Showing papers in "Current protocols in microbiology in 2006"

Influenza: Propagation, Quantification, and Storage

Abstract: Influenza viruses are negative-sense, single-stranded, enveloped RNA viruses belonging to the family Orthomyxoviridae. Three types exist, influenza A, B, and C. All infect humans, but only A and B are major human pathogens. Influenza type A viruses are divided into subtypes based on genetic and antigenic differences in the two surface spike proteins, hemagglutinin (HA) and neuraminidase (NA). The appropriate cell lines to be used for isolation of influenza A or B viruses depend on the clinical information and the host of origin. MDCK cells are the preferred cell line for isolation of human influenza viruses from clinical specimens.

Growing and analyzing biofilms in flow cells.

Abstract: The setup of flow cell systems for the study of microbial biofilms and methods for the analysis of structural biofilm development are described in this unit. Use of flow cells allows direct microscopic investigation of biofilm development. The biofilms in flow cells develop under hydrodynamic conditions, and the environment can be carefully controlled and easily changed. The protocols in this unit include construction of the flow cell and the bubble trap, assembly and sterilization of the flow cell system, inoculation of the flow cells, running of the system, image capture and analysis, and disassembly and cleaning of the system. In addition, embedding and fluorescent in situ hybridization of flow cell-grown biofilms are addressed.

Hamster model of leptospirosis.

Abstract: Animal inoculation remains essential for many aspects of leptospirosis research. Although pathogenic leptospiral strains may be able to infect a wide variety of animals, the Golden Syrian hamster is the preferred model because its susceptibility to infection and the reproducibility of the results. Common applications include determination of strain infectivity, restoring virulence to culture-attenuated strains, assessing usefulness of potential vaccines or diagnostic antigens, and examining pathology of mammalian infection. In many respects, acute leptospirosis in the hamster reproduces the severe form of human leptospirosis (see Commentary). This unit provides a detailed description of the procedures involved in the hamster model of leptospirosis, including housing and handling of hamsters and intraperitoneal challenge (Basic Protocol 1), and monitoring response to challenge (Basic Protocols 2 and 3, and Alternate Protocol). Methods are provided for demonstrating infection, including culture isolation of leptospires from blood and tissues (Basic Protocol 2) and serologic studies and histopathology (Basic Protocol 3). Quantitative PCR is provided as an alternative detection method (Alternate Protocol). Although leptospires disseminate to many organs, the tissues of the liver and kidney are the primary targets of infection. The liver is heavily infected during the initial stage of hematogenous dissemination. Persistent infection occurs primarily in the kidneys of animals that survive acute disease. Infection of the renal tubules leads to shedding of infectious organisms in the urine, which is the primary mode of transmission in nature. A safety policy memorandum is provided (see Strategic Planning) that should be read and signed by all staff. In the Commentary section, a detailed rationale is presented for use of hamsters as an animal model of leptospirosis. The effects of challenge dose on the hamster model, LD50, in vitro passage of leptospiral strains, and immunological maturity are discussed. Alternative animal models are evaluated with reference to the concept of accidental versus reservoir hosts. The advantages and disadvantages of the hamster model are considered, with reference to use of alternative host animals to address specific research or vaccine-development issues. CAUTION: Pathogenic Leptospira species are Biosafety Level 2 (BSL-2) pathogens. L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii are known to be pathogenic for humans. Certain strains of L. inadai, L. meyeri, L. fainei, and L. alexanderi may also be pathogenic. L. biflexa, L. wolbachii, and L. parva are thought to be nonpathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. CAUTION: Protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals. This experiment requires Animal Biosafety Level 2 (ABSL-2) conditions. Follow all appropriate guidelines for the use and handling of infected animals. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. Fluids and tissues of infected animals are highly infectious. Numerous laboratory-associated infections, including deaths, have been reported (Miller et al., 1987; Pike, 1976). A safety policy memorandum is provided that should be signed by all staff (see Strategic Planning).

Zebrafish and frog models of Mycobacterium marinum infection.

Abstract: Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium-host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.

UNIT 4A.2 Genetic Manipulation of Neisseria gonorrhoeae

Abstract: The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and to the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Techniques are described for genetic manipulation of N. gonorrhoeae, as well as for growth of this fastidious organism. Curr. Protoc. Microbiol. 23:4A.2.1-4A.2.24. © 2011 by John Wiley & Sons, Inc. Keywords: Neisseria gonorrhoeae; natural transformation; electroporation; complementation; genetic transformation; heterologous gene expression

Newcastle Disease Virus: Propagation, Quantification, and Storage

Abstract: Newcastle disease virus (NDV) is a prototype paramyxovirus used to define basic steps in the life cycle of this family of viruses. NDV is also an ideal virus system for elucidating determinants of viral pathogenicity. Some strains of this virus are important agricultural pathogens that cause disease in poultry with a high mortality while other strains are avirulent and used for vaccines. Methods for preparation and titration of virus stocks are essential for all of these purposes. Procedures for growth and purification of NDV stocks in embryonated chicken eggs as well as in tissue culture cells are described. Use of embryonated chicken eggs to grow the virus is the superior method since infectious stocks of all strains of NDV result. Stocks of avirulent NDV prepared in tissue culture are noninfectious. Virus stocks are routinely titered using plaque assays or hemagglutination assays, both of which are described.

Delivery and Expression of Functional Viral RNA Genomes In Planta by Agroinfiltration

Abstract: Agroinfiltration is a simple, efficient, and powerful approach for transient expression of viral genes as well as DNA-based expression of full-length RNA genomes of plant viruses for studies leading to understanding of replication, movement, and assembly. Most importantly, it results in synchronous delivery of Agrobacterium transformants to a majority of cells encompassing the infiltrated area and is therefore ideal for examining the biological activities of viruses having multipartite genomes. Because of the high transformation rate and efficient accumulation of mRNAs, the method is also ideal for analyzing biological activities of viral genomes with defective replication and cell-to-cell movement characteristics. Keywords: Agrobacterium tumefaciens; agroinfiltration; transient expression; expression of functional viral genes; Tiplasmid; viral genomes; RNA

Transmission electron microscopy.

Abstract: Transmission electron microscopy has long been an important analytical tool in the field of microbiology. This unit describes preparation techniques for examining particulate samples as well as samples presenting more complex ultrastructural considerations that require analysis in thin sections. Negative staining is a useful technique for routine examination of particulate samples in suspension ranging from bacteria to purified macromolecules. In order to investigate the relationships between microbes and the environments with which they interface, fixed samples can be prepared for imaging in sections of 60- to 90-nm thickness. Due to the many steps in sample preparation for ultrastructural analysis of thin-sectioned samples, the major steps in the process are divided into fixation and initial processing of samples for thin sectioning, the embedment of samples into a plastic resin for sectioning, ultramicrotomy, and staining of samples. Procedures for immunolocalization of antigens in negatively stained and thin-sectioned preparations are also considered.

Human adenoviruses: propagation, purification, quantification, and storage.

Abstract: Detailed protocols are described for the propagation of adenoviruses (Ads) and adenovirus (Ad) vectors and their purification by CsCl equilibrium density gradient centrifugation. A discussion of monolayer and spinner cell culture techniques suitable, respectively, for small- and large-scale growth of adenoviruses is provided. Protocols for cloning into and growth of Ad replication-deficient vectors using a convenient commercially available system are described. Lastly, time-tested plaque titration protocols for the accurate and convenient measurement of the infectivity of adenoviruses and adenovirus vectors are provided in detail.

Isolation and Cloning of Small RNAs from Virus‐Infected Plants

Abstract: RNA silencing is an evolutionarily conserved, RNA-mediated, regulatory system of eukaryotic organisms that also acts as an antiviral system in plants and animals. A defining feature of RNA silencing is the presence of 21- to 26-nucleotide small RNAs corresponding to the silencing target sequence, which in virus-infected plants are derived from the viral genome. The virus-derived small RNAs have a nonrandom distribution along the viral genome, suggesting hotspots for viral small-RNA generation. The isolated small RNAs can be used either as probes for hybridization studies or for directional cloning in order to get detailed information about their sizes, origins, and functions. This unit describes an isotope-free small-RNA cloning procedure that utilizes unmodified small RNAs and is routinely used to characterize small RNAs from various plant tissues.

LacZ-based detection of acyl-homoserine lactone quorum-sensing signals.

Abstract: Many bacteria use quorum-sensing regulatory systems to monitor their population density and to coordinate their genetic expression under certain conditions. Acyl-homoserine lactones are often produced by many Gram-negative bacteria and serve as quorum-sensing signal molecules. This unit describes two commonly used methods to detect acyl-homoserine lactones from laboratory or environmental bacterial cultures. Keywords: quorum sensing; acyl-homoserine lactone; LacZ reporter; Agrobacterium tumefaciens

Using Viral Vectors to Silence Endogenous Genes

Abstract: Virus-induced gene silencing (VIGS) is a fast but transient method for knocking down expression of endogenous genes in plants. Replicating plant viruses activate a defense mechanism called post-transcriptional gene silencing (PTGS), which protects the plant by silencing viral transcripts. VIGS of endogenous genes is accomplished by inserting a gene of interest into a viral vector. When the virus replicates in the plant, PTGS silences both the viral genome and the corresponding endogenous gene. The most robust and widely implemented VIGS system uses tobacco rattle virus (TRV) vectors and N. benthamiana as the plant host. This unit will explain how to introduce TRV-based VIGS vectors into N. benthamiana plants by two methods: syringe infiltration or the Agrobacterium drench method. Furthermore, it will provide two alternate protocols optimized for VIGS in tomato plants: spray inoculation and vacuum infiltration.

Common Bacterial Culture Techniques and Media

Abstract: This appendix provides general techniques, equipment, and media used for the growth of many commonly encountered bacteria. For specific growth conditions, readers should refer to units regarding the laboratory maintenance of the organism of interest. Keywords: bacterial medium; bacterial culture techniques; bacterial culture equipment

Murine Models of Streptococcus pyogenes Infection

Abstract: This unit describes procedures for testing virulence of Streptococcus pyogenes in mice. S. pyogenes is an important human pathogen and causes one of the most common childhood diseases. The syndromes that result from S. pyogenes infection are diverse, ranging from mild, superficial throat or skin infection to severe, invasive disea/se that is often lethal. Thus, a greater understanding of the virulence factors of this bacterium and development of modalities to prevent or relieve the infections it causes are important. Since S. pyogenes is a strictly human pathogen (with the exception of a single strain), the value of all animal models is limited. This unit describes a model for long-term throat colonization following the natural route of infection (inhalation), one for pneumonia and systemic dissemination following intratracheal inoculation, and one for systemic dissemination following the more natural route of skin infection. In addition, methods are presented for culturing S. pyogenes from tissues of the infected animal.

Callus Cultures of Arabidopsis

Abstract: Protoplasts are plant cells lacking cell walls. They can be generated from stationary callus cultures derived from Arabidopsis thaliana seedlings. After treatment of the callus with cellulase and pectinase, protoplasts are inoculated with viral RNAs using polyethylene glycol. After various times postinoculation, total RNA is extracted and subjected to electrophoresis on nondenaturing agarose gels for further analysis. Stationary callus cultures are simpler to maintain than more traditional suspension cultures and yield high-quality, uniform protoplasts. Protoplasts prepared in this fashion can also be used for uptake of DNA.

Tissue culture cell assays used to analyze Listeria monocytogenes.

Abstract: This unit describes tissue culture cell assays for analysis of the ability of Listeria monocytogenes to cause intracellular infection. It includes methods for evaluating the organism's ability to invade its host, to escape the primary vacuole formed upon invasion of host cells, to multiply within the cytosol of its host, and to spread from cell to cell without exiting the intracellular milieu. Each step can be evaluated quantitatively and qualitatively. Keywords: invasion; escape from vacuole; intracellular growth; cell-to-cell spread; Listeria

Laboratory Maintenance of Fusobacteria

Abstract: This unit describes routine laboratory handling of fusobacteria. Different media that can be used to grow or enrich Fusobacterium nucleatum and other species of this genus are described. The growth and stock conditions as well as the susceptibility of F. nucleatum to oxygen in a pure culture are also discussed.

UNIT 6A.5 Detection, Isolation, and Identification of Vibrio cholerae from the Environment

Abstract: Recent molecular advances in microbiology have greatly improved the detection of bacterial pathogens in the environment. These improvements and a downward trend in the cost of molecular detection methods have contributed to increased frequency of detection of pathogenic microorganisms where traditional culture-based detection methods have failed. Culture methods also have been greatly improved, and the confluence of the two suites of methods provides a powerful tool for detection, isolation, and characterization of pathogens. While molecular detection provides data on the presence and type of pathogens, culturing methods allow a researcher to preserve the organism of interest for “-omics” studies, such as genomic, metabolomic, secretomic, and transcriptomic analysis, which are rapidly becoming more affordable. This has yielded a clearer understanding of the ecology and epidemiology of microorganisms that cause disease. In this unit, we present commonly accepted methods for isolation, detection, and characterization of V. cholerae, providing more extensive knowledge of the ecology and epidemiology of this organism. This unit has been fully revised and updated from the earlier version with the latest knowledge and additional information not previously included. Curr. Protoc. Microbiol. 26:6A.5.1-6A.5.51. © 2012 by John Wiley & Sons, Inc. Keywords: Vibrio cholera; isolation; identification; detection; characterization

UNIT 8B.1 Laboratory Maintenance of Helicobacter Species

Abstract: Helicobacter species are Gram-negative bacteria that colonize the gastric or intestinal mucosa of many mammalian and avian hosts and induce histologic inflammation. The association of H. pylori with gastritis, peptic ulcer disease, and gastric cancers makes it a significant human pathogen. Animal models for these diseases are being used to explore the pathogenesis of H. pylori infection and in vaccine development. Both bacterial and host factors contribute to Helicobacter pathogenesis; therefore, the microbiology is very important. This unit describes how to culture the most commonly used gastric Helicobacter species, H. pylori, H. mustelae, and H. felis. Curr. Protoc. Microbiol. 24:8B.1.1-8B.1.19. © 2012 by John Wiley & Sons, Inc. Keywords: Helicobacter; growth curve; morphological identification; blood agar preparation; pinch biopsy culture methods

Inoculation of plants using bombardment.

Abstract: This unit describes methods for the construction and use of handheld particle bombardment devices for high-efficiency inoculation of intact plants with nucleic acids and viruses. The devices accelerate heavy metal particles coated with nucleic acids or viruses into plant tissues and are driven by pressurized gas. They are inexpensive to construct and use, and can be assembled in any laboratory. The equipment enables inoculation with full-length infectious cDNA, PCR products, virus from sap or a virus preparation, and in vitro viral transcripts. The inoculation of some phloem-limited RNA viruses is also possible. Additionally, this technology allows for inoculation of large numbers of plants (mass bombardment), inoculation of soft plants that do not survive bombardment inoculation by other means, inoculation in the greenhouse, study of viral recombination in plants, rapid promoter analysis, and monitoring of virus movement using an infectious clone bearing a reporter gene. Keywords: particle bombardment; gene gun; inoculation of soft-tissue plants; gold powder; tungsten powder; RNA viruses

In vitro translation of plant viral RNA.

Abstract: This unit describes the preparation of a wheat germ extract that provides all the soluble components of the plant translational machinery Many RNA plant viruses have positive-strand genomes and the viral RNA serves as messenger RNA (mRNA) The preparation of mRNA by in vitro transcription is also described The translation assay requires optimization of the amount of wheat germ extract, level of mRNA, and the concentration of Mg(2+) and K(+) for each mRNA The translational efficiency of RNAs or mutants may be compared (eg, capped versus uncapped RNAs to measure cap-independent translation) or the amount/size of the protein product may be determined

Human Immunodeficiency Viruses: Propagation, Quantification, and Storage

Abstract: Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long-term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting.

Rapid allelic exchange in enterohemorrhagic Escherichia coli (EHEC) and other E. coli using lambda red recombination.

Abstract: This unit describes an allelic exchange system for enterohemorrhagic E. coli (EHEC), and similar pathogenic species of bacteria. The phage lambda Red recombination system is expressed from a plasmid, inducing a hyper-recombinogenic state where electroporated PCR-generated substrates recombine with the bacterial chromosome at high efficiency. The technique can be used to substitute a drug marker for the gene of interest, or used to generate a clean in-frame deletion of the target gene. Single gene knockouts in EHEC, or deletions of whole pathogenicity islands, can be constructed. A procedure for the preparation of hyper-recombinogenic electrocompetent cells is also described. Besides E. coli K-12 and EHEC, this method has also been used for the construction of gene knockouts in enteropathogenic E. coli (EPEC), enteroaggregative E. coli, and uropathogenic E. coli, as well as Shigella flexneri and Salmonella enterica.

Basic confocal microscopy.

Abstract: This unit introduces the reader to the basic principles of confocal microscopy and the design and capabilities of current confocal microscopes. The advantages and disadvantages of confocal microscopy as compared to other techniques for fluorescence imaging are described. There are also practical guidelines for sample preparation and optimizing imaging parameters and examples of some of the applications of confocal microscopy.

UNIT 15J.1 Human Immunodeficiency Viruses: Propagation, Quantification, and Storage

Abstract: Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long-term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting. Curr. Protoc. Microbiol. 28:15J.1.1-15J.1.24. © 2013 by John Wiley & Sons, Inc. Keywords: HIV-1; HIV-2; retroviruses; primary isolates; T cell line–adapted (TCLA) viruses; molecular clones; reporter viruses; focus-forming unit; radioactive reverse transcriptase assay; p24 ELISA

Biosafety Practices Associated with Potential Agents of Biocrime and Biowarfare

Abstract: Conducting research in a manner that guards against theft and intentional misuse of biological materials requires a process of hazard identification and risk assessment to most effectively identify and implement a risk management plan. Procedures describing physical security, personnel reliability, and material control and accountability define a security plan for this type of research.

UNIT 1A.4 Safe Use of Radioisotopes

Abstract: The use of radioisotopes to label specific molecules in a defined way has greatly furthered the discovery and dissection of biochemical pathways. The development of methods to synthesize such tagged biological compounds inexpensively on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells) require amounts on the order of millicuries. In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the scientist. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses handling, storage, and disposal of the isotopes most frequently used in biological research. Keywords: safety; radioisotopes; metabolic labeling; in vitro kinase reactions; Plexiglas shielding