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This fully updated edition explores the latest techniques to study the challenging, and at times dangerous, genus of bacteria known as mycobacteria with basic methods that are still required for mycobacteriology along with the newer or improved methods that have been developed. The volume features chapters on the basics of DNA isolation, protein isolation, and lipid isolation, as well as more sophisticated techniques for isolation of ribosomes, and continues with sections involving analyzing subcellular fractions, culture methods, sequencing technology, in vitro models, molecular methods, as well as drug discovery applications. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and up-to-date, Mycobacteria Protocols, Fourth Edition serves as an ideal guide for those starting out with their mycobacteria research and also for those who have worked with it for decades.
List of contents
Culturing Mycobacteria.- DNA Isolation from Mycobacteria.- Extraction and Separation of Mycobacterial Proteins.- Lipid and Lipoarabinomannan Isolation and Characterization.- Purification of Hibernating and Active C- Ribosomes from Zinc-Starved Mycobacteria.- Macrophage Infection Models for Mycobacterium tuberculosis.- Novel Single-Cell and High Throughput Microscopy Techniques to Monitor Dictyostelium discoideum-Mycobacterium marinum Infection Dynamics.- Single-Cell Analysis of Mycobacteria Using Microfluidics and Time-Lapse Microscopy.- Measuring Efflux and Permeability in Mycobacteria.- Drug Susceptibility Screening Using In Vitro Models of Hypoxic Non-Replicating Persistent Mycobacteria.- Flow Cytometry Analysis of Mycobacteria and Mycobacteria-Infected Immune Cells.- Electroporation of Mycobacteria.- Gene Switching and Essentiality Testing.- Oligo-Mediated Recombineering and Its Use for Making SNPs, Knockouts, Insertions, and Fusions in Mycobacterium tuberculosis.- Using Proteolytic Hypomorphs to Detect Small Molecule Mechanism of Action.- CRISPR Interference (CRISPRi) for Targeted Gene Silencing in Mycobacteria.- Exploiting Fluorescent Proteins to Understand Mycobacterium tuberculosis Biology.- Metabolic Labeling of Live Mycobacteria with Trehalose-Based Probes.- Identification and Characterization of Mycobacterial Species Using Whole Genome Sequences.- Whole Genome Sequencing of Mycobacterium tuberculosis Directly from Sputum Samples.- Experimental and Computational Workflow for RNA Sequencing in Mycobacterium tuberculosis: From Total RNA to Differentially Expressed Genes.- RNA Sequencing for Transcript 5'-End Mapping in Mycobacteria.- Methods for Proteomic Analyses of Mycobacteria.- Targeted Lipidomics of Mycobacterial Lipids and Glycolipids.- Metabolomics of Mycobacterium tuberculosis.- Determining Minimum Inhibitory Concentrations in Liquid Cultures or on Solid Medium.- A Multi-Stress Model for High Throughput Screening Against Non-Replicating Mycobacterium tuberculosis.- Antimicrobial Susceptibility Testing for Mycobacterium sps in High Throughput Format.- High-Content Analysis Monitoring Intracellular Trafficking and Replication of Mycobacterium tuberculosis Inside Host Cells.- Efficient Measurement of Drug Interactions with DiaMOND (Diagonal Measurement of N-Way Drug Interactions).- Drug Sensitivity Testing of Mycobacterium tuberculosis Growing in a Hollow Fiber Bioreactor.
