of the luminescence operon, enabling real-time monitoring of gene expression and light emission for studies in microbial communication and gene regulation.

Source: Gilmore, S. F., et al. Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development. J. Vis. Exp. (2022)This video demonstrates the cell-free synthesis of nanodisc-stabilized membrane protein antigens using a dual-compartment system. The device maintains optimal conditions for efficient protein synthesis, proper folding, and stable integration into nanodiscs.

Source: Bru, J., et al., Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response. J. Vis. Exp. (2020)This video demonstrates the procedure for analyzing the swarming behavior of Pseudomonas aeruginosa by observing bacterial growth from a central culture toward satellite spots containing the same culture mixed with an antibiotic, using an agar plate setup and time-lapse imaging.

Source: Liu, P. C., et al. Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection. J. Vis. Exp. (2016)This video demonstrates the use of a microfluidic chip to visualize and compare the growth and morphology of wild-type and trimethoprim-resistant E. coli strains under trimethoprim antibiotic stress.

Source: Bhaduri, S., et al., Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii. J. Vis. Exp. (2016)This video demonstrates the method of enriching Sporosarcina pasteurii with urea and calcium chloride to trigger urease activity, leading to the formation of visible calcite crystals through Microbiologically Induced Calcite Precipitation, or MICP.

Source: Figueroa, D. M., et.al. Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization. J. Vis. Exp. (2018)This video demonstrates the production of Escherichia coli spheroplasts by first inhibiting cell division to induce filamentation, followed by enzymatic digestion of the cell wall. The process includes controlled addition of a chelating agent, DNase, and an osmotic stabilizer to ensure proper spheroplast formation 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 establishment and maintenance of a binary culture of Saccharibacteria and its host bacterium by sequentially co-incubating them in host-specific growth medium.

Source: Erdogan, F.,et al., Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation. J. Vis. Exp. (2017)This video demonstrates the procedure for transferring an F plasmid from donor to recipient E. coli cells using a rescue plasmid and selecting transconjugants through dual antibiotic resistance. The process includes mixing bacterial cultures, incubation without shaking, disrupting mating pairs, and plating on selective media to confirm...

Source: Cheng, C., et al. Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate. J. Vis. Exp. (2022)This video demonstrates a method for evaluating the phototoxic effects of flavin mononucleotide (FMN) on pathogenic bacteria. Bacterial cultures are exposed to increasing concentrations of FMN and illuminated with visible light to generate reactive oxygen species (ROS) that damage cellular components. The surviving bacteria are then quantified to assess the...

Source: Gupta, S., et al. Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria. J. Vis. Exp. (2019)This video demonstrates the procedure for culturing Mycobacterium tuberculosis under iron-limited conditions to induce iron stress and promote the secretion of siderophores that support bacterial survival.

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 preparation of a Mycobacterium marinum single-cell suspension from a late-log phase culture for consistent use in infection assays and downstream applications.

Source: Paschke, P., et al. Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria. J. Vis. Exp. (2019).This video demonstrates the procedure for introducing a plasmid carrying a target gene into amoeba cells via electroporation, followed by recovery in a bacterial supplement to enable efficient gene delivery in wild-type amoeba.

Source: Gutiérrez, S., et. al., Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages. J. Vis. Exp. (2017)This video demonstrates the culturing and preparation of Salmonella typhimurium to obtain a clean, metabolically active bacterial suspension suitable for experimental use.

Source: Lin, P., et al. Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion. J. Vis. Exp. (2018).This video demonstrates the preparation of a monoclonal bacterial culture for mineral production through serial dilution and colony isolation on an agar plate. By shaking the plate with glass beads, individual cells are spatially separated to form genetically uniform colonies, which are then transferred to liquid media for growth. The culture with the...

Source: Lin, P., et al. Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion. J. Vis. Exp. (2018).This video demonstrates the procedure for synthesizing rigid metal oxide microcapsules by culturing metal-reducing bacteria with metal ions, followed by sequential purification through sonication, centrifugation, and ethanol washing.

Source: Fuchs, F. M., et al. Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy. J. Vis. Exp. (2017)This video demonstrates the production, purification, and aerosol-based deposition of Bacillus subtilis spores to create a uniform monolayer, enabling downstream applications such as microscopy-based analysis of spore germination, viability, or stress response.

Source: Vieira, J., et al. Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome. J. Vis. Exp. (2021).This video demonstrates the preparation of an artificial sputum medium that mimics the viscosity, nutrient composition, and pH of cystic fibrosis lung sputum. The resulting medium provides a physiologically relevant platform for ex vivo studies.

Source: Maurakis, S., and Cornelissen, C. N. Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands. J. Vis. Exp. (2020)This video demonstrates the procedure for growing Neisseria gonorrhoeae under metal-limited conditions using a defined, metal-depleted medium.

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 method for culturing Streptococcus mitis under microaerophilic conditions using Todd-Hewitt Yeast Extract, or THY agar medium, to prepare bacterial cultures for host-pathogen interaction studies.

Source: Venuprasad, K. et. al., Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation. J. Vis. Exp. (2016)This video demonstrates the culturing of non-typeable Haemophilus influenzae and the preparation of a dose-standardized inoculum for downstream colonization or infection studies.

Source: Stefan, A., et al., The Multifaceted Benefits of Protein Co-expression in Escherichia coli. J. Vis. Exp. (2015)This video demonstrates the procedure for transforming Escherichia coli with a plasmid carrying an antibiotic resistance gene, including cell preparation, electroporation, recovery, and selection of transformants.

Source: Jiang, H., Ho, M. Isolation and Characterization of Intact Phycobilisome in Cyanobacteria. J. Vis. Exp.(2021).This video demonstrates the procedure for culturing cyanobacteria to obtain biomass by growing cells under continuous illumination with carbon dioxide supply and nutrient-rich conditions, followed by harvesting via centrifugation.

Source: Wiegand, D. J., Lee, et al., Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens. J. Vis. Exp. (2019)This video demonstrates the cultivation and harvesting of Vibrio natriegens, a fast-growing bacterium, under aerated conditions to prepare bacterial pellets for crude extract generation.

Source: Wiegand, D. J., et al. Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens. J. Vis. Exp. (2019).This video demonstrates the preparation of a cell extract from Vibrio natriegens, a rapidly growing bacterium known for its high protein synthesis rate. The bacterial pellet is washed and resuspended in lysis buffer before sonication on ice to prevent cellular degradation. The lysate is centrifuged to separate insoluble cell debris, and the cell extract is...

Source: Wiegand, D. J., et al. Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens. J. Vis. Exp. (2019).This video demonstrates the use of a bacterial cell-free protein expression system based on Vibrio natriegens crude lysate to perform coupled in vitro transcription and translation. It highlights the preparation of a master mix with essential components and the step-by-step synthesis of functional proteins from a DNA template.

Source: Jian, X., et al., Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC). J. Vis. Exp. (2022).This video demonstrates the use of a microbial microdroplet culture system to drive adaptive evolution of bacteria toward methanol tolerance. By mixing engineered bacterial cultures with a high-methanol stress medium and segmenting them into microdroplets, the system selects for mutants that exhibit improved growth under methanol stress. Through...

Source: Weber, N., et al., Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna. J. Vis. Exp. (2022)This video describes a procedure for performing natural transformation in filamentous cyanobacteria using plasmid DNA to achieve stable GFP expression and antibiotic resistance through homologous recombination.

Source: Wagner, A. O.,et al., Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions. J. Vis. Exp. (2019)This video demonstrates the preparation and inoculation of anaerobic cultures using oxygen-free sludge under strictly controlled, deoxygenated conditions.

Source: Lea-Smith, D. J., et al. Generation of Marked and Markerless Mutants in Model Cyanobacterial Species. J. Vis. Exp. (2016)The video demonstrates a method for generating antibiotic-marked mutants in cyanobacteria. The protocol begins with culturing cyanobacteria, followed by centrifugation and washing steps that preserve surface pili. A plasmid carrying an antibiotic resistance gene flanked by homologous sequences is then introduced. During incubation, the plasmid is internalized via pili...

Begin with an anaerobic digester, a sealed, oxygen-free reactor seeded with a synthetic sludge that mimics the settled solids from wastewater.The sludge contains mesophilic anaerobic microbes, a mixed microbial community.These microbes thrive at moderate temperatures and drive the digestion of the sludge’s organic matter.Continuously mix the sludge to sustain the bacterial activity and promote digestion.Later, add bioplastics, which are biodegradable polymers, into the sludge.During incubation,...

Begin with a turbidostat, a device that maintains a constant cell density in culture.The device contains a vessel with a bacterial culture, which is continuously aerated by stirring and maintained at a constant temperature.A laser diode emits a light beam that is split into two paths.One path is directed to a reference sensor to monitor laser intensity fluctuations, while the other passes through the culture.Bacterial cells scatter and absorb light, decreasing the transmitted intensity, which...

Begin with a strain of Pseudomonas aeruginosa bacteria, isolated from oil-contaminated soil, in a medium containing a nitrogen source and an excess of edible oil as the carbon source.This strain synthesizes polyhydroxyalkanoate (PHA), a long-chain polymer useful in producing bioplastics.Incubate under agitation to facilitate aeration and promote bacterial growth.Initially, the medium supplies both carbon and nitrogen sources to support bacterial proliferation and generate high biomass.After...

Begin with a culture of Alishewanella, a Gram-negative, salt-tolerant bacterium.Next, streak the culture onto agar plates containing increasing concentrations of sodium chloride to simulate varying levels of saline stress.Overlay each plate with a layer of unsolidified medium to prevent oxygen diffusion and maintain the culture in an oxygen-limited environment.Seal and incubate the plates at an optimum temperature to support bacterial growth.During incubation, the bacterium exhibits varying...

Source: Arias, S. L., et al. Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles. J. Vis. Exp. (2016).This video demonstrates the production of nanocellulose by culturing cellulose-producing bacteria in a nutrient medium containing mannitol. The bacteria metabolize mannitol into glucose, which is then polymerized and extruded as glucan chains that crystallize into cellulose sheets, forming a three-dimensional matrix at the air–liquid interface. The...

Source: Brumfield, K. D. et. al., Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (2017)This video demonstrates the growth of bacterial cultures in a liquid medium. Shaking promotes aeration and distributes nutrients evenly, supporting exponential growth and resulting in a uniform, metabolically active suspension suitable for downstream applications.

Source: Schumacher, D., et al. 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 preparation and imaging of Myxococcus xanthus, a slow-growing soil bacterium. The culture is grown in a medium, applied to a coverslip with fluorescent microspheres, and overlaid with a pre-warmed agarose pad to support hydration and nutrient supply. This setup enables long-term, time-lapse...

Source: Yang, Z., et al. A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.. J. Vis. Exp. (2018)This video demonstrates the selective enrichment and identification of Salmonella using xylose lysine deoxycholate (XLD) and chromogenic agar. Selenite broth promotes Salmonella growth, while XLD agar reveals black-centered colonies through hydrogen sulfide production. Chromogenic agar confirms the identity via enzyme-mediated pigment formation, enabling species-level...

Source: Jeong, H., et. al. Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat. J. Vis. Exp. (2016).This video demonstrates the selective isolation and growth of stress-adapted bacteria from a culture exposed to prolonged stress conditions. The culture is plated on stressor-containing agar, and a single colony is selected to inoculate a liquid medium with the stressor. Absorbance is measured at regular intervals to monitor the growth and confirm the successful...

Source: Villela, H. D. M., et al. Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation. J. Vis. Exp. (2019).This video demonstrates the isolation of pollutant-degrading marine bacteria through serial dilution, colony selection, and growth in EE2-containing medium. By using EE2 as the sole carbon source, the protocol identifies bacterial strains capable of metabolizing this environmental pollutant.

Source: Villela, H. D. M., et al. Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation. J. Vis. Exp. (2019)This video demonstrates the selective isolation of oil-degrading marine bacteria by enriching seawater samples on oil-based media, enabling the recovery of pure strains with potential applications in crude oil bioremediation and environmental cleanup.

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 an inducible expression system to identify where the bacterial effector protein CaeB disrupts the host apoptotic cascade. It outlines the steps for transfecting CaeB-expressing cells with a tetracycline-responsive system to induce pro-apoptotic gene expression in order to identify the point of...

Source: Wei, D., et al., Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock. J. Vis. Exp. (2017)This video demonstrates the microbial conversion of bamboo-derived sugars into 2,3-butanediol by culturing Klebsiella pneumoniae in a hydrolysate-based medium under microaerobic and mildly acidic conditions.

Source: Hemmerich, J., et al. Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology. J. Vis. Exp. (2017).This video demonstrates the use of an automated microbioreactor to monitor bacterial growth and fluorescent protein secretion in response to varying media formulations. A genetically modified bacterium expressing a secretory fluorescent protein is cultured under antibiotic selection and inoculated into nutrient-varied wells. Real-time optical...

Source: Lea-Smith, D. J., et al. Generation of Marked and Markerless Mutants in Model Cyanobacterial Species. J. Vis. Exp. (2016)This video demonstrates the cryopreservation of cyanobacterial strains by combining concentrated cell suspensions with glycerol or DMSO and storing them at low temperatures, enabling long-term maintenance of viable cultures for photosynthesis and genetic studies.

Source: Arias, S. L., et al. Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles. J. Vis. Exp. (2016)This video demonstrates the in situ synthesis of magnetite nanoparticles within a bacterial nanocellulose pellicle produced by Gluconacetobacter xylinus. It explains how magnetic bacterial nanocellulose is generated through iron ion infusion, alkaline precipitation, and purification.

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 syringe-based pressure method for extracting apoplastic bacteria, which proliferate and form microcolonies within the apoplast, the intercellular space between plant cells. The method yields debris-free bacterial samples suitable for downstream single-cell analysis.

Source: Becker, B., et al. A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast. J. Vis. Exp. (2017)This video demonstrates the inducible expression of His-tagged ricin toxin A chain (RTA) in Escherichia coli. It outlines the steps involved in the selective culturing of bacteria transformed with a recombinant expression plasmid, induction of His-tagged RTA expression, and preparation of the bacteria for further...

Source: Pokhrel, A., et al. Assaying for Inorganic Polyphosphate in Bacteria. J. Vis. Exp. (2019).This video demonstrates a colorimetric assay to measure inorganic polyphosphate (polyP) in bacterial samples. The procedure involves enzymatically converting polyP into free phosphate, which then reacts with molybdate and ascorbic acid to produce a blue-colored complex. By comparing the absorbance of this complex to a standard curve of known phosphate concentrations, the polyP content in bacterial...

Source: Choi, H. J., et al. Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads. J. Vis. Exp. (2022)This video demonstrates the method of selectively separating Gram-negative bacteria from a mixed bacterial sample using aptamer-coated microbeads and acoustic forces within a microfluidic chip.

Source: Brodl, E., et al. In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria. J. Vis. Exp. (2018).This video demonstrates the measurement and correlation of bioluminescence with cell density in transformed bacteria. A single bacterial colony is used to initiate a liquid culture. The culture is placed in a well at the desired cell concentration. An antibiotic and inducer are added to express the gene required for bacterial bioluminescence. Readings...

Source: Koeth, L. M., et. al., A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically. J. Vis. Exp. (2015).This video demonstrates the broth microdilution method for determining the minimum inhibitory concentration (MIC) of a lipoglycopeptide antibiotic against Gram-positive bacteria. It covers bacterial colony isolation, dilution, inoculation of microplate wells with varying antibiotic concentrations, and verification of culture...

Source: Zhang, X. et. al., A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection. J. Vis. Exp. (2019)This video demonstrates a zebrafish embryo model for studying infections linked to implanted biomaterials. Injecting Staphylococcus aureus with polystyrene microspheres enhances bacterial survival, confirming the infection-promoting role of implanted materials.

Source: Yeung, M., et al. Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species. J. Vis. Exp. (2016)This video demonstrates a culture-based method for identifying Vibrio species, highlighting chromogenic agar as a more accurate alternative to the commonly used thiosulfate-citrate-bile salts-sucrose or TCBS agar. Chromogenic agar provides superior differentiation of Vibrio strains by detecting species-specific...

Source: Al Ahmar, R., et al. Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate. J. Vis. Exp. (2020)This video demonstrates a colorimetric assay to quantify alginate produced by Pseudomonas aeruginosa, using carbazole to detect uronic acids after acid hydrolysis. The method enables comparative analysis of alginate production normalized to bacterial biomass in research and clinical studies.

Source: Al Ahmar, R., et al. Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate. J. Vis. Exp. (2020)This video demonstrates a colorimetric immunoassay to quantify alginate, a protective exopolysaccharide produced by Pseudomonas aeruginosa. The procedure involves coating a microplate with the sample, applying specific antibodies, and using an enzyme-substrate reaction to generate a measurable color change. A standard curve is then used to determine the...

Source: Hassan, J. A., et al. High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay. J. Vis. Exp. (2020).This video demonstrates a method to screen for bacterial resistance in different tomato strains using ten-day-old seedlings inoculated with a bacterial suspension. It explains how bacteria invade through stomata, deliver effector proteins to suppress plant immunity, and how plant responses differ between susceptible and resistant...

Source: Anderson, Q. L. et al. A High-throughput, High-content, Liquid-based C. elegans Pathosystem. J. Vis. Exp. (2018)This video demonstrates a high-throughput screening method using Caenorhabditis elegans to study gene knockdowns during host-pathogen interactions. It outlines the procedure for preparing knockdown worms and transferring them to assay plates to evaluate the effects of pathogen-induced worm killing.

Source: Cayron, J., and Lesterlin, C. Multi-scale Analysis of Bacterial Growth Under Stress Treatments. J. Vis. Exp. (2019)This video demonstrates the procedure for assessing E. coli filamentation and DNA replication under cell division-inhibiting antibiotic treatment using plating to measure viability and flow cytometry to analyze cell size and DNA content.

Source: Cheng, C., et al. Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate. J. Vis. Exp. (2022).In this video, a buffered reaction mixture containing L-methionine, nitro blue tetrazolium (NBT), and flavin mononucleotide (FMN) is irradiated with violet light to induce reactive oxygen species (ROS) production. This leads to bacterial inactivation and the formation of spectrometrically detectable formazan.

Source: Weber, N. et. al., Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna. J. Vis. Exp. (2022)This video demonstrates a focused LED-based assay for studying phototaxis in filamentous cyanobacteria. The bacteria migrate toward the light source, forming a dense accumulation at the illuminated center, which is visualized and quantified through pixel intensity analysis of the captured image.

Source: Balczon, R., et al. Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa. J. Vis. Exp. (2018)This video demonstrates the protocol to quantify amyloids in culture supernatants from pulmonary endothelial cells infected with virulent bacteria, using thioflavin T, a fluorescent dye that binds to amyloid beta-sheet structures. An increase in fluorescence compared to baseline indicates the presence of amyloids in the culture...

Source: Speare, L., et al. Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates. J. Vis. Exp. (2019)This video demonstrates a co-incubation assay used to study how bacterial strains compete in a shared environment. It is widely applied in microbiology to identify mechanisms, such as contact-dependent or contact-independent killing, that drive these interactions.

Source: Venkateswaran, S., et al. High-throughput Identification of Bacteria Repellent Polymers for Medical Devices. J. Vis. Exp. (2016)This video demonstrates a high-throughput method to identify bacteria-repellent polymers using a microarray slide exposed to mixed bacterial cultures. Bacterial binding is assessed by DAPI fluorescence, where reduced signal indicates polymers with minimal surface attachment.

Source: Barber, A. E., et al. Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue. J. Vis. Exp. (2019).This video demonstrates the detection of specific analytes using a bacterial biosensor. A bacterial culture carrying a plasmid with a red fluorescent protein (RFP) gene under an analyte-sensitive promoter is taken. After thoroughly wiping the test surface, a small section of the wipe is immersed in the culture and incubated. The analyte...

Source: Augimeri, R. V. et. al., Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria. J. Vis. Exp. (2016)This video demonstrates the effect of ethylene generated by 2-chloroethylphosphonic acid (CEPA) on bacterial growth and cellulose production. Ethylene activates signaling pathways that stimulate cellulose synthesis, resulting in enhanced pellicle formation. These results highlight the bacterial response to ethylene.

Source: Cayron, J., et al. Multi-scale Analysis of Bacterial Growth Under Stress Treatments. J. Vis. Exp. (2019).This video demonstrates how to prepare and use a microfluidic plate for imaging bacterial cells under stress conditions. It outlines the process of introducing a bacterial culture and a stress-inducing antibiotic medium, followed by cell loading and microscopic setup. Time-lapse imaging in phase-contrast mode is then used to observe the development of elongated, filamentous cells...

Source: Sekar, A., et. al., A Novel Method to Determine the Longitudinal Antibacterial Activity of Drug-Eluting Materials. J. Vis. Exp. (2023)This video demonstrates a luminescence-based assay for assessing the antibacterial efficacy of an antibiotic-loaded polymeric strip by quantifying ATP-driven luminescence, which decreases in bacterial cultures exposed to the antibiotic.

Source: Zhu, W., & Chu, W. Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria. J. Vis. Exp. (2022)This video demonstrates a sensitive visual method for the rapid detection of hydrogen sulfide–producing bacteria. A color change from yellow to black indicates bacterial activity through the formation of bismuth sulfide.

Source: Bennett, J. A., et al. Visual and Microscopic Evaluation of Streptomyces Developmental Mutants. J. Vis. Exp. (2018)This video demonstrates the cultivation and phenotypic comparison of wild-type and mutant Streptomyces coelicolor strains. It outlines procedures for minimizing spontaneous mutations, preventing cross-contamination, and promoting bacterial growth through proper plating techniques. Finally, the video highlights visual differences in colony morphology to illustrate the...

Source: Francis, M. B., et al. Detecting Cortex Fragments During Bacterial Spore Germination. J. Vis. Exp. (2016).This video demonstrates a colorimetric method to detect cortex degradation during bacterial spore germination by measuring the release of reducing sugars over time. Using N-acetylglucosamine standards and time-point samples, the assay quantifies sugar concentration through a purple chromogenic reaction, indicating the extent of cortex hydrolysis.

Source: Stenzler, B. R. et. al., An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium. J. Vis. Exp. (2018)This video demonstrates the use of genetically engineered bacterial biosensors to accurately measure the bioavailable fraction of mercury ions under anaerobic conditions. By varying the concentration of magnesium sulfate, the assay ensures that the biosensor selectively responds to free mercury ions, as indicated by changes in fluorescence intensity.

Source: Vidilaseris, K., et al. Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors. J. Vis. Exp. (2019)This video demonstrates a colorimetric assay to screen inhibitors of bacterial membrane-bound pyrophosphatase (mPPase) by quantifying orthophosphate release. The method enables identification of compounds that inhibit mPPase from Thermotoga maritima, a potential antimicrobial drug target.

Source: Sarathy, J. P. et al. An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions. J. Vis. Exp. (2017)This video demonstrates an in vitro assay for measuring drug binding to caseum from tuberculosis lesions using rapid equilibrium dialysis. It outlines the steps for preparing caseum suspensions, performing dialysis, and calculating the unbound drug fraction to predict drug penetration into caseum.

Source: Rugjee, K. N. et. al., Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa. J. Vis. Exp. (2016)This video demonstrates the generation of a cyclic-di-GMP-responsive reporter strain in Pseudomonas aeruginosa using electroporation. Successful transformation is confirmed through antibiotic selection and fluorescence-based...

Source: Rugjee, K. N., et al. Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa. J. Vis. Exp. (2016).This video demonstrates the screening of cyclic di-GMP modulators using genetically modified bacteria that express green fluorescent protein (GFP) under the control of a c-di-GMP-sensitive promoter. Fluorescence is measured in a multi-well plate containing different test compounds. Compounds that inhibit c-di-GMP...

Source: Chapman, I. J., et al., Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry. J. Vis. Exp. (2016)This video demonstrates the method for distinguishing live and dead Microcystis aeruginosa cells by combining fluorescent probe labeling with flow cytometric analysis of phycocyanin autofluorescence and probe signal.

Source: Nickling, J. H., et.al. Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids. J. Vis. Exp. (2018)This video demonstrates an agar well diffusion assay used to evaluate the antimicrobial activity of nisin variants. It shows how Lactococcus lactis expressing the membrane-bound peptidase NisP activates nisin precursors produced in E. coli, resulting in growth inhibition halos. The size of these halos reflects the bioactivity of each nisin variant.

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 a whole-cell electrochemical method to assess how proton transfer affects extracellular electron transport (EET) in electroactive bacteria. By replacing protons with deuterons using deuterium oxide, the method measures changes in current to reveal a kinetic isotope effect, highlighting the role of...

Source: Dodge, R., et al. Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome. J. Vis. Exp. (2023)This video demonstrates the quantification of colony-forming units (CFUs) of gut-associated bacteria in Drosophila flies using a serial dilution and plating approach. Homogenized fly samples containing gut bacteria are serially diluted, plated on agar, and incubated to allow colony growth. Images of the plates are then analyzed using software to align,...

Source: Maurakis, S., et al. Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands. J. Vis. Exp. (2020).This video demonstrates a metal-limited bacterial growth assay to study the role of metal-responsive genes. Wild-type and mutant bacteria grown in metal-depleted medium are incubated with metal-ligand, and their growth is monitored using a plate reader. Under metal-depleted conditions, the wild-type bacteria...

Source: Maurakis, S., and Cornelissen, C. N. Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands. J. Vis. Exp. (2020)This video demonstrates the procedure for detecting surface metal transporters in Neisseria gonorrhoeae grown under metal-limited conditions using a dot blot assay, involving ligand binding, antibody detection, and colorimetric visualization.

Source: Chen, Y., et al., Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems. J. Vis. Exp. (2019)This video demonstrates a method to quantify mistranslation in Mycobacterium smegmatis using an inducible GFP and secreted mutant luciferase reporter system. It shows how luminescence resulting from mistranslation is measured and compared to GFP fluorescence to evaluate the impact of a test molecule on translational accuracy.

Source: Gilmore, S. F. et al. Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development. J. Vis. Exp. (2022)This video demonstrates the use of Western blotting to evaluate the structural integrity of the major outer membrane protein (MOMP) within nanoparticle-based vaccine complexes. It outlines the key steps involved in protein transfer, antibody labeling, and fluorescence imaging.

Source: Stefan, A., et al. The Multifaceted Benefits of Protein Co-expression in Escherichia coli. J. Vis. Exp. (96), (2015).This video demonstrates the evaluation of mutation frequency in bacteria using a beta-glucosidase assay. Bacterial cultures expressing a proofreading-deficient DNA polymerase are collected over successive generations. Replication errors caused by reduced proofreading may activate a suppressed beta-glucosidase gene. After centrifugation and permeabilization, a substrate is...

Source: Jian, X. et. al., Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC). J. Vis. Exp. (2022)This video demonstrates the use of a microfluidic droplet-based platform for real-time monitoring of bacterial growth. By generating uniform droplets containing bacterial suspensions and guiding them through a detection pathway, the system enables continuous optical measurement of cell density to construct a growth curve.

Take a culture of Corynebacterium glutamicum, transformed with a plasmid carrying a fluorescent reporter gene downstream of the ribonucleic acid anti-terminator or RAT sequence, in a multi-well plate.During transcription, the RAT-RNA forms a stem-loop structure that blocks RNA polymerase, halting reporter gene expression.The plasmid also constitutively expresses LicV, a fusion protein comprising the RAT-RNA-binding protein, LicT, and the blue-light-sensitive VVD domain.In darkness, LicV remains...

Begin with a slurry containing bacterial cells treated with silver nanoparticles suspended in an exopolysaccharide matrix.Add a phosphate buffer to the slurry to maintain a stable pH.Next, introduce a solution containing glucose and a soluble redox indicator, then incubate the mixture. The redox indicator acts as an electron acceptor.During incubation, glucose and the redox indicator enter metabolically active bacterial cells.The cells utilize glucose during respiration, generating...

Take serial dilutions of a bacterial culture and add aliquots into calorimetric vials containing growth medium.Load the sealed vials into the multi-well plate of an isothermal microcalorimeter to measure metabolic heat production.The device is held at a constant temperature to ensure the detected heat originates solely from bacterial metabolism.As bacteria metabolize nutrients and proliferate, they release heat that sensors detect in real time.In parallel, incubate identical vials in an...

Source: Ali, I., et al. High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health. J. Vis. Exp. (2022)This video demonstrates a high-throughput fluorescent assay to evaluate how gut bacteria affect stress resilience in Caenorhabditis elegans. By tracking blue fluorescence from ruptured gut granules, the assay reveals earlier death signals in worms exposed to test bacteria.

Source: Sutlief, A. L., et al. Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System. J. Vis. Exp. (2019).This video demonstrates the preparation of a microfluidic plate for shear stress studies in bacterial cultures. After priming the channels with media, bacterial cells are introduced into the experimental channel and incubated for attachment. Residual media and culture are removed to prepare the system for subsequent shear...

Source: Brumfield, K. D., et al. Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (2017)This video demonstrates a soft agar assay using triphenyltetrazolium chloride to assess and compare the motility of Vibrio cholerae strains by visualizing metabolic activity through red zone formation around actively migrating cells.

Source: Brumfield, K. D., et al., Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (2017).This video demonstrates the Voges-Proskauer (VP) test to differentiate among three biotypes of Vibrio cholerae: classical, wild-type El Tor, and El Tor variant. Each culture is inoculated into a glucose-containing medium, incubated, and treated with α-naphthol and potassium hydroxide to detect the presence of acetoin. A...

Source: Mettrick, K. A., Lawrence, et al., Inducing a Site-Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System. J. Vis. Exp. (2016)This video demonstrates site-specific replication fork blockage in Escherichia coli using arabinose-induced expression of yellow fluorescent protein-tagged TetR that binds chromosomal tetO arrays.

Source: Domesle, K. J., et al. Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation. J. Vis. Exp. (2020).This video demonstrates the loop-mediated isothermal amplification (LAMP) technique for detecting a target bacterial gene by amplifying DNA at a constant temperature. The reaction mixture is prepared by adding an isothermal master mix, specific primers for the target gene, and samples containing DNA templates. The...

Source: Aigrain, L., et al. Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation. J. Vis. Exp. (2015)The video demonstrates the delivery of fluorescent biomolecules into electrocompetent microbial cells using electroporation. The process begins with the introduction of fluorophore-tagged DNA or proteins into a chilled cell suspension. A high-voltage pulse is then applied to create temporary membrane pores, enabling biomolecule entry into the...

Source: Kurokawa, M., et al. Precise, High-throughput Analysis of Bacterial Growth. J. Vis. Exp. (2017)This video demonstrates the high-throughput quantification of bacterial growth using a microplate reader. It outlines the steps involved in culture preparation, optical density measurement, and analysis of growth dynamics.

Source: Dahl, J. et al. Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo. J. Vis. Exp. (2016)This video demonstrates a fluorescence spectrophotometer-based assay to evaluate chaperone activity on a substrate protein under acid and heat stress. It outlines the steps for monitoring substrate unfolding and aggregation through light scattering, comparing conditions with and without the chaperone.

Source: Dahl, J., et al. Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo. J. Vis. Exp. (2016)This video demonstrates the use of recombinant E. coli cultures to evaluate the protective effect of an acid-activated chaperone during acid stress. Bacterial growth monitoring reveals enhanced survival in chaperone-expressing cultures compared to controls, highlighting the role of chaperones in protein stabilization under stress conditions.

Source: Stenum, T. S., et al. Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli. J. Vis. Exp. (2017)This video demonstrates the use of gel electrophoresis and northern blotting to detect and compare the charging levels of transfer RNAs in E. coli by separating aminoacylated and deacylated forms.

Source: Gulla, S., et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis. J. Vis. Exp. (2019).This video demonstrates the procedure for separating fluorescently labeled variable number tandem repeat (VNTR) amplicons, derived from a fish-pathogenic bacterium, using capillary electrophoresis to generate size-specific electropherogram profiles for bacterial identification.

Source: Hockett, K. L., and Baltrus, D. A. Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds. J. Vis. Exp. (2017)This video demonstrates the procedure for detecting bacteriocin-mediated killing by spotting supernatant from an antibiotic-induced donor strain onto a soft agar overlay containing a test strain. Clear zones formed after incubation indicate bacterial inhibition.

Source: Cabeen, M. T., et al. Single-cell Microfluidic Analysis of Bacillus subtilis. J. Vis. Exp. (2018)The video demonstrates the use of a microfluidic device to observe bacterial growth at the single-cell level. It showcases how non-motile bacterial cells, confined within micro-scale trenches, are continuously nourished and imaged under a fluorescence microscope. The setup enables real-time visualization of cell division and lineage tracking across multiple generations.

Source: Scheidweiler, D., et al. Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales. J. Vis. Exp. (2020).This video demonstrates the use of a microfluidic device to quantify bacterial motility through a porous matrix composed of randomly arranged pillars. Fluorescently labeled bacteria are introduced into the device, imaged via microscopy as they move through the porous matrix into the observation channel, and analyzed...

Source: Kim, H. et. al., Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System. J. Vis. Exp. (2016)This video demonstrates a genetic screening method to detect enzyme expression using metagenomic fosmid libraries and chromogenic substrates. Flow cytometry identifies and isolates single cells producing colored products, enabling high-throughput selection of active enzyme-expressing cells.

Source: Samuels, D. J., et al. A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus. J. Vis. Exp. (2017)This video demonstrates the use of liquid chromatography–mass spectrometry to identify and quantify intracellular metabolites from S. aureus. It outlines the steps involved in metabolite extraction, peptide normalization, liquid chromatography, and mass spectrometric analysis.