Current Protocols in Microbiology

Current Protocols in Microbiology

Online ISBN: 9780471729259

DOI: 10.1002/9780471729259

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  1. Foreword
  2. Preface
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Technologies
    1. Introduction
    2. Section A Safety
      1. UNIT 1A.1 Biosafety: Guidelines for Working with Pathogenic and Infectious Microorganisms
      2. UNIT 1A.2 Biosafety Practices Associated with Potential Agents of Biocrime and Biowarfare
      3. UNIT 1A.3 Safe Use of Hazardous Chemicals
      4. UNIT 1A.4 Safe Use of Radioisotopes
      5. UNIT 1A.5 Biosafety Oversight and Compliance: What do you Mean, I have to Fill Out Another Form?!
    3. Section B Biofilms
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        UNIT 1B.1 Growing and Analyzing Static Biofilms
      2. UNIT 1B.2 Growing and Analyzing Biofilms in Flow Chambers
      3. UNIT 1B.3 Growing and Analyzing Biofilms in Fermenters
      4. UNIT 1B.4 Preparing for Biofilm Studies in the Field
      5. UNIT 1B.5 Growing Oral Biofilms in a Constant Depth Film Fermentor (CDFF)
    4. Section C Quorum Sensing
      1. UNIT 1C.1 Methods for Analysis of Bacterial Autoinducer-2 Production
      2. UNIT 1C.2 LacZ-Based Detection of Acyl-Homoserine Lactone Quorum-Sensing Signals
      3. UNIT 1C.3 Detection and Analysis of Quorum-Quenching Enzymes Against Acyl Homoserine Lactone Quorum-Sensing Signals
    5. Section D PCR Techniques
      1. UNIT 1D.1 Example of Use of TaqMan Real-Time RT-PCR to Analyze Bacterial Gene Transcript Levels: Haemophilus influenzae
      2. UNIT 1D.2 Using Host 28S Ribosomal RNA as a Housekeeping Gene for Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR) in Virus-Infected Animal Cells
      3. UNIT 1D.3 Example of Real-Time Quantitative Reverse Transcription–PCR (Q-RT-PCR) Analysis of Bacterial Gene Expression during Mammalian Infection: Borrelia burgdorferi in Mouse Tissues
    6. Section E Genomic Methods
      1. UNIT 1E.1 Characterization of Bacteria in Mixed Biofilm Communities Using Denaturing Gradient Gel Electrophoresis (DGGE)
      2. UNIT 1E.2 Simultaneous Isolation of Ixodidae and Bacterial (Borrelia spp.) Genomic DNA
      3. UNIT 1E.3 Genome-Wide Fitness and Genetic Interactions Determined by Tn-seq, a High-Throughput Massively Parallel Sequencing Method for Microorganisms
      4. UNIT 1E.4 Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers
      5. UNIT 1E.5 Using QIIME to Analyze 16S rRNA Gene Sequences from Microbial Communities
      6. UNIT 1E.6 Analyzing Arthropods for the Presence of Bacteria
      7. UNIT 1E.7 Gene Identification in Prokaryotic Genomes, Phages, Metagenomes, and EST Sequences with GeneMarkS Suite
      8. UNIT 1E.8 Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing
      9. UNIT 1E.9 Obtaining High Quality DNA from Diverse Clinical Samples
      10. UNIT 1E.10 Investigation of Viral and Host Chromatin by ChIP-PCR or ChIP-Seq Analysis
      11. UNIT 1E.11 CLIP-seq to Identify KSHV ORF57-Binding RNA in Host B Cells
      12. UNIT 1E.12 Virus Hunting: Discovery of New Episomal Circular Viruses by Rolling Circle Techniques
      13. UNIT 1E.13 Using KBase to Assemble and Annotate Prokaryotic Genomes
      14. UNIT 1E.14 Computational Methods for Human Microbiome Analysis
    7. Section F Protein and Proteomic Methods
      1. UNIT 1F.1 Identification of Novel DNA-Binding Proteins Using DNA-Affinity Chromatography/Pull Down
      2. UNIT 1F.2 2-D Gel-Based Proteomic Approaches to Antibiotic Drug Discovery
      3. UNIT 1F.3 Quantitative Proteomic Analysis of the Cell Envelopes and Native Membrane Vesicles Derived from Gram-Negative Bacteria
      4. UNIT 1F.4 Biochemical Analysis of Microbial Rhodopsins
    8. Section G Bioremediation
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        UNIT 1G.1 Biological Sand Filters: Low-Cost Bioremediation Technique for Production of Clean Drinking Water
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        UNIT 1G.2 Bioremediation of Turbid Surface Water Using Seed Extract from the Moringa oleifera Lam. (Drumstick) Tree
  5. Chapter 2 Microscopy and Imaging
    1. Introduction
    2. Section A Light Microscopy
      1. UNIT 2A.1 Proper Alignment and Adjustment of the Light Microscope
      2. UNIT 2A.2 Total Internal Reflection Fluorescence (TIRF) Microscopy
    3. Section B Electron Microscopy
      1. UNIT 2B.1 Transmission Electron Microscopy
      2. UNIT 2B.2 Scanning Electron Microscopy
    4. Section C Other Imaging Methods
      1. UNIT 2C.1 Basic Confocal Microscopy
      2. UNIT 2C.2 Atomic Force Microscopy (AFM)
      3. UNIT 2C.3 Whole-Body Imaging of Infection Using Fluorescence
      4. UNIT 2C.4 Whole-Body Imaging of Infection Using Bioluminescence
      5. UNIT 2C.5 Use of Image-Based Flow Cytometry in Bacterial Viability Analysis Using Fluorescent Probes
  6. Chapter 3 Alpha Proteobacteria
    1. Introduction
    2. Section A Rickettsiales
      1. UNIT 3A.1 Laboratory Maintenance of Ehrlichia chaffeensis and Ehrlichia canis and Recovery of Organisms for Molecular Biology and Proteomics Studies
      2. UNIT 3A.2 Laboratory Maintenance of Anaplasma phagocytophilum
      3. UNIT 3A.3 Isolation and Molecular Detection of Ehrlichia from Vertebrate Animals
      4. UNIT 3A.4 Transfection of Wolbachia pipientis into Drosophila Embryos
      5. UNIT 3A.5 Laboratory Maintenance of Rickettsia rickettsii
      6. UNIT 3A.6 Electrotransformation and Clonal Isolation of Rickettsia Species
    3. Section B Brucella
      1. UNIT 3B.1 Laboratory Maintenance of Brucella abortus
    4. Section C Bartonella
      1. UNIT 3C.1 Laboratory Maintenance of Bartonella quintana
    5. Section D Rhizobiales
      1. UNIT 3D.1 Laboratory Maintenance of Agrobacterium
      2. UNIT 3D.2 Genetic Manipulation of Agrobacterium
      3. UNIT 3D.3 Phenotypic Analyses of Agrobacterium
      4. UNIT 3D.4 Isolation and Cultivation of Agrobacterium Species from Natural Sources
    6. Section E Rhodospirillales
      1. UNIT 3E.1 Azospirillum brasilense, a Beneficial Soil Bacterium: Isolation and Cultivation
      2. UNIT 3E.2 Azospirillum brasilense: Laboratory Maintenance and Genetic Manipulation
  7. Chapter 4 Beta Proteobacteria
    1. Introduction
    2. Section A Neisseria gonorrhoeae
      1. UNIT 4A.1 Laboratory Maintenance of Neisseria gonorrhoeae
      2. UNIT 4A.2 Genetic Manipulation of Neisseria gonorrhoeae
      3. UNIT 4A.3 Isolation of Cell Envelopes and Naturally Released Membrane Vesicles of Neisseria gonorrhoeae
    3. Section B Bordetella
      1. UNIT 4B.1 Laboratory Maintenance of Bordetella pertussis
    4. Section C Burkholderia
      1. UNIT 4C.1 Burkholderia thailandensis: Growth and Laboratory Maintenance
      2. UNIT 4C.2 Burkholderia thailandensis: Genetic Manipulation
  8. Chapter 5 Enteric Gamma Proteobacteria
    1. Introduction
    2. Section A Escherichia coli
      1. UNIT 5A.1 Interaction of Enterohemorrhagic Escherichia coli (EHEC) with Mammalian Cells: Cell Adhesion, Type III Secretion, and Actin Pedestal Formation
      2. UNIT 5A.2 Rapid Allelic Exchange in Enterohemorrhagic Escherichia coli (EHEC) and Other E. coli Using λ Red Recombination
      3. UNIT 5A.3 Genotyping Escherichia coli O157:H7 for Its Ability to Cause Disease in Humans
      4. UNIT 5A.4 Growth and Maintenance of Escherichia coli Laboratory Strains
    3. Section B Yersinia
      1. UNIT 5B.1 Laboratory Maintenance and Characterization of Yersinia pestis
      2. UNIT 5B.2 Isolation and Confirmation of Yersinia pestis Mutants Exempt from Select Agent Regulations
      3. UNIT 5B.3 Extraction, Purification, and Identification of Yersiniabactin, the Siderophore of Yersinia pestis
  9. Chapter 6 Nonenteric Gamma Proteobacteria
    1. Introduction
    2. Section A Vibrionales
      1. UNIT 6A.1 Growth and Laboratory Maintenance of Vibrio cholerae
      2. UNIT 6A.2 Genetic Screens and Biochemical Assays to Characterize Vibrio cholerae O1 Biotypes: Classical and El Tor
      3. UNIT 6A.3 Vibriocidal Assays to Determine the Antibody Titer of Patient Sera Samples
      4. UNIT 6A.4 Vibrio cholerae: Measuring Natural Transformation Frequency
      5. UNIT 6A.5 Detection, Isolation, and Identification of Vibrio cholerae from the Environment
      6. UNIT 6A.6 Infant Rabbit Model for Diarrheal Diseases
    3. Section B Moraxella
      1. UNIT 6B.1 Laboratory Maintenance of Moraxella catarrhalis
    4. Section C Coxiella
      1. UNIT 6C.1 Laboratory Maintenance of Coxiella burnetii
      2. UNIT 6C.2 Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole
      3. UNIT 6C.3 Measuring pH of the Coxiella burnetii Parasitophorous Vacuole
    5. Section D Pasturella
      1. UNIT 6D.1 Laboratory Growth and Maintenance of Haemophilus influenzae
    6. Section E Pseudomonas
      1. UNIT 6E.1 Growth and Laboratory Maintenance of Pseudomonas aeruginosa
    7. Section F Stenotrophomonas
      1. UNIT 6F.1 Laboratory Culture and Maintenance of Stenotrophomonas maltophilia
      2. UNIT 6F.2 Genetic Manipulation of Stenotrophomonas maltophilia
    8. Section G Acinetobacter
      1. UNIT 6G.1 Laboratory Maintenance of Acinetobacter baumannii
      2. UNIT 6G.2 Genetic Manipulation of Acinetobacter baumannii
      3. UNIT 6G.3 Mouse Models of Acinetobacter baumannii Infection
  10. Chapter 7 Delta Proteobacteria
    1. Introduction
    2. Section A Myxococcaceae
      1. UNIT 7A.1 Myxococcus xanthus Growth, Development, and Isolation
    3. Section B Bdellovibrionales
      1. UNIT 7B.1 Isolation and Classification of Bdellovibrio and Like Organisms
      2. UNIT 7B.2 Laboratory Maintenance of Bdellovibrio
      3. UNIT 7B.3 Lyophilization of Bdellovibrio bacteriovorus 109J for Long-Term Storage
  11. Chapter 8 Epsilon Proteobacteria
    1. Introduction
    2. Section A Campylobacter
      1. UNIT 8A.1 Growth and Laboratory Maintenance of Campylobacter jejuni
      2. UNIT 8A.2 Genetic Manipulation of Campylobacter jejuni
      3. UNIT 8A.3 Experimental Chick Colonization by Campylobacter jejuni
    3. Section B Helicobacter
      1. UNIT 8B.1 Laboratory Maintenance of Helicobacter Species
  12. Chapter 9 Firmicutes (Low G+C Gram Positive)
    1. Introduction
    2. Section A Clostridiae
      1. UNIT 9A.1 Laboratory Maintenance of Clostridium difficile
      2. UNIT 9A.2 Genetic Manipulation of Clostridium difficile
      3. UNIT 9A.3 Therapeutic Approaches for Clostridium difficile Infections
    3. Section B Listeria
      1. UNIT 9B.1 Animal Models of Listeria Infection
      2. UNIT 9B.2 Listeria monocytogenes: Cultivation and Laboratory Maintenance
      3. UNIT 9B.3 A Mouse Model of Foodborne Listeria monocytogenes Infection
      4. UNIT 9B.4 Tissue Culture Cell Assays Used to Analyze Listeria monocytogenes
      5. UNIT 9B.5 Isolation of Listeria monocytogenes from Food and Water: Official and Experimental Protocols
    4. Section C Staphylococcus
      1. UNIT 9C.1 Growth and Laboratory Maintenance of Staphylococcus aureus
      2. UNIT 9C.2 Laboratory Maintenance of Methicillin-Resistant Staphylococcus aureus (MRSA)
      3. UNIT 9C.3 Genetic Manipulation of Staphylococcus aureus
      4. UNIT 9C.4 Tissue Culture Assays Used to Analyze Invasion by Staphylococcus aureus
    5. Section D Streptococcus
      1. UNIT 9D.1 Adult Zebrafish Model of Streptococcal Infection
      2. UNIT 9D.2 Laboratory Growth and Maintenance of Streptococcus pyogenes (The Group A Streptococcus, GAS)
      3. UNIT 9D.3 Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
      4. UNIT 9D.4 Streptococcus pneumoniae Supragenome Hybridization Arrays for Profiling of Genetic Content and Gene Expression
      5. UNIT 9D.5 Murine Models of Streptococcus pyogenes Infection
  13. Chapter 10 Actinobacteria (High G+C Gram Positive)
    1. Introduction
    2. Section A Mycobacterium tuberculosis
      1. UNIT 10A.1 Laboratory Maintenance of Mycobacterium tuberculosis
      2. UNIT 10A.2 Genetic Manipulation of Mycobacterium tuberculosis
      3. UNIT 10A.3 Isolation and Analysis of Mycobacterium tuberculosis Mycolic Acids
      4. UNIT 10A.4 Analyses of Mycobacterium tuberculosis Proteins
      5. UNIT 10A.5 Animal Models of M. tuberculosis Infection
      6. UNIT 10A.6 Acid-Fast Staining and Petroff-Hausser Chamber Counting of Mycobacterial Cells in Liquid Suspension
    3. Section B Mycobacterium marinum
      1. UNIT 10B.1 Laboratory Maintenance of Mycobacterium marinum
      2. UNIT 10B.2 Zebrafish and Frog Models of Mycobacterium marinum Infection
    4. Section C Mycobacterium smegmatis
      1. UNIT 10C.1 Laboratory Maintenance of Mycobacterium smegmatis
    5. Section D Mycobacterium abscessus
      1. UNIT 10D.1 Laboratory Maintenance of Mycobacterium abscessus
      2. UNIT 10D.2 Genetic Manipulation of Mycobacterium abscessus
    6. Section E Streptomyces
      1. UNIT 10E.1 Laboratory Maintenance of Streptomyces Species
      2. UNIT 10E.2 Laboratory Maintenance of Streptomyces argillaceus and Streptomyces griseus
      3. UNIT 10E.3 Genetic Manipulation of Streptomyces Species
      4. UNIT 10E.4 Isolation of Streptomyces Species from Soil
    7. Section F Nocardia Species
      1. UNIT 10F.1 Laboratory Maintenance of Nocardia Species
      2. UNIT 10F.2 Genetic Manipulation of Nocardia Species
    8. Section G Micromonosporaceae Species
      1. UNIT 10G.1 Actinomadura Species: Laboratory Maintenance and Ribosome Engineering
    9. Section H Pseudonocardiaceae Species
      1. UNIT 10H.1 Saccharopolyspora Species: Laboratory Maintenance and Enhanced Production of Secondary Metabolites
  14. Chapter 11 Chlamydiae
    1. Introduction
    2. Section A Chlamydia trachomatis
      1. UNIT 11A.1 Cultivation and Laboratory Maintenance of Chlamydia trachomatis
      2. UNIT 11A.2 Sphingolipid Trafficking and Purification in Chlamydia trachomatis–Infected Cells
      3. UNIT 11A.3 Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis
    3. Section B Chlamydia pneumoniae
      1. UNIT 11B.1 Cultivation and Laboratory Maintenance of Chlamydia pneumoniae
  15. Chapter 12 Spirochetes
    1. Introduction
    2. UNIT 12.1 Analysis of Bacterial Membrane Proteins Produced During Mammalian Infection Using Hydrophobic Antigen Tissue Triton Extraction (HATTREX)
    3. Section A Treponema pallidum and Related Treponemes
      1. UNIT 12A.1 Isolation and Laboratory Maintenance of Treponema pallidum
    4. Section B Oral Treponemes
      1. UNIT 12B.1 Laboratory Maintenance of Treponema denticola
      2. UNIT 12B.2 Genetic Manipulation of Treponema denticola
    5. Section C Lyme Disease Borrelia
      1. UNIT 12C.1 Laboratory Maintenance of Borrelia burgdorferi
      2. UNIT 12C.2 Methods of Identifying Membrane Proteins in Spirochetes
      3. UNIT 12C.3 Cultivation of Borrelia burgdorferi in Dialysis Membrane Chambers in Rat Peritonea
      4. UNIT 12C.4 Genetic Transformation of Borrelia burgdorferi
      5. UNIT 12C.5 Molecular Typing of Borrelia burgdorferi
    6. Section D Brachyspira
      1. UNIT 12D.1 Isolation and Maintenance of Brachyspira Species
    7. Section E Leptospira
      1. UNIT 12E.1 Laboratory Maintenance of Pathogenic Leptospira
      2. UNIT 12E.2 Hamster Model of Leptospirosis
      3. UNIT 12E.3 Rat Model of Chronic Leptospirosis
      4. UNIT 12E.4 Genetic Manipulation of Leptospira biflexa
      5. UNIT 12E.5 Leptospirosis Serodiagnosis by the Microscopic Agglutination Test
      6. UNIT 12E.6 Leptospira: Molecular Detection of Pathogenic Species in Natural Sources
    8. Section F Hard Tick Relapsing Fever Borrelia
      1. UNIT 12F.1 Laboratory Cultivation and Maintenance of Borrelia miyamotoi
  16. Chapter 13 Other Eubacteria
    1. Introduction
    2. Section A Fusobacteria
      1. UNIT 13A.1 Laboratory Maintenance of Fusobacteria
    3. Section B Flavobacterium
      1. UNIT 13B.1 Laboratory Maintenance of Flavobacterium psychrophilum and Flavobacterium columnare
    4. Section C Bacteroides
      1. UNIT 13C.1 Laboratory Maintenance and Cultivation of Bacteroides Species
      2. UNIT 13C.2 Genetic Manipulation of Porphyromonas gingivalis
  17. Chapter 14 DNA Viruses
    1. Introduction
    2. Section A Pox Viruses
      1. UNIT 14A.1 Myxoma Virus: Propagation, Purification, Quantification, and Storage
      2. UNIT 14A.2 Production of Myxoma Virus Gateway Entry and Expression Libraries and Validation of Viral Protein Expression
      3. UNIT 14A.3 Preparation of Cell Cultures and Vaccinia Virus Stocks
      4. UNIT 14A.4 Generation of Recombinant Vaccinia Viruses
      5. UNIT 14A.5 Natural Vaccinia Virus Infection: Diagnosis, Isolation, and Characterization
      6. UNIT 14A.6 ELISA for Molluscum Contagiosum Virus
    3. Section B Papillomaviruses
      1. UNIT 14B.1 Cell Culture Assay for Transient Replication of Human and Animal Papillomaviruses
      2. UNIT 14B.2 Analyzing the Human Papillomavirus (HPV) Life Cycle in Primary Keratinocytes with a Quantitative Colony-Forming Assay
      3. UNIT 14B.3 Using Organotypic (Raft) Epithelial Tissue Cultures for the Biosynthesis and Isolation of Infectious Human Papillomaviruses
      4. UNIT 14B.4 Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments
      5. UNIT 14B.5 Production of Furin-Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1- or L2-Specific Antibodies
      6. UNIT 14B.6 Construction of a Transcription Map for Papillomaviruses using RACE, RNase Protection, and Primer Extension Assays
      7. UNIT 14B.7 Detection of Papillomavirus Gene Expression Patterns in Tissue Sections
      8. UNIT 14B.8 Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle
      9. UNIT 14B.9 Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples
      10. UNIT 14B.10 Analysis of Human Papillomavirus Genome Replication Using Two- and Three-Dimensional Agarose Gel Electrophoresis
    4. Section C Adenoviruses
      1. UNIT 14C.1 Human Adenoviruses: Propagation, Purification, Quantification, and Storage
    5. Section D Adeno-Associated Viruses
      1. UNIT 14D.1 Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors
      2. UNIT 14D.2 Gene Transfer in the Lung Using Recombinant Adeno-Associated Virus
      3. UNIT 14D.3 Gene Transfer in Skeletal and Cardiac Muscle Using Recombinant Adeno-Associated Virus
      4. UNIT 14D.4 Retinal Gene Delivery by rAAV and DNA Electroporation
      5. UNIT 14D.5 Gene Transfer to the CNS Using Recombinant Adeno-Associated Virus
      6. UNIT 14D.6 Gene Transfer in the Liver Using Recombinant Adeno-Associated Virus
    6. Section E Herpesviruses
      1. UNIT 14E.1 Herpes Simplex Virus: Propagation, Quantification, and Storage
      2. UNIT 14E.2 Epstein-Barr Virus (EBV): Infection, Propagation, Quantitation, and Storage
      3. UNIT 14E.3 Human Cytomegalovirus: Propagation, Quantification, and Storage
      4. UNIT 14E.4 Human Cytomegalovirus: Bacterial Artificial Chromosome (BAC) Cloning and Genetic Manipulation
      5. UNIT 14E.5 Quantitative Analysis of HSV Gene Expression during Lytic Infection
      6. UNIT 14E.6 Analysis of HSV Viral Reactivation in Explants of Sensory Neurons
      7. UNIT 14E.7 PA-seq for Global Identification of RNA Polyadenylation Sites of Kaposi's Sarcoma–Associated Herpesvirus Transcripts
    7. Section F Polyoma Viruses
      1. UNIT 14F.1 Mouse Polyomavirus: Propagation, Purification, Quantification, and Storage
      2. UNIT 14F.2 In Vitro Replication Assay for Merkel Cell Polyomavirus (MCPyV)
      3. UNIT 14F.3 Epigenetic Analysis of SV40 Minichromosomes
    8. Section G Mimiviridae
      1. UNIT 14G.1 Mimiviruses: Replication, Purification, and Quantification
  18. Chapter 15 RNA Viruses
    1. Introduction
    2. Section A Arenaviruses
      1. UNIT 15A.1 Lymphocytic Choriomeningitis Virus (LCMV): Propagation, Quantitation, and Storage
    3. Section B Alphaviruses
      1. UNIT 15B.1 Sindbis Virus: Propagation, Quantification, and Storage
    4. Section C Reoviruses
      1. UNIT 15C.1 Mammalian Reoviruses: Propagation, Quantification, and Storage
      2. UNIT 15C.2 Avian Reoviruses: Propagation, Quantification, and Storage
      3. UNIT 15C.3 Culturing, Storage, and Quantification of Rotaviruses
      4. UNIT 15C.4 Bluetongue Virus (BTV): Propagation, Quantification, and Storage
      5. UNIT 15C.5 Separation and Isolation of BTV dsRNA Segments and Viral Proteins
      6. UNIT 15C.6 Rotaviruses: Extraction and Isolation of RNA, Reassortant Strains, and NSP4 Protein
    5. Section D Flaviviruses
      1. UNIT 15D.1 Hepatitis C Virus: Propagation, Quantification, and Storage
      2. UNIT 15D.2 Dengue Virus: Isolation, Propagation, Quantification, and Storage
      3. UNIT 15D.3 Propagation, Quantification, Detection, and Storage of West Nile Virus
      4. UNIT 15D.4 Zika Virus: Quantification, Propagation, Detection, and Storage
    6. Section E Coronaviruses
      1. UNIT 15E.1 Coronaviruses: Propagation, Quantification, Storage, and Construction of Recombinant Mouse Hepatitis Virus
      2. UNIT 15E.2 Growth and Quantification of MERS-CoV Infection
    7. Section F Paramyxoviruses
      1. UNIT 15F.1 Human Parainfluenza Virus Type 3 (HPIV-3): Construction and Rescue of an Infectious, Recombinant Virus Expressing the Enhanced Green Fluorescent Protein (EGFP)
      2. UNIT 15F.2 Newcastle Disease Virus: Propagation, Quantification, and Storage
    8. Section G Orthomyxoviruses
      1. UNIT 15G.1 Influenza: Propagation, Quantification, and Storage
      2. UNIT 15G.2 The Ferret Model for Influenza
      3. UNIT 15G.3 The Mouse Model for Influenza
      4. UNIT 15G.4 Insertion of a GFP Reporter Gene in Influenza Virus
    9. Section H Picornaviruses
      1. UNIT 15H.1 Poliovirus: Generation, Quantification, Propagation, Purification, and Storage
      2. UNIT 15H.2 Poliovirus: Generation and Characterization of Mutants
      3. UNIT 15H.3 RNA Structure Analysis of Viruses Using SHAPE
    10. Section I Bornaviruses
      1. UNIT 15I.1 Avian Bornaviruses: Diagnosis, Isolation, and Genotyping
    11. Section J Retroviruses
      1. UNIT 15J.1 Human Immunodeficiency Viruses: Propagation, Quantification, and Storage
    12. Section K Caliciviruses
      1. UNIT 15K.1 Human Norovirus Detection and Production, Quantification, and Storage of Virus-Like Particles
      2. UNIT 15K.2 Murine Norovirus: Propagation, Quantification, and Genetic Manipulation
  19. Chapter 16 Plant Viruses
    1. Introduction
    2. Section A Plant Propagation for Virus Infection
      1. UNIT 16A.1 Growth Conditions for Plant Virus–Host Studies
      2. UNIT 16A.2 Propagation of Major Plant-Virus Hosts
    3. Section B Plant Inoculation Methods
      1. UNIT 16B.1 Aphid Transmission of Plant Viruses
      2. UNIT 16B.2 Delivery and Expression of Functional Viral RNA Genomes In Planta by Agroinfiltration
      3. UNIT 16B.3 Inoculation of Plants Using Bombardment
      4. UNIT 16B.4 Fungal Transmission of Plant Viruses
      5. UNIT 16B.5 Testing of Transmission of Tobraviruses by Nematodes
      6. UNIT 16B.6 Mechanical Inoculation of Plant Viruses
    4. Section C Methods of Virus Detection
      1. UNIT 16C.1 Reverse Transcription-Polymerase Chain Reaction-Based Detection of Plant Viruses
    5. Section D Preparation and Inoculation of Protoplasts
      1. UNIT 16D.1 Callus Cultures of Arabidopsis
      2. UNIT 16D.2 Preparation and Inoculation of Mesophyll Protoplasts from Monocotyledenous and Dicotyledenous Hosts
      3. UNIT 16D.3 Preparation and Electroporation of Oat Protoplasts from Cell Suspension Culture
      4. UNIT 16D.4 Transfecting Protoplasts by Electroporation to Study Viroid Replication
    6. Section E Methodologies to Extract and Study Viral RNA
      1. UNIT 16E.1 Extracting Viral RNAs from Plant Protoplasts
      2. UNIT 16E.3 Northern Analysis of Viral Plus- and Minus-Strand RNAs
    7. Section F Cloning Full-Length RNA Viruses
      1. UNIT 16F.1 Cloning of Large Positive-Strand RNA Viruses
      2. UNIT 16F.3 Rapid Full-Length Cloning of Nonpolyadenylated RNA Virus Genomes
    8. Section G Viroids
      1. UNIT 16G.1 Identification of Viroids by Gel Electrophoresis
    9. Section H RNA Silencing
      1. UNIT 16H.2 Isolation and Cloning of Small RNAs from Virus-Infected Plants
    10. Section I Viral Vector Methodologies
      1. UNIT 16I.1 Use of Potato Virus X (PVX)–Based Vectors for Gene Expression and Virus-Induced Gene Silencing (VIGS)
      2. UNIT 16I.4 Using Vectors Derived from Tomato Bushy Stunt Virus (TBSV) and TBSV Defective Interfering RNAs (DIs)
      3. UNIT 16I.5 Using Satellite Tobacco Mosaic Virus Vectors for Gene Silencing
      4. UNIT 16I.6 Using Viral Vectors to Silence Endogenous Genes
    11. Section J Expression of Viruses in Yeast
      1. UNIT 16J.1 A High-Throughput Approach for Studying Virus Replication in Yeast
    12. Section K Translation of Plant Viral RNA
      1. UNIT 16K.1 In Vitro Translation of Plant Viral RNA
      2. UNIT 16K.2 In Vivo Translation Studies of Plant Viral RNAs Using Reporter Genes
  20. Chapter 17 Anti-Infectives
    1. Introduction
    2. UNIT 17.1 Characterization of the In Vitro Activity of Novel Lipoglycopeptide Antibiotics
    3. UNIT 17.2 Structure-Based Approaches to Antibiotic Drug Discovery
    4. UNIT 17.3 Novel Approaches to Bacterial Infection Therapy by Interfering with Cell-to-Cell Signaling
    5. UNIT 17.4 Identification of Small-Molecule Scaffolds for P450 Inhibitors
    6. UNIT 17.5 Cell-Based Hepatitis C Virus Infection Fluorescence Resonance Energy Transfer (FRET) Assay for Antiviral Compound Screening
    7. UNIT 17.6 Laboratory Detection of Vancomycin Nonsusceptible Staphylococcus aureus
    8. UNIT 17.7 Screening of Hepatitis C Virus Inhibitors Using Genotype 1a HCV Replicon Cell Lines
    9. UNIT 17.8 HT-SPOTi: A Rapid Drug Susceptibility Test (DST) to Evaluate Antibiotic Resistance Profiles and Novel Chemicals for Anti-Infective Drug Discovery
    10. UNIT 17.9 Aminoglycoside-Enabled Elucidation of Bacterial Persister Metabolism
  21. Chapter 18 Vaccines
    1. UNIT 18.1 Progress in Vaccine Development
    2. UNIT 18.2 Newcastle Disease Virus-Like Particles: Preparation, Purification, Quantification, and Incorporation of Foreign Glycoproteins
    3. UNIT 18.3 DNA Immunization
    4. UNIT 18.4 Western Blot Assay for Quantitative and Qualitative Antigen Detection in Vaccine Development
  22. Chapter 19 Mycology
    1. Section A Plant-Associated Fungi
      1. UNIT 19A.1 Detection and Isolation of Epichloë Species, Fungal Endophytes of Grasses
  23. Chapter 20 Parasitic Protozoa
    1. Section A Blastocystis
      1. UNIT 20A.1 Blastocystis: Isolation, Xenic Cultivation, and Cryopreservation
      2. UNIT 20A.2 Molecular Identification and Subtype Analysis of Blastocystis
    2. Section B Cryptosporidium
      1. UNIT 20B.1 Cryptosporidium: Identification and Genetic Typing
      2. UNIT 20B.2 Generating and Maintaining Transgenic Cryptosporidium parvum Parasites
    3. Section C Toxoplasma
      1. UNIT 20C.1 Toxoplasma gondii: Laboratory Maintenance and Growth
      2. UNIT 20C.2 Purification Toxoplasma gondii Tissue Cysts Using Percoll Gradients
    4. Section D Sarcocystis
      1. UNIT 20D.1 Isolation, Culture and Cryopreservation of Sarcocystis species
  24. Chapter 21 Protists
    1. Section A Oomycetes
      1. UNIT 21A.1 Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9
  25. Appendix 1 Useful Information and Data
    1. APPENDIX 1A Abbreviations Used in this Manual
    2. APPENDIX 1B Resources for International Biosafety Guidelines and Regulations
  26. Appendix 2 Commonly Used Reagents and Equipment
    1. APPENDIX 2A Commonly Used Reagents
    2. APPENDIX 2B Standard Laboratory Equipment
    3. APPENDIX 2C Where to Find Recipes for Media
  27. Appendix 3 Commonly Used Techniques
    1. APPENDIX 3A Assays for Total Protein
    2. APPENDIX 3B Diagnosis and Treatment of Mycoplasma-Contaminated Cell Cultures
    3. APPENDIX 3C Differential Staining of Bacteria: Gram Stain
    4. APPENDIX 3D Direct PCR of Intact Bacteria (Colony PCR)
    5. APPENDIX 3E Preliminary Staining of Bacteria: Simple Stains
    6. APPENDIX 3F Preliminary Staining of Bacteria: Negative Stain
    7. APPENDIX 3G Differential Staining of Bacteria: Flagella Stain
    8. APPENDIX 3H Differential Staining of Bacteria: Acid Fast Stain
    9. APPENDIX 3I Differential Staining of Bacteria: Capsule Stain
    10. APPENDIX 3J Differential Staining of Bacteria: Endospore Stain
    11. APPENDIX 3K Fluorescent Staining of Bacteria: Viability and Antibody Labeling
    12. APPENDIX 3L Generation of Transformation Competent E. coli
    13. APPENDIX 3M SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins
    14. APPENDIX A.3N Care and Handling of Laboratory Mice
  28. Appendix 4 Commonly Used Methods for Cell Culture
    1. APPENDIX 4A Common Bacterial Culture Techniques and Media
    2. APPENDIX 4B Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVEC)
    3. APPENDIX 4C Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
    4. APPENDIX 4D Aseptic Technique
    5. APPENDIX 4E Growth and Maintenance of Vero Cell Lines
    6. APPENDIX 4F Anaerobic Cell Culture
    7. APPENDIX 4G Growth and Maintenance of Quail Fibrosarcoma QM5 Cells
    8. APPENDIX 4H Growth and Maintenance of Baby Hamster Kidney (BHK) Cells
    9. APPENDIX 4I Growth and Maintenance of Chick Embryo Fibroblasts (CEF)
    10. APPENDIX 4J Growth and Maintenance of Mosquito Cell Lines
    11. APPENDIX A.4K A Simple and Low-Cost Procedure for Growing Geobacter sulfurreducens Cell Cultures and Biofilms in Bioelectrochemical Systems

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