Current Protocols in Microbiology

Table of Contents

• Chapter 1 Emerging Technologies
  • Introduction
  • Section A Safety
    • Unit 1A.1 Biosafety: Guidelines for Working with Pathogenic and Infectious Microorganisms
    • Unit 1A.2 Biosafety Practices Associated with Potential Agents of Biocrime and Biowarfare
    • Unit 1A.3 Safe Use of Hazardous Chemicals
    • Unit 1A.4 Safe Use of Radioisotopes
  • Section B Biofilms
    • Unit 1B.1 Growing and Analyzing Static Biofilms
    • Unit 1B.2 Growing and Analyzing Biofilms in Flow Chambers
    • Unit 1B.3 Growing and Analyzing Biofilms in Fermenters
    • Unit 1B.4 Preparing for Biofilm Studies in the Field
    • Unit 1B.5 Growing Oral Biofilms in a Constant Depth Film Fermentor (CDFF)
  • Section C Quorum Sensing
    • Unit 1C.1 Methods for Analysis of Bacterial Autoinducer‐2 Production
    • Unit 1C.2 LacZ‐Based Detection of Acyl‐Homoserine Lactone Quorum‐Sensing Signals
    • Unit 1C.3 Detection and Analysis of Quorum‐Quenching Enzymes Against Acyl Homoserine Lactone Quorum‐Sensing Signals
  • Section D PCR Techniques
    • Unit 1D.1 Example of Use of TaqMan Real‐Time RT‐PCR to Analyze Bacterial Gene Transcript Levels: Haemophilus influenzae
    • 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
    • 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
  • Section E Genomic Methods
    • Unit 1E.1 Characterization of Bacteria in Mixed Biofilm Communities Using Denaturing Gradient Gel Electrophoresis (DGGE)
    • Unit 1E.2 Simultaneous Isolation of Ixodidae and Bacterial (Borrelia spp.) Genomic DNA
    • Unit 1E.3 Genome‐Wide Fitness and Genetic Interactions Determined by Tn‐seq, a High‐Throughput Massively Parallel Sequencing Method for Microorganisms
    • Unit 1E.4 Preparing DNA Libraries for Multiplexed Paired‐End Deep Sequencing for Illumina GA Sequencers
    • Unit 1E.5 Using QIIME to Analyze 16S rRNA Gene Sequences from Microbial Communities
    • Unit 1E.6 Analyzing Arthropods for the Presence of Bacteria
  • Section F Proteomic Methods
    • Unit 1F.1 Identification of Novel DNA‐Binding Proteins Using DNA‐Affinity Chromatography/Pull Down
    • Unit 1F.2 2‐D Gel‐Based Proteomic Approaches to Antibiotic Drug Discovery
  • Section G Bioremediation
    • Unit 1G.1 Biological Sand Filters: Low‐Cost Bioremediation Technique for Production of Clean Drinking Water
    • Unit 1G.2 Bioremediation of Turbid Surface Water Using Seed Extract from Moringa oleifera Lam. (Drumstick) Tree
• Chapter 2 Microscopy and Imaging
  • Introduction
  • Section A Light Microscopy
    • Unit 2A.1 Proper Alignment and Adjustment of the Light Microscope
    • Unit 2A.2 Total Internal Reflection Fluorescence (TIRF) Microscopy
  • Section B Electron Microscopy
    • Unit 2B.1 Transmission Electron Microscopy
    • Unit 2B.2 Scanning Electron Microscopy
  • Section C Other Imaging Methods
    • Unit 2C.1 Basic Confocal Microscopy
    • Unit 2C.2 Atomic Force Microscopy (AFM)
    • Unit 2C.3 Whole‐Body Imaging of Infection Using Fluorescence
    • Unit 2C.4 Whole‐Body Imaging of Infection Using Bioluminescence
    • Unit 2C.5 Use of Image‐Based Flow Cytometry in Bacterial Viability Analysis Using Fluorescent Probes
• Chapter 3 Alpha Proteobacteria
  • Introduction
  • Section A Rickettsiales
    • Unit 3A.1 Laboratory Maintenance of Ehrlichia chaffeensis and Ehrlichia canis and Recovery of Organisms for Molecular Biology and Proteomics Studies
    • Unit 3A.2 Laboratory Maintenance of Anaplasma phagocytophilum
    • Unit 3A.3 Isolation and Molecular Detection of Ehrlichia from Vertebrate Animals
    • Unit 3A.4 Transfection of Wolbachia pipientis into Drosophila Embryos
    • Unit 3A.5 Laboratory Maintenance of Rickettsia rickettsii
  • Section B Brucella
    • Unit 3B.1 Laboratory Maintenance of Brucella abortus
  • Section C Bartonella
    • Unit 3C.1 Laboratory Maintenance of Bartonella quintana
  • Section D Rhizobiales
    • Unit 3D.1 Laboratory Maintenance of Agrobacterium
    • Unit 3D.2 Genetic Manipulation of Agrobacterium
    • Unit 3D.3 Phenotypic Analyses of Agrobacterium
    • Unit 3D.4 Isolation and Cultivation of Agrobacterium Species from Natural Sources
• Chapter 4 Beta Proteobacteria
  • Introduction
  • Section A Neisseria gonorrhoeae
    • Unit 4A.1 Laboratory Maintenance of Neisseria gonorrhoeae
    • Unit 4A.2 Genetic Manipulation of Neisseria gonorrhoeae
  • Section B Bordetella
    • Unit 4B.1 Laboratory Maintenance of Bordetella pertussis
• Chapter 5 Enteric Gamma Proteobacteria
  • Introduction
  • Section A Escherichia coli
    • Unit 5A.1 Interaction of Enterohemorrhagic Escherichia coli (EHEC) with Mammalian Cells: Cell Adhesion, Type III Secretion, and Actin Pedestal Formation
    • Unit 5A.2 Rapid Allelic Exchange in Enterohemorrhagic Escherichia coli (EHEC) and Other E. coli Using λ Red Recombination
    • Unit 5A.3 Genotyping Escherichia coli O157:H7 for Its Ability to Cause Disease in Humans
    • Unit 5A.4 Growth and Maintenance of Escherichia coli Laboratory Strains
  • Section B Yersinia
    • Unit 5B.1 Laboratory Maintenance and Characterization of Yersinia pestis
    • Unit 5B.2 Isolation and Confirmation of Yersinia pestis Mutants Exempt from Select Agent Regulations
    • Unit 5B.3 Extraction, Purification, and Identification of Yersiniabactin, the Siderophore of Yersinia pestis
• Chapter 6 Nonenteric Gamma Proteobacteria
  • Introduction
  • Section A Vibrionales
    • Unit 6A.1 Growth and Laboratory Maintenance of Vibrio cholerae
    • Unit 6A.2 Genetic Screens and Biochemical Assays to Characterize Vibrio cholerae O1 Biotypes: Classical and El Tor
    • Unit 6A.3 Vibriocidal Assays to Determine the Antibody Titer of Patient Sera Samples
    • Unit 6A.5 Detection, Isolation, and Identification of Vibrio cholerae from the Environment
  • Section B Moraxella
    • Unit 6B.1 Laboratory Maintenance of Moraxella catarrhalis
  • Section C Coxiella
    • Unit 6C.1 Laboratory Maintenance of Coxiella burnetii
  • Section D Pasturella
    • Unit 6D.1 Laboratory Growth and Maintenance of Haemophilus influenzae
  • Section E Pseudomonas
    • Unit 6E.1 Growth and Laboratory Maintenance of Pseudomonas aeruginosa
• Chapter 7 Delta Proteobacteria
  • Introduction
  • Section B Bdellovibrionales
    • Unit 7B.1 Isolation and Classification of Bdellovibrio and Like Organisms
    • Unit 7B.2 Laboratory Maintenance of Bdellovibrio
• Chapter 8 Epsilon Proteobacteria
  • Introduction
  • Section A Campylobacter
    • Unit 8A.1 Growth and Laboratory Maintenance of Campylobacter jejuni
    • Unit 8A.2 Genetic Manipulation of Campylobacter jejuni
    • Unit 8A.3 Experimental Chick Colonization by Campylobacter jejuni
  • Section B Helicobacter
    • Unit 8B.1 Laboratory Maintenance of Helicobacter Species
• Chapter 9 Firmicutes (Low G+C Gram Positive)
  • Introduction
  • Section A Clostridiae
    • Unit 9A.1 Laboratory Maintenance of Clostridium difficile
    • Unit 9A.2 Genetic Manipulation of Clostridium difficile
  • Section B Listeria
    • Unit 9B.1 Animal Models of Listeria Infection
    • Unit 9B.4 Tissue Culture Cell Assays Used to Analyze Listeria monocytogenes
  • Section C Staphylococcus
    • Unit 9C.1 Growth and Laboratory Maintenance of Staphylococcus aureus
    • Unit 9C.2 Laboratory Maintenance of Methicillin‐Resistant Staphylococcus aureus (MRSA)
    • Unit 9C.4 Tissue Culture Assays Used to Analyze Invasion by Staphylococcus aureus
  • Section D Streptococcus
    • Unit 9D.1 Adult Zebrafish Model of Streptococcal Infection
    • Unit 9D.5 Murine Models of Streptococcus pyogenes Infection
• Chapter 10 Actinobacteria (High G+C Gram Positive)
  • Introduction
  • Section A Mycobacterium tuberculosis
    • Unit 10A.1 Laboratory Maintenance of Mycobacterium tuberculosis
    • Unit 10A.2 Genetic Manipulation of Mycobacterium tuberculosis
    • Unit 10A.3 Isolation and Analysis of Mycobacterium tuberculosis Mycolic Acids
    • Unit 10A.4 Analyses of Mycobacterium tuberculosis Proteins
    • Unit 10A.5 Animal Models of M. tuberculosis Infection
    • Unit 10A.6 Acid‐Fast Staining and Petroff‐Hausser Chamber Counting of Mycobacterial Cells in Liquid Suspension
  • Section B Mycobacterium marinum
    • Unit 10B.1 Laboratory Maintenance of Mycobacterium marinum
    • Unit 10B.2 Zebrafish and Frog Models of Mycobacterium marinum Infection
  • Section C Mycobacterium smegmatis
    • Unit 10C.1 Laboratory Maintenance of Mycobacterium smegmatis
  • Section D Mycobacterium abscessus
    • Unit 10D.1 Laboratory Maintenance of Mycobacterium abscessus
    • Unit 10D.2 Genetic Manipulation of Mycobacterium abscessus
  • Section E Streptomyces
    • Unit 10E.1 Laboratory Maintenance of Streptomyces Species
    • Unit 10E.2 Laboratory Maintenance of Streptomyces argillaceus and Streptomyces griseus
    • Unit 10E.3 Genetic Manipulation of Streptomyces Species
    • Unit 10E.4 Isolation of Streptomyces Species from Soil
• Chapter 11 Chlamydiae
  • Introduction
  • Section A Chlamydia trachomatis
    • Unit 11A.1 Cultivation and Laboratory Maintenance of Chlamydia trachomatis
    • Unit 11A.2 Sphingolipid Trafficking and Purification in Chlamydia trachomatis–Infected Cells
  • Section B Chlamydia pneumoniae
    • Unit 11B.1 Cultivation and Laboratory Maintenance of Chlamydia pneumoniae
• Chapter 12 Spirochetes
  • Introduction
  • Unit 12.1 Analysis of Bacterial Membrane Proteins Produced During Mammalian Infection Using Hydrophobic Antigen Tissue Triton Extraction (HATTREX)
  • Section A Treponema pallidum and Related Treponemes
    • Unit 12A.1 Isolation and Laboratory Maintenance of Treponema pallidum
  • Section B Oral Treponemes
    • Unit 12B.1 Laboratory Maintenance of Treponema denticola
    • Unit 12B.2 Genetic Manipulation of Treponema denticola
  • Section C Lyme Disease Borrelia
    • Unit 12C.1 Laboratory Maintenance of Borrelia burgdorferi
    • Unit 12C.2 Methods of Identifying Membrane Proteins in Spirochetes
    • Unit 12C.3 Cultivation of Borrelia burgdorferi in Dialysis Membrane Chambers in Rat Peritonea
    • Unit 12C.4 Genetic Transformation of Borrelia burgdorferi
  • Section D Brachyspira
    • Unit 12D.1 Isolation and Maintenance of Brachyspira Species
  • Section E Leptospira
    • Unit 12E.1 Laboratory Maintenance of Pathogenic Leptospira
    • Unit 12E.2 Hamster Model of Leptospirosis
    • Unit 12E.3 Rat Model of Chronic Leptospirosis
    • Unit 12E.4 Genetic Manipulation of Leptospira biflexa
• Chapter 13 Other Eubacteria
  • Introduction
  • Section A Fusobacteria
    • Unit 13A.1 Laboratory Maintenance of Fusobacteria
  • Section B Flavobacterium
    • Unit 13B.1 Laboratory Maintenance of Flavobacterium psychrophilum and Flavobacterium columnare
  • Section C Bacteroides
    • Unit 13C.1 Laboratory Maintenance and Cultivation of Bacteroides Species
    • Unit 13C.2 Genetic Manipulation of Porphyromonas gingivalis
• Chapter 14 Animal DNA Viruses
  • Introduction
  • Section A Pox Viruses
    • Unit 14A.1 Myxoma Virus: Propagation, Purification, Quantification, and Storage
    • Unit 14A.2 Production of Myxoma Virus Gateway Entry and Expression Libraries and Validation of Viral Protein Expression
  • Section B Papillomaviruses
    • Unit 14B.1 Cell Culture Assay for Transient Replication of Human and Animal Papillomaviruses
  • Section C Adenoviruses
    • Unit 14C.1 Human Adenoviruses: Propagation, Purification, Quantification, and Storage
  • Section D Adeno‐Associated Viruses
    • Unit 14D.1 Production and Discovery of Novel Recombinant Adeno‐Associated Viral Vectors
    • Unit 14D.2 Gene Transfer in the Lung Using Recombinant Adeno‐Associated Virus
    • Unit 14D.3 Gene Transfer in Skeletal and Cardiac Muscle Using Recombinant Adeno‐Associated Virus
    • Unit 14D.4 Retinal Gene Delivery by rAAV and DNA Electroporation
    • Unit 14D.5 Gene Transfer to the CNS Using Recombinant Adeno‐Associated Virus
    • Unit 14D.6 Gene Transfer in the Liver Using Recombinant Adeno‐Associated Virus
  • Section E Herpesviruses
    • Unit 14E.1 Herpes Simplex Virus: Propagation, Quantification, and Storage
    • Unit 14E.2 Epstein‐Barr Virus (EBV): Infection, Propagation, Quantitation, and Storage
    • Unit 14E.3 Human Cytomegalovirus: Propagation, Quantification, and Storage
    • Unit 14E.4 Human Cytomegalovirus: Bacterial Artificial Chromosome (BAC) Cloning and Genetic Manipulation
• Chapter 15 Animal RNA Viruses
  • Introduction
  • Section A Arenaviruses
    • Unit 15A.1 Lymphocytic Choriomeningitis Virus (LCMV): Propagation, Quantitation, and Storage
  • Section B Alphaviruses
    • Unit 15B.1 Sindbis Virus: Propagation, Quantification, and Storage
  • Section C Reoviruses
    • Unit 15C.1 Mammalian Reoviruses: Propagation, Quantification, and Storage
    • Unit 15C.2 Avian Reoviruses: Propagation, Quantification, and Storage
    • Unit 15C.3 Culturing, Storage, and Quantification of Rotaviruses
    • Unit 15C.4 Bluetongue Virus (BTV): Propagation, Quantification, and Storage
    • Unit 15C.5 Separation and Isolation of BTV dsRNA Segments and Viral Proteins
  • Section D Flaviviruses
    • Unit 15D.1 Hepatitis C Virus: Propagation, Quantification, and Storage
    • Unit 15D.2 Dengue Virus: Isolation, Propagation, Quantification, and Storage
  • Section E Coronaviruses
    • Unit 15E.1 Coronaviruses: Propagation, Quantification, Storage, and Construction of Recombinant Mouse Hepatitis Virus
  • Section F Paramyxoviruses
    • 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)
    • Unit 15F.2 Newcastle Disease Virus: Propagation, Quantification, and Storage
  • Section G Orthomyxoviruses
    • Unit 15G.1 Influenza: Propagation, Quantification, and Storage
    • Unit 15G.2 The Ferret Model for Influenza
    • Unit 15G.3 The Mouse Model for Influenza
    • Unit 15G.4 Insertion of a GFP Reporter Gene in Influenza Virus
  • Section H Picornaviruses
    • Unit 15H.1 Poliovirus: Generation, Quantification, Propagation, Purification, and Storage
    • Unit 15H.2 Poliovirus: Generation and Characterization of Mutants
  • Section J Retroviruses
    • Unit 15J.1 Human Immunodeficiency Viruses: Propagation, Quantification, and Storage
• Chapter 16 Plant RNA Viruses
  • Introduction
  • Section A Plant Propagation for Virus Infection
    • Unit 16A.1 Growth Conditions for Plant Virus–Host Studies
    • Unit 16A.2 Propagation of Major Plant‐Virus Hosts
  • Section B Plant Inoculation Methods
    • Unit 16B.1 Aphid Transmission of Plant Viruses
    • Unit 16B.2 Delivery and Expression of Functional Viral RNA Genomes In Planta by Agroinfiltration
    • Unit 16B.3 Inoculation of Plants Using Bombardment
    • Unit 16B.4 Fungal Transmission of Plant Viruses
    • Unit 16B.5 Testing of Transmission of Tobraviruses by Nematodes
    • Unit 16B.6 Mechanical Inoculation of Plant Viruses
  • Section C Methods of Virus Detection
    • Unit 16C.1 Reverse Transcription‐Polymerase Chain Reaction‐Based Detection of Plant Viruses
  • Section D Preparation and Inoculation of Protoplasts
    • Unit 16D.1 Callus Cultures of Arabidopsis
    • Unit 16D.2 Preparation and Inoculation of Mesophyll Protoplasts from Monocotyledenous and Dicotyledenous Hosts
    • Unit 16D.3 Preparation and Electroporation of Oat Protoplasts from Cell Suspension Culture
    • Unit 16D.4 Transfecting Protoplasts by Electroporation to Study Viroid Replication
  • Section E Methodologies to Extract and Study Viral RNA
    • Unit 16E.1 Extracting Viral RNAs from Plant Protoplasts
    • Unit 16E.3 Northern Analysis of Viral Plus‐ and Minus‐Strand RNAs
  • Section F Cloning Full‐Length RNA Viruses
    • Unit 16F.1 Cloning of Large Positive‐Strand RNA Viruses
    • Unit 16F.3 Rapid Full‐Length Cloning of Nonpolyadenylated RNA Virus Genomes
  • Section G Viroids
    • Unit 16G.1 Identification of Viroids by Gel Electrophoresis
  • Section H RNA Silencing
    • Unit 16H.2 Isolation and Cloning of Small RNAs from Virus‐Infected Plants
  • Section I Viral Vector Methodologies
    • Unit 16I.1 Use of Potato Virus X (PVX)–Based Vectors for Gene Expression and Virus‐Induced Gene Silencing (VIGS)
    • Unit 16I.4 Using Vectors Derived from Tomato Bushy Stunt Virus (TBSV) and TBSV Defective Interfering RNAs (DIs)
    • Unit 16I.5 Using Satellite Tobacco Mosaic Virus Vectors for Gene Silencing
    • Unit 16I.6 Using Viral Vectors to Silence Endogenous Genes
  • Section J Expression of Viruses in Yeast
    • Unit 16J.1 A High‐Throughput Approach for Studying Virus Replication in Yeast
  • Section K Translation of Plant Viral RNA
    • Unit 16K.1 In Vitro Translation of Plant Viral RNA
    • Unit 16K.2 In Vivo Translation Studies of Plant Viral RNAs Using Reporter Genes
• Chapter 17 Anti‐Infectives
  • Introduction
  • Unit 17.1 Characterization of the In Vitro Activity of Novel Lipoglycopeptide Antibiotics
  • Unit 17.2 Structure‐Based Approaches to Antibiotic Drug Discovery
  • Unit 17.3 Novel Approaches to Bacterial Infection Therapy by Interfering with Cell‐to‐Cell Signaling
  • Unit 17.4 Identification of Small‐Molecule Scaffolds for P450 Inhibitors
  • Unit 17.5 Cell‐Based Hepatitis C Virus Infection Fluorescence Resonance Energy Transfer (FRET) Assay for Antiviral Compound Screening
  • Unit 17.6 Laboratory Detection of Vancomycin Nonsusceptible Staphylococcus aureus
  • Unit 17.7 Screening of Hepatitis C Virus Inhibitors Using Genotype 1a HCV Replicon Cell Lines
• Appendix 1 Useful Information and Data
  • Appendix 1A Abbreviations Used in this Manual
  • Appendix 1B Resources for International Biosafety Guidelines and Regulations
• Appendix 2 Commonly Used Reagents and Equipment
  • Appendix 2A Commonly Used Reagents
  • Appendix 2B Standard Laboratory Equipment
• Appendix 3 Commonly Used Techniques
  • Appendix 3A Assays for Total Protein
  • Appendix 3B Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures
  • Appendix 3C Differential Staining of Bacteria: Gram Stain
  • Appendix 3D Direct PCR of Intact Bacteria (Colony PCR)
  • Appendix 3E Preliminary Staining of Bacteria: Simple Stains
  • Appendix 3F Preliminary Staining of Bacteria: Negative Stain
  • Appendix 3G Differential Staining of Bacteria: Flagella Stain
  • Appendix 3H Differential Staining of Bacteria: Acid Fast Stain
  • Appendix 3I Differential Staining of Bacteria: Capsule Stain
  • Appendix 3J Differential Staining of Bacteria: Endospore Stain
  • Appendix 3K Fluorescent Staining of Bacteria: Viability and Antibody Labeling
  • Appendix 3L Generation of Transformation Competent E. coli
  • Appendix 3M SDS‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE) of Proteins
• Appendix 4 Commonly Used Methods for Cell Culture
  • Appendix 4A Common Bacterial Culture Techniques and Media
  • Appendix 4B Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVEC)
  • Appendix 4C Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
  • Appendix 4D Aseptic Technique
  • Appendix 4E Growth and Maintenance of Vero Cell Lines
  • Appendix 4F Anaerobic Cell Culture
  • Appendix 4G Growth and Maintenance of Quail Fibrosarcoma QM5 Cells
  • Appendix 4H Growth and Maintenance of Baby Hamster Kidney (BHK) Cells
  • Appendix 4I Growth and Maintenance of Chick Embryo Fibroblasts (CEF)
  • Appendix 4J Growth and Maintenance of Mosquito Cell Lines

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