SALMONELLA : methods and protocols.

Preface for Third Edition of Salmonella Book in the Series Methods in Molecular Biology
Contents
Contributors
Chapter 1: Next-Day Salmonella spp. Detection Method Based on Real-Time PCR for Foods
	1 Introduction
	2 Materials
		2.1 Eighteen-Hour Enrichment
		2.2 Salmonella DNA Extraction from Foods
		2.3 Salmonella-Specific Real-Time PCR
	3 Methods
		3.1 Preparation of Enrichment Broth
		3.2 Nonselective Enrichment of Salmonella in  Food
		3.3 Preparation for Bacterial DNA Extraction
		3.4 Bacterial DNA Extraction: Rapid Lysis
		3.5 Bacterial DNA Extraction Using Chelex 100 Resin
		3.6 Detection of Salmonella spp. by Real-Time PCR (See Note 1)
	4 Notes
	References
Chapter 2: Isolation of Salmonella spp. from Animal Feed
	1 Introduction
	2 Materials
		2.1 Sample Preparation Supplies
		2.2 Pre-enrichment Supplies (See Note 1)
		2.3 Enrichment Supplies
		2.4 Streaking on Selective Agar Supplies
		2.5 Presumptive Positive Colony Isolation Supplies
		2.6 Serotyping from SMA Plate Supplies
	3 Methods
		3.1 Initiation of Samples (See Note 1)
		3.2 Pre-enrichment
		3.3 Enrichment
		3.4 Streaking of Selective Agar
		3.5 Presumptive Positive Colony Isolation
		3.6 Serotyping with SMA Plate
	4 Notes
	References
Chapter 3: Investigating Outbreaks of Salmonella Typhimurium Using Case-Control Studies, with a Reference to the One Health Ap...
	1 Infectious Disease Outbreaks
	2 Practical Aspects of Detecting and Investigating Outbreaks of Foodborne Diseases
	3 Methods
		3.1 Steps Within the Case-Control Study
		3.2 Two Salmonella Typhimurium Outbreaks Investigated by Case-Control Studies
			3.2.1 A Small Localized Outbreak Linked to Consumption of Smoked Salami
			3.2.2 A Long-Lasting National Outbreak Caused by Several Pork Products
	4 Notes
	References
Chapter 4: Detection of Salmonella by the 3M Molecular Detection Assays: MDS Method
	1 Introduction
	2 Materials
	3 Methods
		3.1 Pre-enrichment Stage
		3.2 DNA Denaturation Stage
		3.3 Ligation of DNA Strands with Known Primers
		3.4 Reading the Results
	4 Notes
	References
Chapter 5: CRISPR Typing of Salmonella Isolates
	1 Introduction
	2 Materials
		2.1 Polymerase Chain Reaction (PCR)
		2.2 Gel Electrophoresis
		2.3 Amplicon Purification
	3 Methods
		3.1 PCR
		3.2 Gel Electrophoresis
		3.3 PCR Purification
		3.4 Sequencing and CRISPR Analysis
	4 Notes
	References
Chapter 6: CRISPR Typing of Salmonella Isolates from Different Resources
	1 Introduction
	2 Materials
	3 Methods
		3.1 Bacterial Genome Extraction
			3.1.1 Whole-Genome Sequencing
			3.1.2 PCR Amplification of CRISPR Loci and Sequencing
		3.2 Sequence Analysis
		3.3 Cluster Analysis
	4 Notes
	References
Chapter 7: Immunomagnetic Separation of Salmonella with Tailored Magnetic Micro- and Nanocarriers
	1 Introduction
	2 Materials
		2.1 Covalent Immobilization of Antibodies on Tosyl-Activated Magnetic Microparticles
		2.2 Covalent Immobilization of Antibodies on Carboxyl Magnetic Nanoparticles
		2.3 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by ELISA
		2.4 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by Bradford
		2.5 Immunomagnetic Separation of the Bacteria on Micro- and Nano-Sized Magnetic Particles
		2.6 Evaluation of IMS Efficiencies by Microbiological Culture Techniques
		2.7 Evaluation of IMS Efficiencies by Scanning Electron Microscopy
		2.8 Evaluation of IMS Efficiencies by Confocal Microscopy
	3 Methods
		3.1 Covalent Immobilization of Antibodies on Tosyl-Activated Magnetic Microparticles
		3.2 Covalent Immobilization of Antibodies on Carboxyl Magnetic Nanoparticles
		3.3 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by ELISA
		3.4 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by Bradford
		3.5 Immunomagnetic Separation of the Bacteria on Micro- and Nano-Sized Magnetic Particles
		3.6 Evaluation of IMS Efficiencies by Microbiological Culture Techniques
		3.7 Evaluation of IMS Efficiencies by Scanning Electron Microscopy
		3.8 Evaluation of IMS Efficiencies by Confocal Microscopy
	4 Notes
	References
Chapter 8: Self-Labeling Enzyme Tags for Translocation Analyses of Salmonella Effector Proteins
	1 Introduction
		1.1 Live-Cell Imaging Techniques for Effector Proteins
		1.2 Self-Labeling Enzyme Tags
		1.3 Super-resolution Applications
		1.4 Effector Proteins of Salmonella enterica
	2 Materials
		2.1 Host Cell Infection
		2.2 Labeling Reagents
		2.3 Confocal Laser Scanning Microscopy (cLSM)
		2.4 Tracking and Localization Microscopy (TALM)
		2.5 Direct Stochastic Optical Reconstitution Microscopy (dSTORM)
	3 Methods
		3.1 Seeding of Cells
		3.2 Confocal Laser Scanning Microscopy (cLSM) of SLE-Tagged SPI1-T3SS Effector Proteins
		3.3 Confocal Laser Scanning Microscopy of (cLSM) SLE-Tagged SPI2-T3SS Effector Proteins
		3.4 Tracking and Localization Microscopy (TALM) of SLE-Tagged SPI2-T3SS Effector Proteins
		3.5 Direct Stochastic Optical Reconstitution Microscopy (dSTORM) of SLE-Tagged SPI2-T3SS Effector Proteins
	4 Notes
	References
Chapter 9: Smartphone-Based Paper Microfluidic Immunoassay of Salmonella and E. coli
	1 Introduction
	2 Materials
		2.1 Material  List
		2.2 Preparation of Solutions
		2.3 Washing Processes
		2.4 Target Bacterial Samples (See Note 1)
		2.5 Antibody-Conjugated Particles
		2.6 BSA Conjugated Particles
		2.7 Paper Microfluidic Chip Fabrication
			2.7.1 Mask Printing
			2.7.2 Paper Microfluidics Chip Treatment (See Note 10)
			2.7.3 Loading Antibody-Conjugated PS Particles to Paper  Chip
	3 Methods
		3.1 Collect and Prepare Samples
		3.2 Optimization of Mie Scatter Detection
		3.3 Set Up Staging Area and Smartphone
		3.4 Capture Background Signal
		3.5 Loading Samples on a Paper  Chip
		3.6 Obtain Sample Signals
		3.7 Smartphone Analysis (See Notes 29, 30, and 31)
	4 Notes
	References
Chapter 10: Correlative Light and Scanning Electron Microscopy to Study Interactions of Salmonella enterica with Polarized Epi...
	1 Introduction
		1.1 Specific Advantages of Fluorescence and Electron Microscopy
		1.2 The Power of CLSEM for Studies of Salmonella Adhesion and Invasion
	2 Materials
		2.1 Cells
		2.2 Media
		2.3 Reagents
		2.4 Materials
		2.5 Software
	3 Methods
		3.1 Preparation of Grids in Cell Culture Dishes
		3.2 Cell Seeding
		3.3 Live Cell Imaging Setup of Spinning Disk Confocal Microscope (SDCM)
		3.4 Fixation and Drying
		3.5 Imaging Setup of the Scanning Electron Microscope for Correlative  Work
	4 Notes
	References
Chapter 11: Production of Murine Macrophages from Hoxb8-Immortalized Myeloblasts: Utility and Use in the Context of Salmonella...
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Antibodies
	3 Methods
		3.1 Production of ERHBD-Hoxb8 Retrovirus
		3.2 Preparation and Stimulation of Bone Marrow Cells
		3.3 Infection of Stimulated Bone Marrow Cells by the ERHBD-Hoxb8 Retrovirus
		3.4 Emergence of Immortalized Hoxb8-Myeloblast
		3.5 Differentiation of Hoxb8-Myeloblast in Hoxb8-Macrophages
		3.6 Characterization of Hoxb8-Myeloblast Cells and Hoxb8-Macrophages
	4 Notes
	References
Chapter 12: In Vitro Evaluation of Anti-biofilm Agents Against Salmonella enterica
	1 Introduction
	2 Materials
	3 Methods
		3.1 General Biofilm Culture Methods
			3.1.1 Rapid Attachment Assay for Culture of Non-typhoidal Salmonella
			3.1.2 Adaptations and Special Culture Conditions
		3.2 Screening for Anti-biofilm Compounds
			3.2.1 Biofilm Inhibition Assay
			3.2.2 Delayed Addition Assay
			3.2.3 Disruption Assay
			3.2.4 Combination Assays
				The Checkerboard Assay
				Fractional Inhibitory Concentration Index
		3.3 Biofilm Analysis
			3.3.1 Crystal Violet Assay
			3.3.2 Confocal Microscopy
			3.3.3 Viable Cell Enumeration
	4 Notes
	References
Chapter 13: Generation of Random luxCDABE Transcriptional Fusions in the Genome of Salmonella enterica
	1 Introduction
	2 Materials
		2.1 Generation of Random lux Fusions
		2.2 Screen for SPI1 and SPI2 Related Fusions
		2.3 Localization of Mini-Tn5luxCDABE Insertions
	3 Methods
		3.1 Generation of Random lux Fusions
		3.2 Screen for SPI1 and SPI2 Related Fusions
		3.3 Localization of Mini-Tn5luxCDABE Insertions
	4 Notes
	References
Chapter 14: Vaccine Based on Outer Membrane Vesicles Using Hydrogels as Vaccine Delivery System
	1 Introduction
	2 Materials
		2.1 Bacterial Growth and Antigen Extraction
		2.2 Protein and Lipopolysaccharide Content Determination
		2.3 SDS Polyacrylamide Gel Components
		2.4 Immunoblotting Components
		2.5 Hydrogel Formulation
		2.6 Hydrogel Characterization
	3 Methods
		3.1 Bacterial Strain and Growth Conditions
		3.2 Antigenic Extraction (HE Membrane Vesicles) (Fig. 1)
		3.3 Characterization of the Antigenic Extracts
		3.4 Preparation and Characterization of Hydrogels
		3.5 Characterization of Hydrogels
	4 Notes
	References
Chapter 15: Detection and Characterization of Salmonella enterica Serotypes by Simple PCR Technologies
	1 Introduction
	2 PCR Methods for Detection and Characterization of Salmonella spp.
		2.1 Primer and TaqMan Probe Design
		2.2 Laboratory Procedure for TaqMan Probe Real-Time (RT)-PCR Detection
		2.3 RT-PCR for Salmonella Detection Using Disc-Capture  DNA
			2.3.1 DNA Extraction for RT-PCR Using the Capture Disk DNA Isolation Kit (Gentra Systems)
			2.3.2 A RT-PCR for Detecting Salmonella from Capture Disc  DNA
			2.3.3 Analysis of PCR Products
		2.4 PCR Serotyping of Salmonella spp.
		2.5 Multiplex PCR for Discriminating salmonella Serovars Typhi, from Paratyphi Serovars
		2.6 Primers for Serotyping Salmonella Species
			2.6.1 Example: Multiplex PCR to Identify Salmonella Typhimurium and Variants, Salmonella Enteritidis and Salmonella Dublin
			2.6.2 Example: Detection of Salmonella Stanleyville
	3 Antimicrobial Resistance in Salmonella spp.: Molecular Detection of Key Phenotypes
		3.1 Screening for Extended Spectrum Beta-Lactamase (ESBL) Genes
		3.2 Salient Features of CTX-M- Type ESBLs
	4 Notes
	References
Chapter 16: Determination of Antimicrobial Resistance of Salmonella in Pork
	1 Introduction
	2 Materials
		2.1 Disk Diffusion
		2.2 Broth Microdilution
		2.3 Agar Dilution
	3 Methods
		3.1 Disk Diffusion (Fig. 1)
		3.2 Broth Microdilution Method
		3.3 Agar Dilution Method (Fig. 5)
	4 Notes
	References
Chapter 17: Molecular Typing of Salmonella by Pulsed-Field Gel Electrophoresis
	1 Introduction
	2 Materials
	3 Methods
		3.1 Bacterial Culture
		3.2 PFGE Plug Preparation
		3.3 Lysis of Cells in 1% SKG:1% SDS Agarose Plugs
		3.4 Washing of Plugs
		3.5 Restriction Digestion of DNA in Plugs with Xba I
		3.6 Casting Agarose Gel
		3.7 Electrophoresis
		3.8 Image Acquisition
	4 Notes
	References
Chapter 18: Isolation and Identification of Salmonella in Pork
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sample Collection
		3.2 Pre-enrichment
		3.3 Selective Enrichment
		3.4 Plating Out on Selective Solid Media
		3.5 Confirmation
	4 Notes
	References
Index