Abstract: Publisher Summary The selection of a reasonable range of biochemical tests to be performed as a routine for bacterial identification and characterization is dependent upon the source of the material, the diversity of the bacteria likely to be encountered, and the aim of the investigation. The Gram staining procedure, which is usually done at an early stage in the investigation, allows observations on size, shape, and arrangement of cells, and gives provisional information on spore formation. In the tests depending upon the detection of end-products of bacterial decomposition of a substrate incorporated in the test culture medium, the ability of the organism to grow in the test medium without the added substrate should normally first be confirmed before a negative result is recorded. The development of acidity or alkalinity is frequently demonstrated by a color change produced by a pH indicator incorporated in the test medium. The chapter discusses the oxygen requirement for the utilization of the compound. It describes the utilization of nitrogenous compounds. The chapter also discusses the decomposition of large compounds.

Abstract: Publisher Summary Chemical extraction methods for microbial cells may be used for various purposes The substances being extracted may be required in their native state and specialized methods are required to maintain this native state The main purpose of the extraction and subsequent fractionation may be in the estimation of the component substances and the prime consideration is to achieve as quantitative a yield as possible The chapter discusses extraction methods starting with whole cells without prior separation into different morphological components It focuses on chemical extraction and fractionation of the prokaryotic cell, but most of the methods are applicable to cells of eukaryotic micro-organisms In micro-organisms as in other cells, the soluble, intracellular material contains a wide variety of intermediary metabolites and related compounds These range from free amino acids, sugar phosphates, and sugar nucleotides to relatively complex coenzymes such as the folate derivatives These compounds are present at the highest concentration in cells in the exponential phase of growth Growth at lowered incubation temperatures may also lead to their accumulation

Abstract: Publisher Summary This chapter presents the general cultural concepts related to fungal culture media. Agar is made in many countries, some of which are self-supporting in production or are nearly so. The type of seaweed from which agar is produced is different in every country. Agars of different origin differ considerably in chemical composition and also to a greater or lesser extent in gelling capacity, melting point, hardness (percentage needed for a certain set), and viscosity. The differences depend on the type of seaweed used. Most countries use mixtures comprised of proportions of different species, the time of harvesting (condition of the weed), and the weather conditions of each individual year on which the growth of the weed will depend. Differences also arise in processing, which entail cleaning, weather bleaching, pounding, boiling, blending, acidification during boiling, addition of previous boilings, chemical bleaching, straining, setting, alternate freezing and thawing, and drying. The selection of a satisfactory medium for stimulating growth and sporulation of a particular fungus can only be found by test. A few general principles that may guide ones choice are also presented.

Abstract: Publisher Summary This chapter discusses the assays of enzymes of the tricarboxylic acid and glyoxylate cycles. The photometric and spectrophotometric assay methods combine the most desirable advantages: simplicity, rapidity, sensitivity, specificity, and the fact that they can be used to assay relatively crude preparations of each enzyme. The aerobic oxidation of acetate to carbon dioxide is catalyzed by a series of discrete enzymatic reactions collectively referred to as the “tricarboxylic acid cycle.” The purified enzyme is conveniently assayed by determining the formation of citrate from oxaloacetate and acetyl-S-CoA either colorimetrically or enzymatically.

Abstract: Publisher Summary Cytological studies of the cells of fungi have revealed the presence of a cell wall surrounding the fungal protoplasm Recent electron photomicrographs indicate that the wall is composed of an outer dense layer about 005μ thick and a less dense layer of about 03μ The inner part of the wall may in turn be subdivided into about three layers Cell walls are composed of polymers of glucose and mannose, more rarely galactose, with smaller amounts of protein, lipid, and chitin A number of workers have isolated the cell walls by mechanical disintegration of the fungal cells and subsequent centrifugation and washing Selective digestion of the cell wall by enzyme systems has been effected in several species of yeasts and filamentous fungi, resulting in the liberation of the fungal protoplasm in an intact and spherical form Electron microscopy observations on ultrathin sections of these bodies did not show any structure corresponding to the wall of the cell The occurrence of forms of fungi resembling protoplasts under particular cultural conditions, or under abnormal conditions, is well known From current studies, it would appear that unbalanced growth can occur Under many conditions, suppression of wall synthesis may be effected without concomitant inhibition of protoplasmic growth A variety of lytic organisms, mainly bacteria and actinomycetes of the soil, are able to excrete all kinds of cell wall digestive enzymes into the surrounding media

Abstract: Publisher Summary This chapter describes physical and chemical methods for disintegrating microbes for preparing biologically active fractions. Direct measurement of cell disintegration can be sometimes dispensed with and the criteria of yield of the desired enzyme activity be relied on, especially when one of the chemical methods of extraction is used. In any comparative work and where control or location is studied by means of a physical method of disruption or with spheroplasts, some method of assessing disintegration is essential. Estimation of cell disintegration by measuring the release of typical cell components can be made quantitatively more precise than direct counting. There is some difficulty in interpretation and in obtaining a normal zero time and 100% standards. For instance, the measurement of the release of soluble protein is often used. This depends partly on the degree of disruption, but also on the method used to separate soluble protein from insoluble debris.

Abstract: Publisher Summary The past four decades have produced a wealth of information concerning the metabolic pathways utilized by microorganisms to degrade aromatic compounds Although organisms from many different groups are capable of degrading the benzene ring, it is notably species from the genus Pseudomonas that have yielded much of the present information Catechol and protocatechuic acid are central intermediates in the degradation of many different aromatic substrates The further metabolism of these two compounds may precede either by fission between the hydroxyl groups or between the bond adjacent to one of the hydroxyl group Not all aromatic compounds are aztabolized through catechol or protocatechuic acid The true metabolic intermediate compounds in an aromatic pathway may act as inducers for enzymes unrelated to those used for the degradation of growth substrate One of the most useful techniques in the study of aromatic metabolism is the method of simultaneous adaptation The majority of enzymes concerned in the degradation of the benzene nucleus are synthesized in response to the presence of an aromatic substrate

Abstract: Publisher Summary 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.

Abstract: Publisher Summary This chapter presents the methods for the preparation of a wet mount of bacterial cells. It is used extensively both for the observation of bacterial motility and to prepare micro-organisms for phase contrast observation. Bacterial stains are of two kinds: (1) those that are no more than histochemical reactions, such as the Gram stain and the acid-fast stain, which are of diagnostic significance, telling the observer how the bacterial cell reacts to certain chemical manipulations but giving no real information concerning the structure of the cell and (2) those that are designed to throw some light on the nature of cell component structures. The wet mount has two main functions in microbiology: to determine whether or not a micro-organism is motile and to present unstained material for phase contrast microscopy. The method of preparing the mount is similar for both purposes and the two are often combined.

Abstract: Publisher Summary The cell wall of a microbe may be defined as the principal structure of the cell responsible for mechanical rigidity and shape, and essential to the normal functioning of the cell. In most organisms, with the exception of gram-negative bacteria, there are clear morphological, functional, and chemical differences between the cytoplasmic membrane and the cell wall that lies immediately outside this membrane. In these cases the cell wall can usually be mechanically separated from the cytoplasmic membrane and therefore may be considered as a separate morphological entity. The cells of gram-negative bacteria have more complicated multilayered walls that have external lipoprotein-containing layers having a morphological similarity to a “unit membrane,” in addition to the internal “unit” membrane bordering the cytoplasm. It is not yet certain whether these two membrane systems are separate entities with different structures and functions.

Abstract: Publisher Summary This chapter discusses the assay of enzymes representative of metabolic pathways There has been continued interest in assessing the kind and relative contribution of the various pathways of carbohydrate metabolism in microorganisms These metabolic pathways were established mainly during the 1950s and few changes have been made since that time except for embellishments, such as the steps in the modification of a variety of carbohydrates and polyols for entry into the central pathways The necessity of preserving enzyme activity during preparation of the extract is a major problem Another problem is the fact that such extracts are very crude and present the maximum possibility of side reactions, which can obscure the enzyme activity under test The probability of a major role of an enzyme is strengthened when its presence is influenced by physiological conditions The most convincing evidence involves showing the induction of an enzyme in response to the relevant growth substrate, or the predicted change in physiological behaviour of a culture due to loss of an enzyme by mutation The chapter concludes with a discussion of glucose and gluconate dehydrogenases

Abstract: Publisher Summary This chapter presents the techniques for microscopic preparation. In fungi, as with other organisms, microscopic preparations must be made for the study of detail and for accurate measurements of spores and other structures. The nature of the fungus itself and its host, if any, determine the method to be employed in making the preparation. Thus, internal plant-parasites are often examined by techniques appropriate to the host; external parasites may be embedded in a film of cellulose acetate and stripped off the epidermis of the host. Saprophytic moulds are often grown on the microscope slide on which they are to be examined. The chapter presents the methods particularly suitable for superficial parasites such as Erysiphaceae and Microthyriaceae as well as for saprophytes on leaf surfaces. Although pieces of fungi growing superficially on leaves or other surfaces may be scraped off with a needle and mounted, it is often preferable to remove them in more or less intact sheets. This is easily done either by embedding them in, or sticking them to, transparent films. Slide cultures provide an admirable method for the examination of moulds. In these techniques, the fungus is induced to grow on a small quantity of agar medium on a microscope slide and the agar is then discarded leaving the sporing structures and aerial mycelium more or less firmly adhering to the glass. This is particularly useful for moulds such as Penicillium and Aspergillus, whose conidiophores and chains of conidia are very fragile and do not lend themselves to teasing.

Abstract: Publisher Summary This chapter describes the principle of free-flow electrophoresis. “Continuous deflection electrophoresis” is a method of separation, based on a different principle and also suitable for preparative problems. In this electrophoretic procedure the mixture to be separated is injected continuously in a fine stream into the medium—that is flowing uniformly at right angles to the electric field. Molecules and particles with differing charge are then deflected from the direction of flow at an angle that depends on the rate of flow of the medium and the electrophoretic mobility of the particle. As it is possible to apply this principle to a buffer film that flows vertically, continuous free-flow electrophoresis can be applied both for analysis and preparation to a whole spectrum of materials from low-molecular substances to coarse particles. The method thus permits not only the separation of cells and subcellular particles or organelles, but also the separation and preparative isolation of their enzyme systems without loss of activity.

Abstract: Publisher Summary This chapter describes techniques involving compounds known as “optical brightening agents.” Brighteners are strongly fluorescent when excited by ultraviolet radiation and are accepted and retained as dyes by a wide variety of organic structures. As industrial applications have expanded, especially for the treatment of wool, cellulose, and synthetic fibers, many chemical derivatives, largely of unknown properties, have become available for biological investigations at low cost. Industrial samples are not usually pure and may contain several chemically related components. They are also associated with diluents such as sodium sulfate, sodium carbonate or urea, often in large proportions. An important feature of fluorescent brighteners is their general lack of toxicity to micro-organisms and, as far as is known, to other cells. When added to nutrient media, they can impart fluorescence to the growth of many organisms. This is particularly evident when brighteners are incorporated into solid media. The ensuing colonies, when examined by ultraviolet light, can be shown to be largely composed of stained organisms although a proportion, as found with the direct-staining technique, are almost non-fluorescent.

Abstract: Publisher Summary One of the weaknesses of numerical analysis is that most of the physiological and biochemical tests give no information on the underlying enzymic mechanisms. This chapter describes simple biochemical tests, performed with resting cells; giving a reasonable certainty that only one enzyme is involved. Owing to the large number of enzymes in the cell, their complex interactions, and permeability factors, resting cells can be used only in a limited number of cases for the reliable identification of single enzymes. In most cases, it is necessary to disrupt the cell structure, to work with cell-free extracts and to impose conditions such that only a selected enzyme system will be detected. Several enzyme reactions are complex, time consuming, or require expensive or unusual equipments. Some of these have been developed in the laboratory; they concern the detection of particulate and of soluble, coenzyme-linked dehydrogenases, as well as the enzyme systems of the Entner–Doudoroff pathway. These enzymes are presented in the chapter as examples of the many services that can still be rendered to bacterial taxonomy.

Abstract: Publisher Summary This chapter describes the preservation of fungi. It is desirable to retain some form of reference material, both for use during the work and later as a permanent record. This material should be maintained both as living cultures and dried reference material. If the fungi have been used in the production of an antibiotic or for other biochemical research, it would be desirable or perhaps essential to retain it in the living form and in such a condition that its physiological activity remains constant: If the work is of a taxonomic nature it might be sufficient or only possible (many fungi have not yet been grown in artificial culture) to retain a record of its structure by means of slides and dried specimens. The aim of the curator of a culture collection is to maintain the organisms alive and healthy in a condition as near as possible, both physically and physiologically, to their condition at the time of deposit. In some cases it is necessary to work on the cultures before incorporation to find the best methods for maintaining them. The running of the collection and the methods of maintenance employed are designed to minimize the hazards to which cultures are exposed. Sensitivity to pH and osmotic pressure must also be considered. Most fungi grow at a pH 5-7. There are a few fungi termed “osmophiles” that thrive on media with a high osmotic pressure, which can be produced by increasing the sugar or salt concentration of the media.

Abstract: Publisher Summary This chapter describes the procedures used for the assay of enzymes of CO2 metabolism in microorganisms. The description of the assay methods for each enzyme is preceded by a brief summary, which includes the species distribution of the enzyme, the probable physiological role of the enzyme (where appropriate), and some relevant properties. The species distribution indicates some types of microorganisms in which the enzyme has been found as a possible guide to further studies. The summary of the properties of the enzyme is in most instances confined to a description of the requirement for dissociable cofactors and of the effect—if any—of metabolites, which are neither substrates nor products of the reaction on the rate of catalysis. The assay procedures described have been devised in most cases for the enzyme in a given microorganism. The procedures have been modified from the original description when necessary, such that standard total volumes of 1 ml and 3 ml are used for spectrophotometric and manometric procedures respectively. These standard volumes are adopted for convenience and have no especial significance.

Abstract: Publisher Summary This chapter describes the methods that are applicable to the cultivation of the common moulds and other fungi imperfecti, which are for the most part conidial states of ascomycetes. The methods used for the isolation of fungi and for their cultivation depend largely upon their environment in nature. For isolation, fungi can be roughly divided into the parasitic and saprophytic species, although this distinction is not clear-cut. There is a close relationship between the genetic complement, the health and environment of the host, and the parasitism of many fungi. In general, it is easier to isolate the saprophytic moulds than specific animal or plant pathogens. In either case, isolation is made easier if the fungus is producing either sexual or asexual fructifications so that the isolation can be made from single spores. This makes purification easier and simplifies later handling and identification of the fungus. In the cultivation of fungi the dependence of the growth form on the nature and concentration of the constituents of the nutrient media should also be appreciated. For identification and maintenance, fungi are normally grown on solidified media as sporulation, which is usually stimulated by this form of growth. When fungal mycelium is required as opposed to spores, it is preferable to grow fungi in liquid cultures where sporulation is generally suppressed.

Abstract: Publisher Summary The reactions between antigens and antibodies are of several different types and have many applications in the biological sciences. This chapter discusses the use of antigen–antibody reactions at the research level, with particular emphasis on microbiological applications. Some details of appropriate injection schedules for the preparation of antisera of various types are also included. Antibodies (immunoglobulins) are produced by warm-blooded animals, and by some poikilothermous vertebrates maintained at temperatures of 19°C and above in response to parenteral experiences, natural or artificial, of antigens—substances of high molecular weight. The antibodies produced in response to an antigen combine almost specifically with that antigen. They are all members of the globulin class of proteins, but antibodies to a particular antigen may be divisible into immunoglobulins of very different molecular weight.

Abstract: Publisher Summary This chapter discusses the inhibitors of electron transport and oxidative phosphorylation. For the investigation of the function and properties of the respiratory chain in mammalian systems—that is, in mitochondria, inhibitory agents play a major role. These substances can act either on electron transport, energy transfer, or the coupling between phosphorylation and electron transfer. Because an overlapping of the effects of certain inhibitors is frequently observed, the classification of the various substances as uncoupling agents or energy transfer-inhibitors is somewhat superficial, but useful. These can be classified either by their site of action or by their chemical nature. The use of inhibitors is a useful means of elucidating pathways of electron transport and oxidative phosphorylation in microorgamisms. The composition and function of bacterial cytoplasmic membrane systems shows that their properties are in general similar to those of mitochondria.

Abstract: Publisher Summary Slime mould is a group of micro-organisms having as a characteristic phase in their life-cycle the plasmodium, an irregular mass of protoplasm containing many nuclei but not sub-divided into cells. Their life-cycle also includes an amoeboid phase, the amoebae being capable of developing flagella under appropriate conditions. The Plasmodiophorales are obligate parasites, producing a plasmodium within host cells, and are commonly regarded as fungi. The Acrasiales have an amoeboid phase, the amoebae later undergoing aggregation to form a structure capable of migration and resembling a minute slug. Physarum polycephalum has become more widely known. The plasmodium of this species has been used for studies on the rheological properties of protoplasm, especially protoplasmic streaming. The plasmodium of Ph. polycephalum is bright yellow, may cover several square centimeters and many thousands of nuclei, which in Ph. polycephalum and some other species have been shown to divide synchronously. Myxomycetes obtain nutrients mainly by attacking and digesting other micro-organisms, such as bacteria, yeasts, and fungi. In the active state, they require moisture, are aerobic, and do not usually tolerate near-freezing conditions, although in the resting state cold and dry conditions are tolerated.

Abstract: Publisher Summary This chapter discusses the methods for studying bacterial spores. Spores are characteristically formed by Bacillus and Clostridium species, but also by Sporosarcim , and spores of thermophilic Actinomyces and cysts of Azotobacter have features in common with spores of the Bacillaceae . Sporulation in bacteria has proved to be an excellent model system for studying the control of cellular differentiation, for during sporulation, the vegetative genome is essentially suppressed, and the spore genome, controlling synthesis of numerous new proteins and other easily recognized products and structures, is expressed within a very short period. The experiments described in the chapter reveal the secrets of resistance, dormancy, germination, outgrowth, and sporulation so that, eventually, the yet unexpected methods of controlling spores may become available.

Abstract: Publisher Summary This chapter discusses the evaluation of methods used to determine metabolic pathways. It also discusses the methods used to investigate the main metabolic pathways found in microorganisms. Two distinguishing features of the chemistry of living matter have called for the development of specialized experimental approaches and methods—namely, the extreme rapidity by which reactions of the metabolic network are catalyzed, and by the means by which the regulation of their rates is accomplished. The configuration of an enzyme, and the efficiency of its catalytic centre, may be profoundly altered when metabolites other than substrates or products are bound to the protein molecule. This recognition of allosteric behavior permits a description of mechanisms by which many metabolic pathways may be regulated. The chapter explains the way in which the studies of metabolic pathways in mammals may resemble or differ from those concerned with microbes. With organisms of both kinds, some limited information can be obtained by relating the compounds excreted by the organism to those supplied in its diet. The chapter illustrates two themes for explaining the use of induced auxotrophs in determining biosynthetic. The first is that the complexities of metabolism require experimental findings to be interpreted with caution; and second, that the elucidation of biosynthetic pathways proceeds from an outline of the steps involved, through a more detailed knowledge of their chemistry, to a study of isolated enzymes and the nature of their regulation in the cell.

Abstract: Publisher Summary This chapter discusses centrifugal techniques for the isolation and characterization of subcellular components from bacteria. Centrifugal procedures are applied early in many microbiological investigations. For small volumes of culture fluid the micro-organisms may be harvested in a single batch process in a variety of swing-out or fixed-angle rotors. The majority of manufacturers of centrifuge equipment produce instruments operating at controlled temperatures with rotors of this capacity and capable of developing the necessary gravitational fields to sediment most micro-organisms. For larger volumes of culture fluid, a continuous flow rotor is to be preferred. Such rotors may be of the cream-separator type or a rotor designed or modified for continuous operation in a conventional laboratory centrifuge. In both continuous and batch harvesting, although the fields applied will theoretically sediment all of the organisms from the culture, the yield of cells is sometimes sacrificed to speed the harvest. Developments in the theory of sedimentation and the design of rotors, cells, and accessories have permitted the full exploitation of three basic physical parameters of biological objects to separate and characterize them by the application of centrifugal force. Separations may be achieved (1) on the basis of their size and shape, (2) by their density differences, or (3) by their mass differences.

Abstract: Publisher Summary The atmosphere contains all the major groups of microbes ranging from the algae to the viruses. Outdoors, with the changing seasons of the year, the air contains a varying assemblage of pollen grains, a prolific fungal spora in which all the major groups are well represented, occasional spores of ferns, mosses and algal cells such as those of the terrestrial genus Pleurococcus. The methods and apparatus used to trap airborne microbes and employ either impaction, impingement or sedimentation are described in the chapter. Other principles are utilized such as electrostatic precipitation. However, because before an electrostatic attraction or electron wind can be effective, a volume of air has to be drawn into the sampler. It is for most purposes simpler to utilize the essential suction pump to produce a jet of air and direct this and the suspended particles it entrains on to a prepared surface that retains the particles. The choice of method must be governed by such features as whether sampling is to be undertaken indoors or outdoors, the concentration in which the microbes occur in the air to be sampled, whether it is essential that the microbe be grown for identification and enumeration or whether, as is the case with pollen grains and the spores of certain obligate fungal pathogens of plants, they can be recognized by their morphological features under the microscope.

Abstract: Publisher Summary This chapter describes the methods of preparation, staining, and examination of specimens using fluorescent-labeled antibodies Fluorescent labeling of antibodies was introduced by Coons et al in 1941 They showed that fluorochromes—that is, substances that emit light in the visible region of the spectrum when irradiated with ultraviolet light, could be coupled to antibodies without loss of immunological specificity There are two main methods of applying the technique: direct and indirect A schematic representation of the use of fluorescent-labeled antibodies to locate bacterial cell-wall antigens with these two methods is given in the chapter The fluorescent-labeled antibody technique has found many applications in bacteriology, virology, pathology, mycology, and parasitology, both in the diagnosis of disease—that is, in the identification of pathogens, and in basic research The use of ferritin-labeled antibodies has enabled the electron microscopist to identify areas of specific antigen in ultrastructural studies in much the same way the fluorescent-labeled antibodies have been used to detect specific antigens at lower magnification with the light microscope Ferritin is an iron-containing protein present in many tissues in the body, but most readily prepared from spleen

Abstract: Publisher Summary The technique of disc electrophoresis derives its name from the introduction of disc ontinuities into both gel matrix and buffer systems. Several supporting media have been used for electrophoretic separation of biological compounds and each has its particular advantage. The advantage of using a gel as compared to—for instance, paper, is that the matrix of the gel imparts a separation based on the size of the individual components of the mixture undergoing separation. This was recognized by Smithies who suggested that the remarkable resolution obtained in starch-gel electrophoresis was because of molecular sieving in the gel matrix. Starch gel is a good medium for electrophoresis, particularly in flat blocks. It does suffer from the disadvantage that it is difficult to make gels of large pore size and difficulty is experienced in slicing and handling gels of high-urea content. Starch also has a small number of ionizable groups that cause electroendosmosis during electrophoresis.

Abstract: Publisher Summary Chemical tests have been frequently used in the taxonomy of the basidiomycetous fungi; indeed with careful use and rigorous interpretation they have been utilized to suggest and substantiate possible lines of phylogeny. Many chemical tests simply separate taxa that can be isolated quite easily by conventional characters such as veil and ring structure and viscidness. Thus the pinkish hue is produced when sulphuric acid is run onto the gills of Amanita phalloides and separates it from its close relatives but so can its cap color and veil form; ammonia used in the same way however will allow the olivaceous and yellow forms of the normally purple-capped Russula drimeia to be picked out from amongst the other greenish Russula species. These examples show that in certain cases assistance is offered by carefully selecting the tests to confirm a suspected species' autonomy; they can therefore be useful spot or field tests. The chapter suggests that chemical tests must be critically assessed as to their worthiness, for like other characters a reagent useful in one group of organisms might be of very reduced importance in another, possibly quite closely related, group; one must not allow chemical tests to run away with the taxonomy but they must be integrated into it.

Abstract: Publisher Summary Methods for analyzing soil fungi may be broadly classified as direct and indirect. With direct techniques, attempts are made to establish the presence of fungi and their relationship to the environment by direct observation. This can be done most simply by mounting and staining soil particles and studying them under the microscope for the presence of mycelium or hyphal fragments. The preparation of soil mounts may involve considerable disturbance of the material so that the relationship between the fungus and its environment is destroyed. Other techniques therefore involve processing and sectioning soil so that fungi may be studied in situ with a minimum of disturbance from the original state. Alternatively, known fungi may be added to soil and their behavior studied by direct examination following recovery. Indirect methods of analysis attempt to evaluate the role of a fungus by inference. Quantitative and qualitative estimates of fungi present in a given soil sample are made by processing the soil in such a way as to recover the fungi it contains. No single method recovers more than a small fraction of the spectrum of fungi present and to study a sample adequately may involve processing by a variety of techniques. The simplest method of demonstrating the presence of fungi in soil is by direct microscopic examination of soil particles for the presence of hyphae.