Methods in Microbiology

This chapter discusses a methodology employed for two-dimensional (2-D) crystallization of a soluble protein. The optimization procedure should include the search for—(1) a reliable specimen preparation method to obtain good imaging conditions in the transmission electron microscope (TEM); (2) a suitable heterogeneous nucleation surface (HNS) and precipitant; (3) the optimum initial protein concentration and the precipitant concentration; and (4) the optimum incubation time at possibly ≥25° C. An appropriate precipitant can often be taken from three-dimensional (3-D) crystallization recipes, if available, or it has to be discovered by trial and error. In 3-D crystallization, the protein to be crystallized is the most important prerequisite and should always be as homogeneous as possible to avoid any unspecific events that per se reduce reproducibility. The crystallization method is designed to mediate between the protein solution to be crystallized and the crystallization conditions such as incubation time, incubation temperature, pH value, ionic milieu, and type and concentration of the precipitant.

This chapter focuses on various methods for studying fungi in soil and forest litter. Now, with the focus on nutrient cycling by fungi in whole ecosystems, bulky dead wood is also treated as an important constituent of a forest floor. The current upsurge in mycorrhizal research has also generated new approaches to integrated studies of fungi in soil, litter, and roots. Whatever the shortcomings of old and new methods of studying soil fungi, investigators' claims have tended to become more reliable with wider recognition that the use of sound statistical techniques when designing experiments and handling numerical data is essential. Statistical analyses are not discussed in this chapter, but some useful references are mentioned. The methods considered in the chapter have been divided into four broad categories: observation, isolation, quantification, and measurement of activity identification are also discussed in this chapter.

This chapter discusses the isolation and preparation of lymphocytes from infected animals for in vitro analysis. Lymphocytes are the cells that provide specificity to host defense. Identifying the phenotype and antigen specificity of lymphocytes that have been isolated from animals infected with microbial agents is integral to understand protective adaptive immunity. Lymphocytes can be obtained from a variety of the tissues of infected animals. The numbers of cells that can be recovered from each site vary depending on the type of tissue and the age and physiological status of animals. There are various methods to isolate, purify, and characterize lymphocytes from the tissues of infected animals. Some of these are elegant and sophisticated procedures that rely on expensive instrumentation to yield highly purified and well-characterized cell populations. The chapter focuses largely on simple preparative techniques that can be used by nearly any microbiology laboratory. These techniques will yield populations of lymphocytes suitable for functional assessment in vitro or adoptive transfer to recipient animals in vivo. The chapter discusses the use of adherence to remove mononuclear phagocytes and the use of nylon wool to enrich for T lymphocytes.

This chapter describes the most relevant practical approaches for the identification and characterization of the N-acylhomoserine lactones (AHL) family of quorum sensing signal molecules. The first indication of AHL-mediated gene regulation in a Gram-negative bacterial pathogen is the appearance of a specific phenotype in a cell density dependent fashion. This may be further evidenced by an earlier manifestation of that phenotype, when the organism is grown in the presence of some of its own filtered spent culture supernatant as a potential source of AHLs or indeed other chemically distinct signal molecules. The development of sensitive bioassays for the detection of AHLs has greatly facilitated the screening of micro-organisms for new AHL molecules. N-Acyl-L-homoserine lactones can be prepared by a carbodiimide-mediated acylation of L-homoserine lactone hydrochloride. A typical procedure is explained in the chapter.

Phage typing is one of the standard methods for epidemiological typing. For Pseudomonas aeruginosa, various phage typing procedures have been developed. These phage typing sets have mostly been developed locally. This chapter discusses basic mechanisms involved for phage–host-interaction, and methods used for phage typing of P. aeruginosa. Phages of P. aeruginosa may be isolated from sewage or from lysogenic isolates. Such strains are fairly common. The nucleic acid of the bacteriophages of P. aeruginosa may contain either DNA or RNA. The former are best known. They contain double-stranded DNA. The mole % (G + C) ratio of such phages has been reported as falling in the range 46–55%. The DNA-phages resemble the T-even phages of Escherichia coli in having a head and tail. Most have a morphology corresponding to group A of Bradley and Kay, but group B phage morphology has also been encountered. The tail lengths vary; upon contact with specific bacterial receptors, the sheath of the tails contracts.

This chapter discusses the pathogenic species of Clostridium associated with gas gangrene, botulism, tetanus, and food poisoning. The clostridia are usually Gram-positive and spore-forming catalase-negative anaerobic bacteria. Some species are much more aerotolerant than others, but anaerobic techniques must be applied to assure the isolation of any member of the genus. Clostridium perfringens is often reluctant to produce spores and is non-motile. Some clostridia may only be specifically identified by toxin neutralisation tests—for example, Clostridium botulism and Clostridium tetani. Newer analytical tools—such as gas–liquid chromatography—have enhanced the identification of many anaerobic bacteria by detecting specific organic acids produced by the fermentation of defined carbohydrates. Similarly, toxin typing of clostridia may be of importance in species identification but is less useful for strain differentiation. The serology of other species of Clostridium has been insufficiently developed to permit more than crude typing of organisms.

Cholera is a disease of bacterial origin. It is not only important to recognize the disease clinically, but also to attempt as complete an identification of the causative organism as possible using various epidemiological typing procedures. This chapter explains the practical procedures currently available for the characterization, as well as a working background of the relevant theoretical knowledge. V. cholera is asporogenous, single curved or rigid rod with a single polar flagellum. It is indophenols oxidase positive and produces acid without gas from glucose. The aim of all typing systems is to identify strains within a species with a degree of precision that makes it safe to assume that all the isolates from an epidemic are truly identical and have therefore originated from a single parent strain. Of the three methods of typing V. cholera—serotyping, phage-typing and bacteriocin biotyping—serological typing was the earliest to be developed.

Visible and near-visible light (200-750 nanometers) profoundly influence many aspects of the growth, development, reproduction, and behavior of fungi. In nature, most fungi are exposed during a portion of their life cycle to a little far-ultraviolet radiation (mainly 290-300 nm), and to considerable near-ultraviolet (300-380 nm) and visible (380-750 nm) radiation. Many effects of light on fungi have been reported by numerous investigators. The chapter presents general practical guide for those interested in the effects of light on fungi. Each light-induced phenomenon is described briefly with emphasis on the responsible wavelengths where these are known. New evidence indicates that visible light may be able to induce mutations in some microorganisms. The effect of far-UV on the survival of fungi is dependent on many factors. Spores for example, are much more resistant to the lethal effects of UV than vegetative mycelium, sensitivity of spores is frequently related to color and pigmented spores often survive longer exposures to far-UV than colorless spores; in addition thickness of spore wall may also be involved in the ability of fungi to tolerate radiation. Age can also influence sensitivity of spores and old spores of Aspergillus melleus are more tolerant to far-UV than young spores. The lethal effects of far-UV have many applications in science, industry, and medicine. The low-pressure mercury vapor lamp or germicidal lamp is an inexpensive, efficient, and widely used source of fungicidal UV.

The products of a fermentation process may be few, as in the case of the classical yeast fermentation, or they may be tremendously diverse, which is typical of much bacterial fermentation. Therefore, the analysis of fermentation presents tasks ranging in magnitude from the relatively simple to the highly complex, involving the separation, identification, and quantitation of a broad spectrum of compounds. The development of column, paper, and thin-layer chromatography has had a major impact not only on the separation and identification of fermentation products but also on their quantitative determination. This chapter discusses the analysis of fermentation products, the general principles of fermentation balances, and the apparatus and techniques for carrying out fermentation studies. The chapter also discusses the procedures involved in the separation, identification and quantitative analysis of the various compounds likely to be encountered in fermentation processes. The identification and determination of volatile fatty acids by gas-liquid chromatography is also described.

The identification of microorganisms using conventional procedures is a highly developed routine employing a wide range of morphological, serological, nutritional and biochemical tests. The application of Py-MS to microbiology remains underdeveloped and ill-defined. The advent of low-cost instrumentation should increase the access of microbiologists to the method. Perhaps the most significant obstacles to the more widespread use of Py- MS are the lack of understanding about what pyrolysis mass spectra represent and why they can be used to differentiate organisms. Detailed studies of microbial pyrolysis products by Py-GC-MS techniques are required to identify the cell constituents that contribute to the spectra and hence the differentiation of microorganisms. In the future, Py-MS should be used in tandem with other chemotaxonomic techniques, so that an improved understanding of the chemical basis of differences between spectra can be acquired.

This chapter discusses the classification of Enterobacteriaceae and demonstrates the degree of phenetic relationship between the major taxa as assessed by a collection of clinical isolates. These organisms grow readily, are among the most common isolates studied in medical bacteriology, and cause a range of serious infections (urinary tract infections, wound infections, and septicemia), including epidemic diseases. Escherichia coli and Salmonella species are important tools for the geneticist, molecular biologist, cloner, and biochemist. Computer taxonomy and identification combined with DNA relationships have completely changed the taxonomy. The high phenetic similarity between the species of enterobacteria is related to their wide occurrence and to the fact that they exhibit diverse metabolic activity. In many published studies, one or rather few reference DNAs have been taken to represent one species. Heterogenicity may require more labelled DNAs in taxonomic studies employing DNA hybridization. This would improve on the representativeness of the labelled DNAs. A numerical grouping method, such as principal components analysis, employed to sort out the large body of quantitative data efficiently would serve as a useful tool for such identification schemes. Further phenetic and genetic analysis of this type is being carried out on strains from the typical clinical laboratory and from less traditional sources.

This chapter discusses the principles and procedures for serotyping of Escherichia coli (E. coli) and discusses the morphology and immunochemistry of the surface structures that are important in serotyping. The genus Escherichia is one of the genera of tribus Escherichieae as defined in Bergey's Manual of Determinative Bacteriology. It contains one species E. coli, which consists of Gram-negative, peritrichously flagellated rods that conform to the family Enterobacteriaceae. E. coli can be differentiated from other genera in the tribus Escherichieae by indole production, the fermentation of lactose, negative reactions in KCN, gelatin, and malonate tests. E. coli is methyl red positive, Voges–Proskauer negative, and urease negative. E. coli group is heterogeneous and contains a very high number of stable subtypes. Therefore, it will be a difficult task to command all available methods in a single laboratory, if a complete typing of strains is needed.

Publisher Summary This chapter discusses the analysis of the chemical composition and primary structure of the murein. Murein (peptidoglycan, mucopeptide) is the main cell wall polymer of eubacteria and is common to both Gram-negative and Gram-positive bacteria. There are only a few prokaryotic organisms, such as mycoplasmas and archaebacteria, which lack murein. The glycan moiety is rather uniform and shows only a few variations such as O-acetylation or O-phosphorylation or the exceptional absence of peptide substituents. The peptide moiety of the murein shows, in contrast to the glycan part, a considerable variation. Extensive investigation of the chemical structure of murein has demonstrated the existence of almost 100 different variations of the peptide moiety. Depending on the mode of cross-linking two main groups of murein, named A and B, have been distinguished. The different structure of cell walls of Gram-positive and Gram-negative bacteria necessitates discrete methods for preparing cell walls. The cell walls of Gram-positive bacteria reveal in profile one thick and more or less homogeneous layer, whereas Gram-negative bacteria have thinner, but distinctly layered cell walls with an outer membrane resembling the cytoplasmic membrane in profile.

This chapter describes some microscopical approaches for analyzing the structure of endomycorrhizal infections, identifying modifications in protein expression (enzyme activities) and localizing specific molecules at both the tissue and cellular level. Techniques are chosen to illustrate how they can not only increase the understanding of how endomycorrhizal symbionts interact with each other, but also furnish potential tools for diagnosing the functional state of the symbiosis. Non-destructive observations can be made of vesicular-arbuscular endomycorrhizal infections in root pieces using ultraviolet light. Although this can be useful for selecting materials for biochemical analysis or electron microscope preparation, the technique is limited to young root tissues in which only living arbuscules can usually be detected. The chapter illustrates cytological, histochemical, and immunocytochemical techniques. It provides powerful tools for studying the structure and function of symbionts and their interactions in endomycorrhiza. Knowledge of the physical and chemical nature of cellular and subcellular structures permitting functional compatability in endomycorrhizal associations has been considerably improved and subcellular localization of enzyme activities has given some insight into their physiological significance for the symbiotic condition. Furthermore, by combining immunolocalization with ultra cytoenzymology it is possible to understand how the synthesis and the expression of the molecules involved are regulated.

Electron microscopes (EM) have been routinely used to study plasmid molecular biology since the development of a suitable preparation technique for DNA molecules by the introduction of spreading techniques using basic protein monolayers. There are several reasons for their widespread use in plasmid research: (1) most EM techniques are relatively quick in comparison to other molecular biological methods, (2) their accuracy is only surpassed by DNA sequencing, and (3) one can actually see what happens to the DNA molecules under investigation. Therefore, EM always gives information about single individual molecules, in contrast to DNA gel techniques. This chapter focuses on molecular weight determination, and homo- and heteroduplexing because these methods are based on the same preparation technique for DNA visualization, are relatively simple and can be performed in every EM laboratory without the need for complicated and expensive apparatus and chemicals.

The major difficulty encountered when analyzing plasmid or phage encoded mRNA and polypeptides in whole (normal) bacterial cells is that the majority of these products are masked by those encoded by the host cell's chromosome. This difficulty often remains even when the genes of interest have been amplified by cloning into multicopy vectors. In relatively few cases does a very high rate of expression or the availability of a specific assay (e.g. zymogram staining or specific antisera) allow the experimenter to distinguish between the plasmid and chromosome-encoded products. It is usually necessary to label specifically the plasmid-encoded products in the absence of significant expression from the host cell's chromosome. In recent years, a number of techniques have been developed to achieve this. These include the use of bacterial minicells, the maxicell system, cell-free (in vitro) synthesis, selective expression from ColEl-type plasm ids after prolonged chloramphenicol treatemetn. These techniques have been designed to maximize expression from plasmid DNA while minimizing the level of background expression from the host chromosome or, in the case of the in vitro system, from mis-transcription or mis-translation.

The chapter discusses extremophile micro-organisms respect to life at high temperatures, pressures, extremes of pH, salt concentration, and ionizing radiation. It also discusses their behavioral mechanism. Many of these extremophiles cannot live under the more moderate conditions preferred by most living organisms. The various extremophiles discussed are thermophiles, psychrophiles, acidophiles, alkaliphiles, halophiles, barophiles/piezophiles, and radiation resistant micro-organisms. Many of the thermophiles are associated with volcanic activity. Hot springs, geysers, volcanoes, and deep-sea hydrothermal vents are popular hunting grounds for the search for new types of thermophilic micro-organisms. Psychrophiles are generally defined as such organisms that have their optimum temperature below 15–20oC. Acidic environments are often associated with volcanic activity: hot sulfur springs, mud pots, etc. Another type of low pH environments is that caused by microbial activity. Acidophiles keep their cytoplasm at near-neutral pH values by means of powerful proton pumps in their cytoplasmic membrane, which maintains a proton concentration gradient of four, five, or even more orders of magnitude. Alkaliphilic micro-organisms are widespread in nature. Bacteria (e.g. species of the genus Bacillus) that grow at pH 9–10, while being unable to grow at neutral pH can easily be isolated from soils. Hypersaline environments are easily formed when seawater dries up in coastal lagoons and salt marshes, as well as in manmade evaporation ponds of saltern systems built to produce common salt by evaporation of seawater. Micro-organisms found on terrestrial surfaces, air-borne micro-organisms, and microbes living in the upper layers of the sea and other aquatic environments are exposed to direct sunlight, including a significant amount of potentially harmful ultraviolet radiation. Such organisms have to protect themselves against radiation damage. Protection mechanisms include repair mechanisms for damaged deoxyribo nucleic acid (DNA).

There is little known about bacterial virulence factors except that they are expressed in and required for growth in the host. The focus of this chapter is to identify virulence factors via their expression with emphasis on Salmonella. Regulation of expression of a given virulence factor may take place at several levels-transcription, post-transcription, translation or post-translation. Most often one thinks of transcriptional regulation of virulence factors. There are few examples of regulation at later steps probably because they have not been studied. Almost all analysis of virulence rests on the availability of a good animal model without which virulence factors are identified solely by analogy with genes from other pathogens with no method of verification. An organism that is highly refractory to transformation, such as Chlamydia, is almost as perverse as not having an animal model. Manipulations must be carried out in E. coli or other organism that are easy to manipulate and the genes re-introduced only as a last resort.

This chapter presents a variety of techniques that are currently employed in the analyses of different aspects of plasmid replication in vivo. The chapter describes methods that are primarily useful for the measurement of plasmid copy number and for the determination of the rate of plasmid replication. These two types of measurement allow the characterization of plasmid replication under a variety of experimental conditions. The chapter focuses on DNA–DNA hybridization methods because although are not widely used, they are yet straightforward. In principal, they are more accurate than other techniques because they do not require prior separation of bacterial and plasmid DNA. Later methods that can be used to cure plasmids, and to measure rates of plasmid segregation are described.

Publisher Summary This chapter discusses analysis of isoprenoid quinones. The respiratory quinones represent an important group of isoprenoid lipids that occur in the cytoplasmic membrane of most prokaryotes. Two major structural groups of bacterial isoprenoid quinones can be recognized: the naphthoquinones and benzoquinones. Naphthoquinones can be divided further into two major types: phylloquinones and menaquinones. In choosing the methods to be employed for the extraction and purification of menaquinones, ubiquinones, and related quinones one must take into account the susceptibility of these compounds to degradation. Isoprenoid quinones are quite rapidly photo-oxidized in the presence of oxygen and strong light. They are also particularly susceptible to alkaline conditions (the last limitation rules out alkaline saponification). Thus, it is preferable to conduct extraction and subsequent purification procedures fairly rapidly, avoiding extremes of pH and strong light. The composition of natural mixtures of bacterial quinones can be investigated using partition chromatography. Separation of compounds by partition chromatography is generally on the basis of relative solubilities, which in the case of homologous series such as ubiquinones and menaquinones is determined by the length and degree of hydrogenation of the multiprenyl side chain.

This chapter discusses the serology and epidemiology of Vibrio cholerae and Vibrio mimicus. Although most strains of V. cholerae ferment sucrose, some sucrose-negative strains have also also included in this species. Based on DNA hybridization, these strains, which do not ferment sucrose, have recently been placed in a separate species and the name “Vibrio mimicus” has been proposed for the species. Because of the serological identity and probably similar clinical significance of V. mimicus and V. cholerae, the former is reviewed in the chapter. The strains of V. cholerae and V. mimicus are gram-negative, straight or slightly curved rods with a single polar flagellum. They are facultatively anaerobic and can grow readily on or in ordinary media. Growth occurs between pH 6.0 and 9.6, but is optimal between pH 7.6 and 8.6. Colonies on nutrient agar are usually translucent and amorphous, but sometimes wrinkled or rugose colonies may occur. Broth cultures of these vibrios show moderate turbidity and sometimes pellicle formation, especially in an alkaline broth. They do not require more than trace amounts of NaCl for their growth and, on this basis, can be differentiated from other Vibrio species. The physiological and biochemical characteristics of V. cholerae and V. mimicus based on 751 and 85 strains, respectively, are summarized in a tabulated form in the chapter.

This chapter discusses the antibiotic resistance and prescription practices in developing countries. One of the main concerns in the treatment of infectious diseases worldwide is the emergence and spread of bacterial resistance to antimicrobial drugs. These compounds are designed to inhibit vital functions of bacterial cells, but in turn, bacteria have evolved multiple resistance mechanisms to counteract antibiotic effects. Resistance can arise by chromosomal mutations or it can be acquired by mobile genetic elements, such as plasmids, phages or conjugative transposons. In this case, multiple resistance determinants can be transferred across species and genera, contributing to the spread of resistance determinants. Among the major concerns related to antimicrobial resistance is the misuse of antibiotics as a result of both inadequate prescription and self-medication. Antibiotics are drugs with a major impact, but owing to the continuous rise in drug resistance, considerable economic resources spent on antibiotics are wasted. In order to achieve an effective program to control infectious diseases, the characteristics of antibiotic use in each region, as well as the possible environmental pressures that may exist, must be considered. Designing new antibiotics, taking into account what is known about the resistance mechanisms, is an alternative strategy to obtain drugs with proven antibacterial efficacy and a lesser likelihood of inducing resistance.

This chapter studies animal models and mucosal candida infections. The use of animals models have been invaluable to investigate and understand host defense mechanism protecting against mucosal candidiasis. Experimental animal studies and the deriving considerations strongly suggest that local mucosal immunity is critically important as defense mechanism against Candida infections. Particularly, the role of CD4+ T cells in resistance to mucosal candidiasis is relevant. These cells may act locally and be involved as critical regulators of defined-isotype antibody response by the production of Th1 cytokines. Experimental oral candidiasis has evidenced the role of CD4+ T cells and CD8+T cells together with Th1-type responses in protection against Candida infection. The observations of gastrointestinal candidiasis reproduced in genetically modified mice including cytokine-deficient mice have clarified the role of cytokine-mediated regulation of Th cell development. They have also revealed complex levels of immunoregulation in mucosal candidiasis. The results obtained in experimental Candida vaginitis have clearly evidenced that systemic cell-mediated immunity is not protective against vaginitis while some form of locally acquired mucosal immunity seems to be protective. The result obtained in a rat model of Candida vaginitis demonstrates the presence of protective antibodies against specific virulence factors of the fungus, together with activation of the T cell mucosal compartment.

Cytomegaloviruses (CMVs) are conditional pathogens that are strictly species specific and are usually well controlled in their respective mammalian hosts by the effector mechanisms of both innate and adaptive immunity. Human CMV (hCMV) is mostly acquired perinatally as well as in early childhood and is transmitted, for instance, through breast milk and saliva. Whilst the immune response in an immunocompetent host prevents an overt CMV disease and rapidly terminates the productive acute infection, viral genome is maintained in most tissues for the life span of the infected host in a state known as viral latency. Latency implies that infectious virions are no longer produced so that the host is no longer infectious. Furthermore, CMV diseases with multiple organ manifestations such as interstitial pneumonia, hepatitis, adrenalitis, gastrointestinal disease and bone marrow failure result from primary or recurrent infection of the immunocompromised or immunologically immature host. The chapter also describes murine CMV.

This chapter focuses on the flow cytometric analysis of salmonella-containing vacuoles. Salmonella enterica serovar typhimurium (S. typhimurium) is an enteric pathogen that causes gastroenteritis in humans. In mice, it is the etiologic agent of a systemic infection similar to typhoid fever. Thus it is used as a model to study the pathophysiology of typhoid fever. After uptake, Salmonella resides within a vacuole that successively acquires markers of the early endosomes, the recycling compartment and late endocytic compartments. Salmonella grown under different conditions are needed whether the invasion of phagocytic or non-phagocytic is being used. As Salmonella grown to the log phase induce apoptosis of macrophages, invasion of this cell type requires bacteria grown to stationary phase. Optimized cell invasion is necessary for the analysis of fluorescent vacuoles by flow cytometry. Optimal detection is achieved when vacuoles represent more than 5% of particles. This requires cells infected with between 1 and 10 fluorescent bacteria. Many bacterial pathogens reside within host cells, either transiently or throughout an infection, and this intracellular lifestyle is often a key component to disease.

This chapter discusses some of the methods used for exploiting the vesicular-arbuscular mycorrhizal symbiosis in agriculture. The first step towards application of vesicular-arbuscular mycorrhizal technology is to obtain a good starter culture. Another approach is to isolate spores of vesicular-arbuscular mycorrhizal fungi from soil by wet sieving and decanting technique. Such spores must be checked for the occurrence of mycoparasites. They are then surface sterilized and introduced into pot cultures by the use of the funnel technique. These pot cultures can be maintained in a greenhouse on a suitable host. Techniques are available for the production of vesicular-arbuscular mycorrhizal inoculum in an almost sterile environment through nutrient film techniques, circulation hydroponic culture systems, aeroponic culture systems, root organ culture, and tissue culture. Pot cultures are complex systems comprising host plants, mycorrhizal fungi, soil microflora and microfauna, and supporting soil. Soil cultures can sometimes harbor root pathogens and thus can act as a source of disease. The chapter further explains greenhouse sanitation and mycorrhizal dependency of plants.

This chapter describes protocols for staining of haloarchaea with fluorescent dyes and correlation with CFUs, including improved media for growth of cells from environmental samples. Staining of micro-organisms with fluorescent dyes in the presence of high ionic strength (up to 4.2 M NaCl) is possible. Morphology, size and the presence of nucleoids (which is considered to be indicative of active cells) can be detected in the epifluorescence microscope; an assessment of the intactness or damage of membranes, whether of bacterial or archaeal composition can be made. Extremes of pH do not interfere with the application of the LIVE/DEAD®kit. Staining with DAPI does not distinguish between viable and dead bacterial cells, which are also true for haloarchaea. The LIVE/DEAD®kit is thought to permit a differentiation between active and dead cells. For information about the true status of microbial cells, determination of CFUs is still the most valuable approach, though not always feasible. A useful property of the dyes of the LIVE/DEAD® kit is their noninterference, when used at low concentrations, as for staining with subsequent growth experiments.

This chapter focuses on various methods for the assessment of intracellular bacterial killing and digestion and the mobilization and action of individual anti-microbial agents within professional phagocytes. Essential host defense responses to invading microbes include sequestration of viable organisms to prevent further dissemination and creation of a highly noxious microenvironment to render the targeted microbe non-viable. Professional phagocytes play major roles in these responses. The antimicrobial role of professional phagocytes, especially of macrophages, includes microbial digestion to facilitate disassembly and removal of microbial remnants, down-regulation of the inflammatory response and return of the host to a resting state. Relatively few host antibacterial proteins have actions sufficiently specific to permit product accumulation to serve faithfully as evidence of their interaction with ingested microbes. Prominent among the array of defensive responses mounted by phagocytes is the agonist-dependent generation of reactive oxygen species (ROS) by neutrophils, monocytes, and macrophages. The enzyme responsible for the conversion of molecular oxygen to superoxide anion is a multicomponent complex composed of a heterodimeric membrane protein, flavocytochrome b558, and at least three cytosolic proteins.

This chapter describes the apparatus and methodology for growth, isolation, scale-up of the most stringent anaerobes and the isolation of oxygen-labile biomolecules. Methods described in the chapter are applicable to methanogenic Euryarchaeota, non-methanogenic hyperthermophiles within both the Euryarchaeota and Crenarchaeota, and hyperthemophilic bacteria within the Thermotogales. These methods are also applicable to “nonextremophiles,” such as the iron-reducing and sulfate-reducing bacteria as well as other obligate anaerobes within the bacteria. These anaerobic micro-organisms are ubiquitous in the environment and occur in a large range of “extreme” habitats that include anoxic sewage digestors, mammalian, ruminant and termite digestive tracts, polar lakes and tundra, geothermal submarine vents, calderas and hot springs, deep sea sediments, and deep subsurface rock. Unlike facultative anaerobes and oxygen-tolerant anaerobes, many obligate anaerobes require stringent anaerobic conditions to maintain viability. This is achieved by using oxygen-free gases to prepare anoxic medium with the addition of chemical reducing agents to achieve a low redox potential. Specialized glassware and vessels are utilized to maintain reduced anoxic conditions during growth.

This chapter discusses an overview of testing pathogenicity. Pathogenicity tests are performed for a number of reasons. Attention is primarily focused either on the bacterium or on the host plant. In the former case, the objectives include determination of the pathogenicity of a bacterial isolate from a plant and determination of the plant host range of an isolate. There are many cases known in which production of specific biochemical factors by bacteria is essential for pathogenicity. It may be possible to test directly for the production of such factors without using plants. An important consideration when planning pathogenicity tests is the scale of the experiment. When screening for new bacterial mutants altered in pathogenicity, it may be necessary to test thousands of individual survivors of mutagenesis for altered symptoms induced in plants. In addition, the chapter discusses types of diseases incited by bacteria. Bacterial plant pathogens are classified into a small group of genera. The general types of disease incited can also be placed into a small number of groups. The measurement of bacterial growth within infected tissue is an excellent objective property of pathogenesis.

An introduction to fractionation of bacterial cell envelopes is discussed in this chapter. The molecular investigation of bacterial virulence determinants generally requires localization or separation of cellular components, which in turn rely upon true and reproducible cell fractionation techniques. The first step in any such fractionation is the separation of cells from the culture supernatant. A centrifugation speed and time that will sediment the cells into a tight pellet without causing lysis is essential and can be monitored by comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profile in the cell pellet with that of the supernatant. To analyze the proteins in the supernatant is necessary to concentrate before electrophoresis. Washed bacterial cells can be fractionated in a number of ways, depending upon the end use of their macromolecular components. Cell envelope components can also be fractionated using treatments, such as differential solubilization, but the work should be carried out quickly in the cold using protease inhibitors. Peripheral membrane proteins can be separated by washing membranes with agents, such as sodium chloride.

This chapter discusses an overview of fimbriae; it also discusses its detection, purification, and characterization. Fimbriae are adhesive bacterial surface structures that enable bacteria to target and colonize particular host tissues, due to specific receptor recognition. Fimbrial-associated receptor recognition permits bacteria to adhere to diverse targets ranging from inorganic substances to highly complex biomolecules. This capacity is of paramount importance in bacterial colonization of a given surface, whether inanimate or a particular tissue in a mammalian host. The interplay between a fimbrial adhesin and its cognate receptor plays a significant role in determining host and tissue tropisms in pathogenic bacteria. Consequently, fimbriae are often recognized as virulence factors, and this aspect has spurred intensive research into the molecular biology, biochemistry, and genetics of these surface structures. In general, fimbriae are heteropolymers, consisting of a major structural protein and a small percentage of minor component proteins, one of which is the fimbrial adhesin.

This chapter describes the existing data on the molecular biology of C. jejuni and C. coli, discusses strategies and techniques currently available to investigate the molecular genetic basis of the pathogenesis of C. jejuni, and indicates possible future directions. Further, the chapter explains cloning of campylobacter genes. C. jejuni has a genome of approximately 1700 kb, with an unusually high A+T content of 70%. This compares to a genome size of approximately 4600 kb with an A+T content of 50% in E. coli. The identification and characterization of C. jejuni genes has been severely hampered by the fact that one of the most common strategies used in the study of other bacterial pathogens, transposon mutagenesis, has so far not been successful with C. jejuni. Currently, the cloning and sequencing of around 60 C. jejuni genes is described. Common strategies for the cloning and identification of C. jejuni genes are also explained.

Listeria monocytogenes is a ubiquitous, Gram-positive, non-spore forming, facultative intracellular bacterium that is responsible for infrequent, but often serious, opportunistic infections in humans and animals. Because this pathogen has emerged as an important agent causing foodborne diseases and as a model system for investigation of intracellular bacteria, molecular approaches for the study of L. monocytogenes are of great interest for the identification and analysis of listerial genes. In addition, the chapter explains cloning in Listeria. For isolation of nucleic acids from Listeria spp. complete lysis of the bacteria is essential. In contrast to Gram-negative bacteria, where lysis is easily performed using NaOH/SDS, Gram-positive bacteria are particularly resistant because of the more complex structure of their cell wall peptidoglycan.

This chapter presents some of the basic experimental assays that have been developed or used in laboratories for the study of cholera and related E. coli heat labile enterotoxins, diphtheria toxin, and anthrax toxins. The chapter emphasizes on the importance of experimental systems that provide a high degree of temporal resolution when measuring toxin function. Microscopic techniques for non-polarized cells and monolayers grown on glass or plastic are described. For studies of protein binding, sorting, or intracellular trafficking in polarized epithelial cells, morphological examination of polarized monolayers grown on permeable supports is essential. The chapter briefly discusses the principles of microscopy on polarized monolayers for study of toxin binding or internalization. Bacterial AB toxins are designed to translocate a functional enzyme into the cytosol. Thus, the functional signal induced by membrane translocation of the enzymatic toxin-subunit is magnified tremendously over the signal that can be obtained by direct measurement of the mass of translocated protein. This is one of the key advantages in using bacterial toxins to study membrane translocation and vesicular transport in intact cells. The technologies described in this chapter have been used to identify the mechanisms of internalization and intracellular compartments from which AB-toxins gain entry to the cytosol.

This chapter discusses the methods currently used in the identification and serological and phage typing of Aeromonas species. For convenience, the genus, Aeromonas, is divided into four species: A. hydrophila, A. caviae, A. sobria, and A. salmonicida. The genus, Aeromonas, belongs to the family Vibrionaceae. The members of the genus, Aeromonas, are Gram-negative, rod-shaped bacteria measuring 1–4 by 0.5-1μm. They are motile by polar flagella, generally monotrichous, or non-motile. The aeromonads grow on meat extract media, are facultatively aerobic or anaerobic, and ferment glucose with or without gas production. They reduce nitrate to nitrite and are oxidase positive. The optimum temperature for growth is 28°C. Two clearly distinguished groups are included in the genus, Aeromonas. Psychrophilic and non-motile aeromonads are clustered in the first group, named “A. salmonicida.” The second group is formed of mesophilic and usually motile bacteria. Recent taxonomic studies indicate that the motile aeromonads can be separated into three species—namely, A . hydrophila, A . caviae, and A. sobria.

This chapter discusses isolation and characterization of human epithelial antimicrobial peptides and proteins. Human skin contains a huge number of different antimicrobial peptides and proteins (AMPs). Many AMPs contain high numbers of cysteines, forming in a defined manner disulphide bridges. Recombinant expression of AMPs (example defensins) often generates mixtures of AMPs with different connectivities of the disulphide bridges. For hBD-3, all variants with different cysteine bridges show antimicrobial activity. But only the hBD-3 variant, which shows the connectivity of natural hBD-3, is able to act receptor-dependent as chemotactic and activating factor. Thus, in this case the natural AMP serves as positive control. In skin, nearly all AMPs are produced in the uppermost, fully differentiated epidermal layers (stratum granulosum), where they are stored or secreted. The uppermost epidermal cells are subject to cornification, and at the end these form the stratum corneum (SC), which is a layer of flattened, dead epidermal cells. Thus, the SC, which is easily available in sufficient amounts from the heel revealed to be one of the best sources of human (epithelial) AMPs. Healthy person's derived heel SC is a good source for constitutively produced human AMPs. The chapter further reviews extraction of AMPs from tissue.

This chapter describes the various aspects of DNA vaccines. It also discusses the basic materials and methods involved in preparing and administering DNA vaccines. The preparation of Escherichia coli-derived plasmid DNA expression vectors utilizes standard molecular biology reagents. The gene of interest, or fragment, can be generated by polymerase chain reaction (PCR). Wolff demonstrated the expression of proteins in situ after the administration of plasmid DNA containing the genes encoding those proteins. In some cases, it may be desirable to include only the coding region from the ATG to the termination codon. Sequence verify at least three clones using primers 30–50 bp from the restriction site so that bases within the vector can be read, as well as 150–200 bases within the gene. In this way, the orientation and quality of the PCR-generated gene can be assessed. For the characterization of plasmid DNA vectors prior to immunization of animals, expression can be assessed by transient transfection in vitro and immunoblot analysis. A crude estimation of relative vaccine efficacy can be made by comparing relative expression levels of a particular antigen in vitro.

The genus, Leuconostoc, comprises six species, but there is evidence that these should be reduced to four. If this is accepted, both Leuconostoc dextranicum and L. cremoris will be reduced to the subspecies of L. mesenteroides. In this chapter, the current names are used, but the evidence for the proposed changes in classification is given. Leuconostocs are normally found living in association with vegetable matter with lactose-fermenting species occurring in milk and dairy products. All lactic acid bacteria depend on the fermentation of carbohydrates for energy and all form lactate as a major end-product of the fermentation of glucose. They can be divided into homofermentative and heterofermentative species, depending on the end-products from the fermentation of glucose when they are growing in a good nutritive medium. The homofermentative species use the Embden–Myerhof (EM) glycolytic pathway converting glucose to fructose-1,6-diphosphate (EDP), which is split to glyceraldehyde phosphate. The end-product is two moles of lactic acid for each mole of glucose consumed. Fructose-1,6-diphosphate aldolase is a key enzyme in the EM pathway. Streptococci, pediococci, and many lactobacilli are homofermentative. Some species possess alternative glycolytic pathways, which is used when, for some reason, the EM pathway is suppressed.

Thousands of identification methods for medically important bacteria have been described in the chapter. The overwhelming majority represent modifications of tests discovered in the first half of this century, when the foundation of modern taxonomy was laid. Selection of the best of these methods or replacement of an old test with an improved one requires extensive comparative trials. In choosing methods, an enormous number of commercial reagents, dehydrated or complete media and multitest systems also have to be considered. In general, any method is satisfactory that gives accurate and reproducible results. Tests used in routine clinical and public health bacteriology should be suitable for a tolerably rapid and reliable identification. To determine their characteristics, bacteria must be grown in pure culture, which is a group of organisms that have developed from a single cell or from a single clump of similar cells.

This chapter discusses the analysis of crystalline bacterial surface (S) layers by freeze-etching, metal shadowing, negative staining, and ultrathin sectioning. When using freeze-etching techniques for studying S-layers, the main purpose of the sampling, pretreatment, and cryofixation procedures is to preserve the regular array as closely as possible to that existing in vivo. Most S-layers completely cover the cell surface, leaving no gaps. Some organisms possess more than one S-layer. In such instances the fracturing process may separate adjacent layers. On rod-shaped cells S-layer patterns are generally uniform over large areas of the cylindrical part of the cells. Oblique and square lattices are usually arranged with one axis parallel to the long axis of the cell, but with some organisms a strain-specific skew angle may be observed. Freeze-etching is suitable for evaluating S-layers labeled with morphologically detectable marker molecules. Labeling with polycationic ferritin, a marker for negative surface charges, is used for probing the net charge of native and chemically modified S-layers.

Identification of bacteria is based on results of characterization tests, which place the unknown organism in a defined group. The results of conventional tests applied to clinical isolates are usually not available in less than 2 days from receipt of specimen. A large number of enzyme classes have been studied in bacteria. Each class will be dealt with briefly, followed by the results of application of batteries of enzyme tests to characterization and identification of various bacterial taxa, and problems of handling quantitative enzyme data. This chapter discusses the study of iso-enzymes using electrophoretic techniques, in which enzymes performing the same catalytic activities are compared with regard to their electrophoretic mobility, as well as their substrate, cofactor and inhibitor specificities. Bacterial enzymes may be situated within the cell or secreted into the growth medium. Extracellular enzymes can be measured around bacterial growth on solid medium or in the supernatant of spent liquid medium. Within the prokaryotic cell three different localities have been defined—namely, the periplasm, the cytoplasmic membrane, and the cytoplasm.

The relative biological simplicity of bacterial genomes makes microarray design a straightforward process. Compared to complex eukaryotic genomes, bacteria not having introns or alternative splicing have seldom been annotated to have overlapping genes on opposite strands and they have high gene density. The starting point for constructing whole genome microarrays is the fully sequenced and annotated genome and bioinformatics has a major role to play in the design process. This is particularly true when designing arrays that cover the genomes of multiple strains of a single species, whereby there may be additional genes from multiple strains compared to a sequenced reference strain. Although a truism, it is safe to say that better design aids better data analysis, and this is most important when cross-hybridization among several genes is present. Deconvolution of complex data sets to account for the effect of multiple cross-hybridization on signal intensity will not be straightforward.

This chapter discusses the use of bioinformatics to predict MHC ligands and T-cell and its application to epitope-driven vaccine design. T-cell epitopes derived using bioinformatics are now being used to construct and enhance vaccines. These vaccines are designed to imitate the natural development of cell-mediated immunity after initial exposure to a pathogen. Upon vaccination, epitope-specific memory T-cell clones are generated to respond more rapidly and efficiently upon subsequent exposure to the pathogen. The underlying paradigm is that an effective vaccine is able to elicit a number of epitope-specific memory T cells, which drive the protective immune response upon re-exposure to the pathogen. A similar premise operates for B-cell epitope (antibody)-driven vaccines. The chapter discusses the bioinformatic tools and methods that are currently being used to mine proteomes for T-cell epitopes and being applied to the design of epitope-driven vaccines. It also discusses approaches for identifying T-cell epitopes from protein sequences and in vivo assessment of the immunogenicity of a T-cell vaccine.

The yeast Saccharomyces cerevisiae is a very suitable organism for genetic analysis and now its genome has been completely sequenced; deletion mutants for each gene can be easily established by reverse genetics. Using the very precise recombination apparatus of S. cerevisiae, each gene locus can be replaced by selection markers, such as amino acid auxotrophies, nucleoside auxotrophies, or dominant resistance markers. This allowed the generation of a collection of mutants for each of the approximately 6000 open reading frames (ORFs) within the S. cerevisiae genome. The deletion cassettes can be amplified by polymerase chain reaction from the respective deletion mutant, so that the deletion can be easily introduced into any S. cerevisiae strain of interest. Currently, deletion mutants for about 5900 genes are available from the various S. cerevisiae strain collections. In addition, an increasing number of strains and plasmids can be received where genes are under regulated expression, have affinity tags for easy purification of the respective protein, and are fused to the green fluorescence protein for their easy cellular localization.