Methods for General and Molecular Microbiology, Third Edition

Light microscopy is made easy, interesting, and useful for bacteriological purposes if at least four different kinds of instruments are readily available, permanently set up for work, and maintained in good order. An additional instrument, a stereoscopic “dissecting” microscope, is valuable for undertaking the isolation of bacteria from nature on agar plates when colonies early in growth are small and close together. The chapter outlines the principles of basic microscope. Achieving high resolution as well as freedom from chromatic and spherical aberration requires attention to basic principles. Unfortunately, light of the most effective wavelength (namely, UV light of about 365 nm) is not perceived by the eye and does not pass through glass. The Koehler system of illumination is useful for high-resolution microscopy because it provides appropriate illumination of the field and makes good use of high-quality optics. The chapter outlines the steps allowing achievement of Koehler (nearly optimum) illumination for an oil-immersion objective. The chapter also focuses on dark-field microscopy, phase-contrast microscopy, interference microscopy, fluorescence microscopy, and photomicrography.

Specific morphological details are required to characterize a microorganism; these are usually determined by means of light microscopy, but some require more sophisticated techniques such as laser scanning microscopy or electron microscopy. This chapter concentrates on the general means of characterizing bacteria by light microscopy because it includes the cytological approach to making the best of preparations for study and photomicrography. The field of aerobiology encompasses both indoor and outdoor components and focuses on a wide range of microorganisms, including pathogenic and nonpathogenic varieties. Although the numbers in a unit of volume may be quite small, the simplest approach to air sampling is the use of open petri dishes containing a suitable nutrient agar near a suspected contamination source and is applicable when the concentration of organisms is relatively high. Translational movement of bacteria by flagellar propulsion (swimming) may be observed in wet mounts of specimens by use of in most cases, the low-power or high-dry objectives. To ascertain the presence of flagella in doubtful cases, as well as to determine flagellar distribution (polar, peritrichous, or lateral), staining procedures and electron microscopy may be required. Gram staining is the most important differential technique applied to bacteria. Mature, dormant endospores of bacteria, when viewed unstained, are sharp edged, even sized, and strongly refractile, shining brightly in a plane slightly above true focus. A great number of techniques based on light microscopy are special to particular areas of bacteriology. The chapter gives examples of some special methods which are not routinely used.

This chapter provides a basis for understanding laser scanning microscopy (LSM) and the approaches that may be used to apply high-resolution digital microscopy to the study of bacteria. Additional reviews of image analysis, digital imaging, and LSM for microbiology applications are also provided in this chapter. The major focus of most courses is cell biology, although the information is of use and techniques are broadly transferable across disciplines. In practice, one of the appealing aspects of fluorescence microscopy is the lack of a requirement for fixation, dehydration, and the air drying of samples required by some other techniques. One of the goals of this approach is to examine living bacterial cells, aggregates, and biofilms. Changes in any of the microscope settings will alter the section thickness, the brightness, and the apparent size of the objects being imaged. The major advantage of LSM is the capacity to collect a series of images that allow the user to obtain 3-D spatial information. The chapter gives general protocol and considerations of lectin staining. A number of approaches have been applied to study diffusion in bacterial biofilms and serve to illustrate the application of kinetic analyses using LSM systems. The major limitation of all light microscopic systems and particularly LSM is poor axial resolution. In LSM there are both 2-D- and 3-D-related considerations regarding sampling. Application of LSM techniques results in the creation of vast image and data sets.

This chapter explains some of the mysteries of electron microscopy (EM), and makes the various techniques more user friendly to researchers who have lost the skills and recognized the importance of its use. The first level of ultrastructural information is provided by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Many new and complex derivatives of EM are available, all with impressive and complicated names, e.g., scanning transmission electron microscopy, energy-dispersive X-ray spectroscopy (EDS), electron energy loss spectroscopy (EELS), electron spectroscopic imaging (ESI), cryoTEM, and cryoSEM; often these techniques can be intermixed with one another. The chapter describes a limited number of TEM methods that are considered useful to the worker who has need of basic structural information about microorganisms. Emphasis is placed on the use of negatively stained preparations and thin-sectioned materials for examination by TEM. The chapter outlines a simple plunging freeze-substitution method. Shadowing was among the first techniques used to visualize bacteria by TEM. The chapter presents several methods of using shadowing (e.g., shadow casting, freeze-etching, and rotary shadowing). It also focuses on image acquisition. The main advantage of digital imaging is the amount of time saved. As the price of electron microscope plate film doubles and triples, the charge-coupled device (CCD) option becomes more attractive. The identities and positions of bacterial macromolecules, either within the cytoplasm or on the surfaces of cells, are often matters of importance to research and require appropriate TEM methods. Finally, the chapter provides a catalog of useful items for EM.

In this chapter, some specialized applications are described in which computers are essential for manipulating and enhancing images obtained by using a transmission electron microscope, particularly to understand the tertiary and quaternary structures of biological macromolecules and their complexes. The chapter must be read with some knowledge of the basic principles of transmission electron microscopy (TEM) of biological material. The computational techniques for analyzing and reconstructing macromolecular structures from TEM images are reviewed in monographs. Partial results of single-particle analyses of both frozen-hydrated and negatively stained preparations are illustrated in the chapter. S-layers, the proteinaceous surface arrays found on some gram-positive and gram-negative eubacteria and many archaea, are naturally occurring 2-D crystals that are amenable to image analysis by electron crystallography. TEM, particularly cryoTEM, has become a powerful tool for analyzing the tertiary and quaternary conformations of biological macromolecules and their complexes. Computational image analysis and reconstruction are integral tools in this process. The chapter has illustrated two approaches to manipulating and processing macromolecular TEM images that one would commonly find in the structural and molecular microbiological literature.

The advent of atomic force microscopy (AFM), one technique in a family of new microscopies called scanning probe microscopies, has recently opened a wide range of novel applications for microbiologists. This chapter focuses on the use of AFM in microbiology. Rather than providing an exhaustive review of the literature in this area, the chapter emphasizes methods and gives recommendations for reproducible, reliable experiments. The first section concentrates on sample preparation procedures: selection of appropriate substrates and immobilization protocols available for isolated macromolecules, cell surface layers, and whole cells. The second section deals with the various aspects of AFM imaging: different imaging modes together with common problems and artifacts, imaging parameters, and imaging environments. The third section focuses on AFM force measurements: the principle of force-distance curves and their application to probe molecular interactions and mechanical properties. Finally, isoporous polymer membranes can be used to immobilize large objects such as whole cells. The protocols differ according to the type of sample investigated, i.e., macromolecules, cell surface layers, or whole cells. Probing molecular interactions method has been used to measure the forces between E. coli-coated probes and solids of different surface hydrophobicities.

This chapter presents techniques for the fractionation of cellular components, including organelles and appendages, beginning with external surfaces and ending with internal components. It should be appreciated, however, that many of the techniques described in the chapter, from cell breakage to centrifugation theory, will have similar applications. The factors pertinent to cell fractionation, often dictating the approach needed, are the differences in wall structures and the bonding forces within walls (and associated surface materials) responsible for maintaining cell shape. Purification of the murein-outer membrane complex from the crude envelope fraction by density gradient separation in sucrose is described. Lipopolysaccharide (LPS) isolated by the phenol-water method preserves the reactivity of pseudomonad LPS with antibodies in Western immunoblots and is better in this regard than LPS isolated by the Darveau-Hancock method. In general, the murein portion of the cell wall is first removed to form protoplasts or spheroplasts, which are then lysed to provide either the plasma membrane or the plasma plus outer membranes. A structurally intact, intracytoplasmic membrane array may best be isolated by lysis of osmotically sensitive spheroplasts. The effect of lysis and physiological conditions on the nucleoid is discussed by Korch.

Reactions between antigens (Ags) and antibodies (Abs) are usefully exploited in many areas of life science research. The monoclonal Ab (MAb) technology developed by Köhler and Milstein allows for the production of unlimited quantities of Abs against virtually any molecule. As there is an immense volume of information concerning all aspects of Ag-Ab reactions in the literature, the objective of this chapter is to provide simple and useful protocols and an introduction to some of the more novel techniques. The routes of injection commonly used on rabbits, mice and rats include intradermal (i.d.), subcutaneous (s.c.), intramuscular (i.m.), intraperitoneal (i.p.), and intravenous (i.v.). Nucleic acid (DNA or RNA) immunization and genetic vaccines introduced in the early 1990s are some of the most important discoveries and novel strategies in vaccine development. The essential features of a DNA vaccine are a bacterial plasmid vector engineered to carry a DNA insert encoding the protein immunogen(s) of interest, a eukaryote gene promoter, and a poly(A) site to enable expression of the protein in mammalian cells. The vectors are usually maintained in and purified from Escherichia coli. The polyclonal serum is expressed from the blood clot by centrifugation, and approximately 52% of its volume can be collected as serum. The serum can be used directly in Ag-Ab reactions, absorbed to deplete it of nonspecific or cross-reactive Abs, or fractionated to purify Abs free of other serum proteins.

This chapter talks about four classes of pitfalls in bacterial growth measurement. The change of enteric bacteria from large RNA-rich forms in the exponential phase to small RNA-poor forms in the stationary phase has many of the aspects of differentiation. Evidence of the difficulties involved is the fact that many of the people who helped develop the technique for measuring distribution of cell volumes no longer use it. This article, therefore, only presents the principles, mentions the difficulties and the attempted solutions to these difficulties, and directs the reader to published literature. Three colony count methods are discussed. The first is the spread plate, in which all colonies are surface colonies. The second method is the layered plate. The third method is the pour plate. Flow cytometry has become an extremely powerful method for the studies of many aspects of the biology of eucaryotes, but the methods are only now coming into their own in the study of the biology of procaryotes. The concentration of viable cells can be roughly estimated by the most-probable-number (MPN) method. For growth measurements, the time can be precisely defined, and the major error is in the measurement of the number of cells or other indices of biomass.

Recent intersections between geology and microbiology, as well as environmental engineering and microbiology, have provided even more creative approaches for developing selective media for novel organisms. This chapter places some emphasis on the nutritional needs of some of the broad groups of prokaryotes that have been discovered through using selective media as an enrichment tool. Other important aspects of nutrition that are considered in the chapter are requirements for vitamins, trace minerals, other growth factors, buffers to control pH, sterilization, and preparation of solid media. The essential nutritional requirements for prokaryotes can be divided into two classes: macronutrients and micronutrients. More than 30 elements are considered essential for cell growth; however, there are six nonmetals (carbon, oxygen, hydrogen, nitrogen, sulfur, and phosphorus) and two metals (potassium and magnesium) that constitute 98% of the dry weight of prokaryotic organisms. Sodium is necessary for certain marine bacteria, phototrophs, and some anaerobes. Mixed hydrolysates are processed yeast or meat extracts to supplement the nutritional aspects of peptones by providing some amino acids, carbohydrates, nucleic acid fractions, organic acids, vitamins, and trace minerals lost during their manufacture. It is important to ensure that the blood and serum used in formulating bacteriological media are free of pathogens and potential health risks, especially in light of the recent bovine spongiform encephalopathy outbreaks.

A section of this chapter focuses on the use of enrichment and cultivation procedures with molecular methods, such as wholecell fluorescent in situ hybridization (FISH) or denaturing gradient gel electrophoresis (DGGE), to monitor the progress of an enrichment, to evaluate the presence of contaminants, and to identify new isolates. Biophysical enrichments make use of such conditions as growth temperature, heat treatment, sonic oscillation, or UV irradiation to kill or inhibit the rest of the population. Biological enrichments may make use of specific hosts for selective growth of a particular organism, or they may take advantage of some pathogenic property, such as invasiveness, which the rest of the population does not possess. Bacteria are usually isolated from enrichment cultures by spatially separating the organisms in or on a solid medium and subsequently allowing them to grow into colonies. The chapter is designed to demonstrate the multiplicity and in many instances the considerable ingenuity of enrichment and isolation methods for bacteria by presenting specific selected examples. The buoyant density of bacteria in pure culture and in samples from natural aquatic environments has been studied by density gradient centrifugation in Percoll gradients, and the average density of a representative bacterium is 1.080 pg µm -3. Isolation of Legionella species, particularly those from the environment, can sometimes be facilitated by acidification of samples to pH 2.2, which kills contaminants more quickly than it does the Legionella species.

This chapter talks about the main culture techniques for microbial growth. It first describes the solid, semisolid, biphasic, membrane surface, and immobilized culture techniques. This is followed by laboratory scale liquid culture techniques including specialized liquid cultures such as synchronous and dialysis cultures. Solid media are also used in mass culture, bioautography, and physiological studies of bacterial cells. Solid culture is one of the most useful techniques in the isolation and cultivation from single cells. The solid surface usually is that of an agar or otherwise solidified medium. Semisolid media are also useful in chemotaxis studies. For example, a semisolid medium containing an oxidizable carbon and energy source can be used to investigate positive chemotaxis in Escherichia coli, Vibrio cholerae, Salmonella enterica, and many other species. Laboratory scale liquid cultures provide one of the most common techniques to grow and study the behavior of microorganisms. The section on energetics and stoichiometry describes the theoretical aspects of microbial growth focusing on the electron acceptor and donor and the carbon and nitrogen sources. There are two basic principles underlying dialysis culture. First, it provides a means for achieving substrate limited growth. Second, dialysis culture provides a means for lowering the concentration of a diffusible metabolite product inhibitory to growth; the product in the culture chamber diffuses through the membrane and is diluted in the larger dialysate reservoir, thus relieving the feedback inhibition by the product that normally regulates its production.

This chapter introduces concepts related to energetics, stoichiometry, and kinetics of microbial growth. It consists of two parts: the first part focuses on energetics and stoichiometry, and the second part focuses on kinetics of microbial growth. The section on energetics and stoichiometry includes some basic concepts such as computation of oxidation states and half-reactions that are critical to formulate a stoichiometric equation. The section on kinetics presents equations governing cell growth, focusing on batch cultures and chemostats. All analyses and statements in this chapter assume the existence of a single cell (or spore that could transform into a vegetative state) that contains the enzymatic capacity and information necessary to put the building blocks, electrons, and energy into a copy of itself. In a cell synthesis reaction, the Gibbs standard free energy at pH 7, ΔG r 0′, obtained from the energy-yielding reaction and electrons (fs 0) from the electron donor are used to reduce carbon, nitrogen, and other cell constituents into an oxidation state of the materials found in the cell and to assemble these constituents into cellular macromolecules.

Some physicochemical factors affecting microbial growth are controlled by the constituents of the culture medium (hydrogen ion activity, water activity, osmotic pressure, and viscosity). Others are controlled by the external environment (temperature, oxygen, light, hydrostatic pressure, and magnetic-field strength). This chapter addresses practical aspects of application and control of physicochemical factors. Continuous pH control may be achieved by the automatic addition of acid or base. The effects of hydrostatic pressure on the physiology and metabolism of bacteria are discussed. A number of Pseudomonas and Bacillus species also exhibit a microaerophilic phenotype when growing in low-substrate media. The oxidation-reduction (redox) potential (Eh ) provides a useful scale for measuring the degree of anaerobiosis. Some motile bacteria that possess magnetite- or greigite containing magnetosomes, e.g., Magnetospirillum (formerly Aquaspirillum) magnetotacticum, align with the Earth geomagnetic field, which has strength of about 1 G. As a consequence, they show a biased swimming behavior, and both north-seeking and south-seeking forms are known. This behavior has been termed magnetotaxis and is thought, in part, to help these bacteria (many of which are microaerophilic) orient toward aquatic sediments where dissolved-oxygen concentrations are lower than in surface waters.

In performing phenotypic characterization tests, the organisms used for the inoculation of test media should be from fresh transfers and in good physiological condition. The methods described in this chapter are based primarily on methods developed for the characterization and identification of organisms which have usually been isolated on nutrient-rich media. While zoologists may be content to classify all insects or all vertebrates, prokaryote systematics generally deals with all prokaryotes and in an evolutionary context attempts to extend this study back to the origin of life itself. To test for acetamide hydrolysis, streak the surface of an acetamide agar slant with a sample from a dilute suspension. Incubate for up to 7 days. Care should be taken to ensure that the basal medium to which the carbohydrates are added does not contain fermentable or oxidizable sugars, and control media without any added carbohydrates should always be included during testing of cultures. Methanogenic members of the Archaea also fluoresce under UV light. Some facultative anaerobes, such as Escherichia coli and Staphylococcus aureus, grow aerobically by respiration with oxygen and grow anaerobically by fermentation.

This chapter presents methods that highlight two general areas of symbiosis research: (i) the detection and characterization of environmental symbioses and (ii) elucidation of the molecular and cellular mechanisms of symbiosis in model systems. The methods are described to demonstrate that an organism is symbiotic and examples of techniques used to elucidate the molecular, physiological, cellular, and evolutionary basis of symbiosis. Many symbioses, however, present additional challenges to fulfilling Koch’s postulates. The study of bacteria-phage symbioses is important for our understanding of bacterial evolution and emergence of disease as well as providing powerful experimental models for the study of symbiosis in a test tube. The study of presently uncultured symbioses, for which either cultivation of the symbiont or live maintenance of the host is intractable or has not been attempted, can be accomplished using many recent advances in molecular biology and biochemistry, as well as more traditional methodologies. Molecular characterization of the phallodrine oligochaete Inanidrilus leukodermatus symbiosis, using 16S rRNA (PCR) characterization and fluorescence in situ hybridization (FISH) microscopy, determined that the primary symbiont was a unique member of the γ-Proteobacteria and clustered with other known chemoautotrophic symbionts. Two established models for symbioses are the legume-rhizobia and E. scolopes light organ-Vibrio fischeri, which share all of the above attributes. The chapter describes a few of the many potential monoxenic symbiotic models that contribute greatly to our understanding of the conserved and diverse mechanisms in which symbioses are initiated, maintained, and evolved.

This chapter deals with the basic physical analytical methods and separation procedures of spectrophotometry, electrodes, chromatography, radioactivity, and electrophoresis. It talks about the simple procedures which are appropriate for laboratory classes or are routinely used in a research laboratory. Gas chromatography (GC) is used to monitor production of methane gas, an end product of the consortium growing on the phenol. Additional options for filters are offered by scintillation cocktails that dissolve cellulose acetate, nitrocellulose, and other types of filter membranes, aiding reproducibility and counting efficiency. Even though these formulations are classified as biodegradable, nonradioactive aliquots still need to be disposed of as organic hazardous waste by the proper chemical or radiation safety authorities. Gels used for separations of proteins and short oligonucleotides are usually made of polymerized and cross-linked acrylamide. A particularly effective example of the original method is described by O'Farrell, who separated 1,000 proteins from an Escherichia coli extract. Chromatographic methods have been successfully used in enzyme purification. The chapter attempts to describe analytical and purification methods most commonly used by the microbiologist. There are many other methods that rely on instrumentation that is too expensive to be found in all but a few individual labs.

The methods selected for this chapter are most commonly used in research relating to microbial components and metabolism, but most are also suited to advanced undergraduate and graduate teaching laboratories. The incorporation of radioactively labeled compounds into bacterial cells is used as a means of estimating the rates and amounts of synthesis as well as the distribution of the main small molecule and macromolecule fractions of the cells. Macromolecules are precipitated by cold dilute solutions of trichloroacetic acid. The precipitate fraction of macromolecules is sequentially extracted with organic solvents for lipids, alkali for RNAs, and hot trichloroacetic acid for DNAs; the precipitate remaining after these extractions contains the cell proteins and peptidoglycan. Some macromolecules contain both carbohydrate and noncarbohydrate components (lipopolysaccharides, peptidoglycans, teichoic acids, lipoteichoic acids, teichuronic acids, nucleic acids, glycoproteins). In some cases gas-liquid chromatography can be useful when coupled with mass spectrometry. For nitrite analysis, the most widely accepted method involves modification of the diazotization and coupling reactions. The methods described in this section differ mainly in how the DNA is purified and prepared. A number of short-chain organic acids and alcohols are formed in bacterial cells as intermediates or end products of the citric acid, glycolytic, and other metabolic pathways.

This chapter provides a brief description of the most important principles and approaches used in enzyme activity measurements. In addition, the discussion presents practical information useful to investigators who seek to design enzyme assays for performing meaningful and accurate evaluations of catalytic activities. Many investigators refer to compounds such adenosine 5'-phosphotransferase (ATP), NAD+, and CoA as cellular coenzymes, since they are regenerated by other enzyme systems within the cell, even though they are modified during the specific enzyme reaction. The great majority of enzyme assays are conducted on cell free preparations. This approach allows for careful control of substrate and cofactor concentrations, reaction conditions such as pH and ionic strength, and the use of coupled assays where desired. The following discussion covers only the most basic elements of enzyme kinetics; yet, this information should be adequate for the characterization of most enzymes of microbiological interest. Comparison of concentration versus-substrates plots for enzymes exhibiting Michaelis-Menten kinetics, substrate inhibition, or cooperative behavior. Any microbial enzyme activity of interest can be investigated by using the information provided, which summarize various types of enzyme preparations, high light practical considerations for the design and optimization of an enzyme assay, and detail basic approaches for obtaining and analyzing of enzyme kinetic data.

Permease systems may be energized by ATP, for example, ATP binding cassette systems, or by the proton motive force across the cell membrane. Techniques that require less biomass and are based on use of membrane filters or a high-speed microcentrifuge are described. Changes in growth medium, growth temperature and pH can also be expected to alter permeability, although alterations will generally be less severe than for energized transport coupled to ATP hydrolysis or ∆p across the cell membrane. Harvested cells need to be centrifuged to obtain a tight pellet, and the details of centrifugation depend on the particular organism. The best candidates are the ones used with suspension cells, small solutes such as sucrose or raffinose for which the cells under study do not have significant permeability or transport systems, or non-transported analogues of known substrates. The membrane continuity of vesicles is best tested by assessing the extent of swelling or shrinking in response to changes in osmolality of the suspending medium. The workings of individual transport components, such as permease proteins, antiporters, symporters, or ion translocating ATPases, can often best be studied by isolating the catalysts from cell membranes and incorporating them into liposomes or proteoliposomes.

This chapter provides a brief introduction to assay the activity of respiratory enzymes. Spot assays can facilitate the rapid detection of enzyme activities in whole or permeabilized cells, in subcellular samples, or in chromatography fractions to follow protein purification. In-gel activity stains, or zymograms, offer an alternative approach to detect oxidoreductase enzymes, especially when the level of enzyme activity is at or below the sensitivity limit of a standardized quantitative enzyme assay. Conversely, if the cellular location of the enzyme is unknown, it can be determined experimentally. Finally, knowledge of the enzyme location can be useful in understanding the physiological role(s) of the enzyme in cell metabolism and in energy conservation. This section outlines experimental approaches to determine the cellular location of a redox enzyme following cell fractionation. Since succinate dehydrogenase may be partially deactivated by tightly bound oxaloacetate at its active site, the enzyme requires activation prior to enzyme assay by incubation with either malate or succinate. Prokaryotes exhibit considerable enzymatic diversity with respect to the number and types of cytochrome oxidase enzymes present for reduction of molecular oxygen to water. A section of the chapter describes a number of commonly used assays for anaerobic respiratory enzymes that act on terminal electron acceptors, including nitrate, nitrite, nitric oxide, nitrous oxide, fumarate, TMAO, DMSO, and metal oxides. The activity of NADH-dependent NirB-type enzymes is assayed by monitoring the oxidation of NADH.

This chapter reviews some of the more common carbohydrate fermentation pathways, schematics of the flow of carbon in these pathways, characteristic products produced, typical energy yields, and assay procedures for the key enzymes. Fermentations are classified according to the key fermentation end products of each as exemplified by bacterial ethanolic, homolactic, heterolactic, propionic, mixed-acid, butyrate-butanol, homoacetogenic, and other fermentations. Much of one's understanding of early fermentations came from alcoholic fermentation of sugar by the yeast Saccharomyces cerevisiae. The enterobacteria carry out mixed acid and butanediol fermentation, and this is the basis for the methyl red/Voges Proskauer test that is used to distinguish the genera. Propionate is an end product of many fermentative bacteria. In contrast, in the propionate-succinate pathway, the formation of propionate involves pyruvate and succinate as intermediates and appears to be much more widespread. The phosphorylation of glucose is carried out by either glucokinase or hexokinase. It appears that among the archaea so far examined, the organisms that contain glucokinase appear to use ADP as the phosphoryl donor for the phosphorylation of glucose. However, in the chapter, only the acetate fermentative pathway of hexoses is considered.

There has been a long-standing fundamental and practical interest in microbial metabolism of aromatic compounds. The goal of this chapter is to provide an overview of the generally used, well-established methods that are the hallmark of aromatic metabolism studies. Growing cells in large liquid volumes such as in a fermentor or chemostat is a challenge as well. The high aeration rate of these cultures strips volatile aromatic substrates out of the medium. Toluene and related aromatic compounds have long been used as models for the analysis of aromatic hydrocarbon degradation due to the fact that there are several catabolic pathways known for its degradation, illustrating the many possible ways by which aromatic hydrocarbons can be metabolized. Bacterial metabolism of aromatic hydrocarbons can result in a variety of chemically different compounds, resulting from the ring oxidation and the later ring cleavage steps. These metabolites can be either neutral or charged and either chemically stable or unstable, and thus, care must be taken in the extraction process to stabilize and competently extract the chemicals. If one can identify a cis-dihydrodiol intermediate in culture supernatants, then a priori the pathway proceeds via an initial dioxygenase attack of the aromatic nucleus. However, if one detects only phenolic or dihydroxylated products in the culture medium then it is uncertain whether the catabolic pathway proceeds by an initial dioxygenase or by two initial monooxygenases.

The study of cellulases is important from the standpoint of microbial conversion of biomass to feeds and chemical feed stock. The complex structure of hemicelluloses has dictated a correspondingly diverse array of hemicellulases. The concentration (or actually dilution) of enzyme preparation required to effect this level of depolymerization is converted, through a somewhat indirect procedure, to the cellulase activity in filter paper units (FPU) per milliliter. Rather than being an exact representation of the saccharification process that occurs in simultaneous saccharification and fermentation (SSF), diafiltration saccharification assay (DSA) data were useful for comparison with SSF data in efforts to identify the influences of factors other than product inhibition on the performance of cellulases in SSF. Cellulases may also be detected in slab gels using either Western blotting or enzyme-linked immunosorbent assay, as reported for enzymes from Trichoderma reesei. When preparing enzyme-treated substrates, care must be taken to employ phenolic acid esterase-free cellulases. Hemicellulose-depolymerizing enzymes are divided into three classes; the endoacting, exoacting, and oligomer-hydrolyzing. The process of detecting and verifying exoglucanases (cellobiohydrolases [CBHs] in context of the fungal cellulose systems) has long been controversial. If purified proteins are available, careful comparisons of reducing-sugar yields and fluidity values from carboxymethylcellulose (CMC) hydrolysis as a function of enzyme concentration can be used to judge whether an enzyme is more endoglucanase-like or CBH-like. Recent reviews by Kashyap and Naidu and Panda outline the pectinase enzymes in detail. Ruthenium red staining in plates and zymograms has also been used for assay of pectinase enzymes.

There is increasing worldwide interest in the use of ligninolytic fungi for bioremediation purposes and for biopulping applications. Three families of fungal enzymes, designated lignin-modifying enzymes (LMEs), consist of lignin peroxidases (LiPs), manganese peroxidases (MnPs), and laccases (LACs), and these play a key role in lignin biotransformation. Demethoxylation is the most obvious consequence of attack on lignin by these fungi. Other methods such as nuclear magnetic resonance spectroscopy have also been used to study the degradation of polymeric lignin, but these methods are not easily amenable for detailed physiological and biochemical studies on white rot fungi and their enzymes. The disadvantage in the use of dimeric lignin model compounds is the fact that, unlike the lignin polymer, they can be taken up and metabolized intracellularly by microorganisms, which can make it difficult to determine whether the degradation products observed really reflect actual ligninolytic activity. Therefore, ideally, lignin model compounds should be sufficiently macromolecular but at the same time facilitate efficient product analysis. A heme peroxidase different from other microbial, plant, and animal peroxidases, termed versatile peroxidase (VP), was discovered in Pleurotus and Bjerkandera species. Hydroxylation of both phenolic and nonphenolic lignin resulting in new phenolic substructures on the lignin polymer may make it susceptible to attack by LAC or MnP.

In contrast to gram-negative bacteria, nearly all gram-positive bacteria must be digested with a lytic enzyme before they can be lysed by a detergent. In addition to lysozyme, which is the enzyme most commonly used, several other enzymes are available. These include N-acetylmuramidase, which is isolated from Streptomyces globisporus and also cleaves the muramic acid backbone; lysostaphin, an endopeptidase that is isolated from Staphylococcus sp. strain K-6-WI and is specific for the cross-linking peptides of other staphylococci. There are two approaches available for disrupting recalcitrant bacteria. The first involves making the cells susceptible to one or more lytic enzymes by growing them in the presence of wall-component analogs or antibiotics, and the second involves the use of any of several physical methods for cell disruption. The moles percent G+C content of DNA can be estimated by several different methods. The methods described in this chapter use thermal denaturation, high-performance liquid chromatography (HPLC), or dye-binding fluorimetry. Bacteriologists have been criticized for being a bit sloppy in doing and reporting DNA reassociation experiments, compared with those researchers working with eucaryotes. Although some of this criticism is justified, there are additional reasons for greater variability in bacterial DNA experiments. First, the sheer number of bacteriology laboratories involved in performing DNA reassociation experiments has contributed to the variability of results. Second, the hydroxylapatite (HA) procedure has been used for most eucaryotic studies, whereas all of the procedures discussed in the chapter have been used extensively by bacteriologists.

This chapter describes several of the most common nucleic acid analyses performed in vitro to characterize a cloned DNA segment carrying a gene or genes. Newer polymerases that are used for DNA sequencing include modified T7 phagederived DNA polymerase and a variety of thermostable DNA polymerases such as that from the thermophilic bacterium Thermus aquaticus. The authors have successfully used the kit marketed by U.S. Biochemicals for many years; however, other products may be just as effective. It is strongly recommended that laboratories use kits from U.S. Biochemicals or competing manufacturers for applications in which they need to do manual sequencing and gel electrophoresis. The gel mobility shift assay (also called the gel retardation assay) is based on the differences in the degrees of electrophoretic mobility between nucleic acid fragments and nucleic acid-protein complexes. Although the assay has been used successfully to study binding to RNA, this discussion will be limited to the most common use, the study of binding to dsDNA. The outcome of the assay is the identification of specific DNA targets of the protein of interest. A section reviews ChIP assays from a number of laboratories relevant to prokaryotic systems. Commonly used in vitro techniques described elsewhere in the chapter generally require biochemical purification of a DNA-binding protein of interest and some knowledge concerning the location of its DNA target(s). Once a gene or promoter has been cloned, it is of interest to determine the start site and the size of the RNA transcript.

Spontaneous mutations are mutations that occur in the absence of exogenous causes. Of particular interest are the spontaneous mutation rates of organisms that live in environments so extreme that the coding properties of their DNA should be destroyed. This chapter provides a guide to methods used for calculating mutation rates in the hope that it will be useful to scientists and students who wish to use mutation rates in their research. In addition, the Luria-Delbrück distribution applies to other cases in which a rare initiating event is amplified in a population. There are two basic methods to determine mutation rates: mutant accumulation and fluctuation analysis. All methods to estimate spontaneous mutation rates are based on theoretical models of mutational processes and cell growth. Of the less complicated methods, the Lea-Coulson method of the median and the Jones median estimator are reliable if mutation rates are low to moderate; if mutation rates are very low (m = 1), only the p0 method is applicable. Drake's formula is based on the same assumption as the Luria-Delbrück method of the mean, i.e., that mutations occur only during the deterministic period of mutant accumulation. All the methods for calculating mutation rates from fluctuation tests are discussed and are dependent for their applicability on the model of expansion of mutant clones originally described by Luria and Delbrück and extended by Lea and Coulson.

Transposon mutagenesis has been used to clone genes, to construct reporter gene fusions, to construct correlated physical and genetic maps of cloned DNA segments, to map entire bacterial genomes by pulsed-field gel electrophoresis, to construct conditional mutations with portable promoters, to introduce desired origins of conjugal transfer or replication into chromosomes and plasmids of interest, and to determine the sequence of large DNA regions without the need of sub-or deletion cloning. In this chapter, a subset of these applications and the corresponding experimental protocols are described. Region-directed mutagenesis usually involves the isolation of Tn5 insertion mutations in genes cloned into plasmids in Escherichia coli, followed by the characterization of the mutant phenotype of this (heterologous) host or the organism of origin of the cloned genes after the reintroduction of the Tn5-mutated loci by gene replacement. The chapter focuses on the use of bacteriophage λ and conjugable narrow-host-range or conditionally replicating plasmid vectors to deliver Tn5 and its derivatives and describes experimental protocols that are commonly used to carry out the random mutagenesis of (predominantly gram-negative) organisms.

The elimination (curing) of a genetic trait by treating the bacterial population with chemical or physical curing agents such as acridine dyes, ethidium bromide, sodium dodecyl sulfate (SDS), antibiotics, high temperature, or electroporation indicates that the expression of that genetic trait is linked to the presence of a plasmid. This chapter deals with the isolation, purification, and characterization of bacterial plasmids and also provides some information concerning the novel application of plasmid biology in gene therapy. The chapter provides a representative rather than comprehensive account of techniques and references concerning the biology of plasmids and their use as tools in molecular biology and pharmacology. The lysostaphin and lysozyme method was adapted in large scale isolation methods. The cold alkaline pH method is a scaled-down version of the one described in large scale that has been used to prepare Escherichia coli and Vibrio anguillarum plasmids. The acid phenol method is based on the addition of an acid phenol extraction to the cold alkaline lysis preparation of DNA and has been used very successfully. The boiling method described here is the original method of Holmes and Quigley, used effectively for screening of high-copy-number plasmids. The protocols to isolate linear plasmids involve cell lysis, in some cases followed by CsCl-ethidium chloride gradient purification, and the separation of the linear plasmid from the chromosomal DNA by either sucrose gradient or agarose gel electrophoresis.

One realization that has come from comparing multiple bacterial genome sequences, including multiple isolates from the same species, is that gene transfer is an important force in bacterial genome evolution. In the laboratory gene transfer is essential for the study of bacteria and for learning more about all living organisms. Three processes in bacteria can broadly define the transfer of DNA: transformation, transduction, and conjugation. This chapter focuses on the many genetic tools available to manipulate the genetic content of Escherichia coli. A DNA molecule that does not have its own origin of replication must integrate into either the host chromosome or another autonomously replicating element such as an endogenous plasmid. In E. coli a modified derivative of the bacteriophage T4 offers some advantages for transduction in that it packages twice as much DNA as P1 and also is less sensitive to capsules found on many pathogenic strains of E. coli. Transformation of bacteria by use of either naturally competent organisms, the process of electroporation, or chemical competency relies on the direct uptake of DNA by bacteria. Conjugation can be used as a tool to deliver plasmids that are capable of being stably maintained in the target host or as a tool to deliver “suicide vectors” that cannot replicate in the target host.

This chapter presents an overview of gene transfer systems and their applications in several important groups of gram-positive bacteria. Bacteriophage-mediated generalized transduction is an important method of genetic manipulation that can be accomplished using either lytic or temperate phages. The competent state is transiently induced when a culture of Streptococcus pneumoniae achieves a critical cell density. Whether or not this element retains the ability to transfer itself to other Staphylococci or has become inactivated by mutation remains to be determined. A section focuses on the lactococci and related food-fermenting bacteria. In the case of organisms like Lactococcus lactis, the relative ease of transformation- and conjugation-based transfer methods might account for this trend. The use of the upp gene and the pheS* cassette in the development of a counter selection system for Enterococcus faecalis described elsewhere in the chapter may be adaptable to various LAB strains, although this will need to be tested for each species. The primary focus of the chapter is on advances that have been developed for genetic analysis of Bacillus subtilis and also on some basic protocols for particularly important types of genetic manipulation. In contrast to the gram+ bacteria, members of the genus Mycobacterium belong to the cluster of so-called high-GC gram+ bacteria based on the nucleotide composition of their genomes.

In recent years several laboratories have developed effective plating techniques, identifying genetic markers that do not target cell wall synthesis, fusing archaeal promoters with recombinant genes, and isolating native vectors and promiscuous nonnative vectors. This chapter focuses on tractable systems that are currently available for the Archaea. Due to fundamental differences between gene transfer systems for each archaeal branch, the chapter is divided into three inclusive sections covering the halophilic and methanogenic Euryarchaeota and the hyperthermophilic Crenarchaeota. Despite varying degrees of difficulty growing Archaea, all three systems are routinely used by laboratories conducting research on archaeal genetics and can be mastered by anyone with a fundamental knowledge of microbial genetic techniques. Under low oxygen tension, Halobacterium sp. NRC-1 induces purple membrane patches in the cell membrane and buoyant gas vesicles intracellularly, which increases the availability of light and oxygen and allows a period of light-driven proton pumping and phototrophic growth. Targeted manipulation of the chromosome by directed recombination was recently added to the growing list of approaches for the genetic analysis of Sulfolobus solfataricus. Plasmids that do not replicate in S. solfataricus can be used to introduce DNA into the genome.

Lytic (or virulent) phages usually have only a single outcome upon infection of a bacterial host: reproduction of the virus and lysis of the bacterium. As a consequence, plaques appear as perfectly clear areas of infection, within which all of the bacterial cells are dead. The role of phage genetics in microbiology has expanded with the realization that we understand only a small fraction of bacterial diversity. Microbes such as Escherichia coli and Salmonella have been deeply studied and to great effect, but as our attention has turned to the broader bacterial community, new bacteria have brought new genetic challenges. This chapter describes some common phage methods and approaches that can be applied to virtually any bacterial host. Several collections of mycobacteriophages had been assembled previously with a view to using their host-range specificities for the typing of clinical isolates. One of these phages had been shown to mediate generalized transduction in Mycobacterium smegmatis, and researchers have achieved uptake of phage DNA by M. tuberculosis. The chapter also describes how phages can be isolated, purified, and genomically characterized, how shuttle phasmids can be constructed and utilized, and how phages can be used to construct a variety of genetic tools. The potential for phage genetics in any bacterial host is vast, and the approaches and methods described in the chapter represent just a small part of what bacteriophages have to offer to the microbial genetic engineer.

With more than 100 fully sequenced microbial genome sequences publicly available from many of the deep divisions of bacteria and archaea, protein and DNA sequence similarity searching is one of the most widely used methods for analyzing bacterial genes and genomes. In this chapter, the author focuses on more effective methods for similarity searching with protein and DNA sequences, with the goal of identifying distantly related genes in bacteria and bacterial populations. While strategies for similarity searching are often presented as a process of selecting a sequence comparison program, and possibly the scoring matrix and gap penalties, the chapter provides a different perspective. The chapter summarizes the statistical basis for inferring homology; from this statistical perspective, several strategies emerge for improving the effectiveness of similarity searches. The author says as on 2003, there are two widely used sets of programs for searching protein and DNA sequence databases: BLAST and FASTA. All similarity searching programs are most effective when comparing sequences at the protein or translated-protein level, and modern versions of the BLAST and FASTA programs provide efficient algorithms for translated searches with sequences that may include frameshift errors and termination codons. To test the ability of translated-DNA similarity searching to identify anonymous DNA sequences, the author selected either 10 10,000-nucleotide sequences or 100 1,000-nucleotide sequences at random from the completed Archaeoglobus fulgidus, Escherichia coli O157:H7, or Streptococcus pyogenes genome. These DNA sequences were used to search the nonredundant (NR) database, excluding all sequences from either the phylum or superkingdom.

Phylogenetics is the study of evolutionary relationships among organisms or genes, and phylogenetic analyses aim at estimating or reconstructing the evolutionary relationships among such “operational taxonomic units” (OTUs). These relationships are usually visualized as a phylogenetic tree, which portrays the evolutionary history of the OTUs. In this chapter the OTUs of interest are represented either by DNA or amino acid sequences. A reader with little or no prior experience, however, should be able to use the chapter as a guide to the options available for different types of data as well as to software available for doing the analysis. Importantly, if the rooting aspect of a tree is crucial to resolve a question, it is also likely to be the most unreliable part of the analysis. Halobacteria would remain monophyletic but the Euryarchaeota would become paraphyletic. With the increasing amount of sequences available, mainly from the genome-sequencing projects, it has become increasingly evident that many genes from prokaryotic genomes have different evolutionary relationships due to transfer of genes between lineages or species, a phenomenon termed lateral or horizontal gene transfer (LGT). It is apparent that the range of phylogenetic techniques is considerable, as are the questions that can be addressed using these techniques. It is also clear that phylogenetic analysis cannot be regarded as a "black box" and that each analysis will be "custom made".

Microorganisms are the key to Earth’s habitability. They harvest light energy, produce organic matter, and facilitate the turnover of key bioelements like nitrogen (N), phosphorus (P), and sulfur (S). Furthermore, it now appears that certain ubiquitous marine microorganisms, e.g., Synechococcus, and Prochlorococcus, may have reduced cell quotas of membrane phospholipids, so they would not be accurately represented in the environmental microbial biomass assessment. The correlations between nucleic acid synthesis, protein synthesis, and cell growth are so universally accepted that they lend themselves well to the study of complex microbial assemblages in nature. This chapter focuses on the most basic and most widely used aspect of the environmental microbial nucleotide fingerprint, namely, the measurement of cellular ATP as a biomass indicator. The preferred method of ATP quantification is the firefly bioluminescence reaction, but a variety of analytical techniques are available for either discrete sample or continuous flow analyses. A review of analytical issues concerned with ATP extraction efficiency from soils has recently appeared. ADP and AMP are both quantitatively coextracted with ATP. Despite recent and significant progress, the field of microbial ecology is still "methods-limited" with regard to the most fundamental properties of natural microbial assemblages, namely, biomass and metabolic activity estimation of the total population.

This chapter discusses one potential approach involving genomic characterization of the naturally occurring microbial genomes, without the requirement of isolating individual microbes one by one in pure culture. It describes some of the methodological options and considerations, and presents a few of the protocols that have been used successfully for preparing genomic libraries from natural microbial assemblages. Some of the earliest large-fragment genome libraries from natural microbial populations made use of bacteriophage lambda cloning protocols. Today, advanced vector systems such as fosmids and bacterial artificial chromosomes (BACs) are now in wide use. BAC and fosmid libraries are useful tools that can be used to sidestep many thorny problems that still inhibit the accurate characterization of naturally occurring microbial communities. They represent useful tools for conducting sequence surveys to determine the phylogenetic and functional content and properties inherent in native microbial populations. They can also serve as reagents for gene discovery, gene expression, and functional genomic characterization of naturally occurring microbes, some still resistant to cultivation techniques. BAC and fosmid libraries can also serve as useful reagents in microarray construction and analyses of naturally occurring microbial dynamics and variability.

This chapter presents an updated collection of protocols for the identification of individual microbial cells by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. FISH of whole cells starts with a fixation of the sample containing the target cell types. Fixation stabilizes macromolecules and cytoskeletal structures, thus preventing lysis of the cells during hybridization, and at the same time permeabilizes the cell walls for fluorescently labeled oligonucleotide probes. The fixed cells are transferred onto gelatin-coated slides and incubated in a buffer containing the specific probe at a temperature near but below the melting point of the probe-rRNA hybrid. The subsequent washing step will remove unbound probe and leave only those probe-rRNA pairs intact that have no mismatches in the hybrid. Consequently, only target cells that contain the full signature sequence on their rRNA will be stained. Finally, hybridized cells can be enumerated by epifluorescence microscopy or by flow cytometry.

This chapter provides background information and basic methodologies required to carry out the measurement of rRNA abundance by hybridization with oligonucleotide probes. Much of the discussion in the chapter focuses on hybridization to RNA, but many of the methods presented are equally applicable to DNA. The chapter addresses the extraction of RNA from environmental samples, the design of oligonucleotide probes, probe labeling and hybridization, and methods for calculating rRNA abundance from hybridization data. Several methodological studies have evaluated the use of RNA hybridization in microbial ecology and have contributed to many of the protocols and methodological caveats.

Microbial communities can exhibit an enormous range of complexity, from those with a mere handful of populations to those with thousands of species derived from all three domains of life. For community analysis, genetic fingerprinting techniques allow the comparative profiling of many environmental samples and thus facilitate the spatial and temporal analysis of microbial communities in ecosystems. Two of these approaches, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), are described herein. In reality, complex microbial communities with population richness exceeding several hundred species are beyond single-primer pair PCR analysis. Many would consider the current analytical methods more exploratory in nature, just the beginning to quantitatively address the complex relationships between microbial communities. The marker should be composed of DGGE PCR products, and under no circumstances should size markers be used. T-RFLP has been used as an effective tool in the dissection of microbial communities. The digestions were carried out assuming that PCR products were generated with a labeled 63F bacterial domain-specific primer. Hence, all fragments are derived from the 5'- terminus of bacterial rRNA genes.

Fungi are of fundamental importance in terrestrial ecosystems, and their roles and importance are usually overlooked or underestimated by ecologists who study plants or animals. Filamentous fungi with a habit of exuding adhesive extracellular polysaccharides or mucopolysaccharides are important in soil stabilization through the formation of microaggregates and the binding of aggregates and particles. In the human environment, fungi are important in many food and industrial fermentations, but they also cause food spoilage and grow unwanted in our living and working spaces, creating problems of environmental health. This chapter highlights the fundamentals of discovering and identifying fungi in these diverse environments. The fundamentally different biologies of different groups of fungi mean that no single method will work to discover or isolate all fungi in any material or area. For this reason, some general methods are presented, which users may need to modify to study their fungi of particular interest. Biodiversity of Fungi presents an extensive review of this topic.

The filamentous fungi display remarkable and diverse metabolic pathways and show great versatility in the utilization of sources of carbon, nitrogen, phosphorus, sulfur, and other metabolites and in acquiring essential elements, e.g., iron and potassium. This chapter emphasizes practical aspects, methods, and simple assays for investigating fungal physiology and metabolism and introduces some of the specialized molecular techniques that have proved invaluable in studying this fascinating group of eukaryotic microorganisms. In studies of metabolism and gene expression, it is often necessary to examine RNA and protein synthesis. A number of useful methods for RNA isolation from filamentous fungi have been devised. The ability to readily transform filamentous fungi with exogenous DNA has been revolutionary and provided the essential step required in cloning, characterization, and sophisticated manipulation of individual genes. The fungi have many remarkable characteristics and promise a wealth of exciting new phenomena and discoveries as their biology and genetics are further explored.

The study of filamentous fungi has a long history, covering investigations of their taxonomy, life cycles, physiology, and nutrition. These studies led to the genetic, biochemical, and molecular investigations. The filamentous growth habit of the fungi and their conspicuous, differentiated sexual structures set them apart from other microbial taxa. Methods for cultivating filamentous fungi and for study of their classical molecular genetic mechanisms have also developed in distinct ways from those used for bacteria and yeast. This chapter describes methods drawn largely from the study of the ascomycetes Neurospora crassa and Aspergillus nidulans. Certain features of filamentous fungi require special technical attention in any methodological treatment. These are (i) a filamentous habit, restricting studies of steady-state liquid cultures; (ii) a tough cell wall, requiring harsh cell disruption methods for biochemical and molecular work; (iii) the use of heterokaryons, mycelia having genetically different nuclei in the same cell; and (iv) in many cases, the lack of a natural diploid vegetative phase, for which heterokaryons, partial diploids, or artificial diploids may often substitute.

This chapter describes the principles and detailed methodology for carrying out DNA microarray experiments using the yeast Saccharomyces cerevisiae, and briefly reviews its attendant applications. Although microarray technology is widely used to study the transcriptome and its differential expression, this technology has also been applied in novel ways to study various aspects of cell biology. The two RNA samples, one derived from experimental RNA and the other from the control RNA sample, are converted to cDNA and labeled individually using fluorescent dyes such as cyanin 5 (Cy5) and cyanin 3 (Cy3). Next, the two labeled samples (targets) are combined in equal proportions and hybridized to a single microarray slide, therefore referred to as dual-color hybridization, involving competitive hybridization, i.e., simultaneous hybridization of the reference and experimental targets to a single microarray. The fluorescent signal emanating from each feature on the microarray slide is quantified by scanning the slide in a microarray scanner. The ratio of the fluorescence intensities emanating from the experimental versus control samples is then subjected to extensive statistical analyses to obtain the relative expression level of each gene on the microarray. A careful interpretation of the microarray data is important so that meaningful hypotheses can be generated for designing follow-up experiments. Further refinement of the statistical tools for microarray data analysis, including standardization of data normalization methods, would yield more robust and reliable data and enable comparison of microarray data generated in different laboratories and possibly find use in clinical practice.

Laboratory safety requires an awareness of the possible risks associated with the handling of hazardous materials, knowledge of mechanisms by which exposures may occur, use of safeguards and techniques that reduce the potential for exposures, and vigilance against compromise and error. Laboratory safety is the subject of several federal health and safety regulations and guidelines. The assessment of potential risks used to define classes of experiments assigned to each containment level, and the selection of safe laboratory practices, containment equipment, and facility safeguards that describe the safety requirements for each level were based on the scientific knowledge and experience acquired in bacteriological and microbiological laboratories conducting research and diagnostic studies involving human pathogens. The majority of cases in which the source of infection is identified could have been prevented by regular use of good laboratory safety practices. Several routine operations in the laboratory can easily become the source of laboratory-acquired infections. They require vigilance to guard against compromise and error. This chapter emphasizes the hazards associated with these operations and underscores the precautions necessary to conduct them safely. Chemical disinfection is necessary because the use of pressurized steam, the most reliable method of sterilization, and other physical methods are not normally feasible for disinfection of large spaces, surfaces, and stationary equipment.

The primary aim of culture preservation is to maintain the organism alive, uncontaminated, and without variation or mutation, that is, to preserve the culture in a condition that is as close as possible to the original isolate. The major disadvantages of the serial-transfer technique are the risks of contamination, transposition of strain numbers or designations (mislabeling), selection of variants or mutants, and possible loss of culture, as well as the required storage space. Commercially available strips impregnated with Bacillus subtilis spores are used to check the efficacy of the sterilization equipment and cycle. Many heterotrophic bacteria can be preserved in dried gelatin drops or disk. A variety of bacteria have been successfully preserved by drying on sterile silica gel granules. Freeze-drying (lyophilization) is one of the most economical and effective methods for long-term preservation of bacteria and other microorganisms. Two of the most common methods used are centrifugal freeze-drying and prefreezing. Furthermore, many culture collections serve as centers of expertise for preserving the microbial germplasm and are very useful in training others in the do's and don't's of culture preservation and maintenance.