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Furthermore, the assays may be slightly modified (e.g., optimal growth conditions) to evaluate other bacterial genera.Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from mouse bone marrow are commonly used models for the study of Salmonella infection. However, these models raise technical or ethical issues that highlight the need for alternative methods. This chapter describes a technique for immortalizing early hematopoietic progenitor cells derived from wild-type or transgenic mice and using them to produce macrophages. It validates, through a specific example, the interest of this cellular approach for the study of Salmonella infection.Live cell fluorescence imaging is the method of choice to visualize dynamic cellular processes in time and space, such as adhesion to and invasion of polarized epithelial cells by Salmonella enterica sv. Typhimurium. Scanning electron microscopy provides highest resolution of surface structures of infected cells, providing ultrastructure of the apical side of host cells and infecting Salmonella. Combining both methods toward correlative light and scanning electron microscopy (CLSEM) enables new insights in adhesion and invasion mechanisms regarding dynamics over time, and high spatial resolution with precise time lines. To correlate fast live cell imaging of polarized monolayer cells with scanning electron microscopy, we developed a robust method by using gold mesh grids as convenient CLSEM carriers for standard microscopes. By this, we were able to unravel the morphology of the apical structures of monolayers of polarized epithelial cells at distinct time points during Salmonella infection.Previous studies from our lab have created a simple procedure for single-cell count of bacteria on a paper chip platform using optical detection from a smartphone. The procedure and steps employed are outlined along with the lessons learned and details of certain steps and how the design was optimized. Smartphone optical detection is easy to use, low cost, and potentially field deployable, which can be useful for early and rapid detection of pathogens. Smartphone imaging of a paper microfluidic chip preloaded with antibody-conjugated particles provides an adaptable platform for detection of different bacterial targets. The paper microfluidic chip was fabricated with a multichannel design. Each channel was preloaded with either a negative control of bovine serum albumin (BSA) conjugated particles, anti-Salmonella Typhimurium-conjugated particles with varying amounts (to cover different ranges of assay), or anti-Escherichia coli-conjugated particles. Samples were introduced to the paper microfluidic chip using ge processing algorithm that calculated bacteria concentrations. selleck kinase inhibitor The detection limit was at a single-cell level with a total assay time ranging from 90 to less than 60 s depending on the target.Salmonella enterica is an invasive, facultative intracellular pathogen with a highly sophisticated intracellular lifestyle. Invasion and intracellular proliferation are dependent on the translocation of effector proteins by two distinct type III secretion systems (T3SS) into the host cell. To unravel host-pathogen interactions, dedicated imaging techniques visualizing Salmonella effector proteins during the infection are essential. Here we describe a new approach utilizing self-labeling enzyme (SLE) tags as a universal labeling tool for tracing effector proteins. This method is able to resolve the temporal and spatial dynamics of effector proteins in living cells. The method is applicable to conventional confocal fluorescence microscopy, but also to tracking and localization microscopy (TALM), and super-resolution microscopy (SRM) of single molecules, allowing the visualization of effector proteins beyond the optical diffraction limit.One of the main drawbacks in current methods for bacterium detection is their quantification at very low concentration level in complex specimens. Novel developments that are needed involve solid-phase preconcentration procedures which can be easily integrated with emerging technologies. Here, we describe the immunomagnetic separation (IMS) of Salmonella using magnetic carriers. Nano (300 nm) and micro (2.8 μm) sized magnetic particles are modified with anti-Salmonella antibody to preconcentrate the bacteria from the samples throughout an immunological reaction. The immunomagnetic separation can be easily coupled with downstream characterization and quantification methods, including classical culturing, molecular biology techniques such as PCR, immunoassays, confocal and scanning electron microscopy, and emerging technologies and rapid detection methods including biosensors, lateral flow, and microfluidic devices.CRISPR typing is a newly developed method used to reveal the genetic relationship of bacterial isolates from different resources. For Salmonella, CRISPR typing can not only reveal the phylogenic difference among isolates belonging to the identical serotype, but also show good correspondence with Salmonella serotypes. Here we describe the protocol of CRISPR typing method used in Salmonella, and the approaches to analyze the genetic relationship among different strains.Polymerase chain reaction (PCR) and sequencing-based subtyping tools are useful for rapid analyses of Salmonella isolates. Here we describe the process of clustered regularly interspaced short palindromic repeat-multiple virulence locus sequence typing (CRISPR-MVLST) for Salmonella subtyping.Developed by 3M Company, 3M ™ Molecular Detection Assays-3M MDS-enable detection of Salmonella from advanced isothermal DNA amplification and bioluminescence detection technology. It can be used for a wide variety of products, including poultry, eggs, pet foods, and environmental samples, and results are obtained within about 24 h. In this chapter, all steps of the 3M MDS™ method for detection of Salmonella are described and detailed.