Microscopy in mycological research with especial reference ...formatex.info/microscopy5/book/646-659.pdf · Microscopy in mycological research with especial reference to ultrastructures

Microscopy in mycological research with especial reference to ultrastructures and biofilm studies

Iqbal Ahmad and Mohd Sajjad Ahmad Khan*

Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh-202002 India *Corresponding author’s email: [email protected]

Microscopy is the only technique whereby fungal morphology and its cellular structures can be studied. Our understanding of physiology, intracellular and surface structures of fungi are dependent on microscopic techniques. These techniques involved use of classical light microscopes and electron microscopy. However, advancement of microscopic techniques such as fluorescent microcopy, confocal laser scanning microscopy, atomic force microscopy and others involving integration of automated computer monitoring have aided significantly to the mycological research especially structural alterations and biofilm studies. Growing awareness of heterogeneity in cells of microbial populations has emphasized the importance of advanced microscopy for visualization and understanding of the mechanisms underlying cell-to-cell variation. The main role of these techniques is in examining biological specimens such as: (i) visualizing the microbial cell surface layers and ultrastructures (ii) monitoring conformational changes of individual membrane proteins (iii) examining the morphology of biofilms especially exopolysaccahrides matrix containing proteins, carbohydrates and nucleic acids, with the capability of fully revealing them in three dimension (iv) studying the nanoscale structure of living microbial cells (v) probing the local mechanical properties of cell surface layers of single cells. A large number of critical molecular and cellular events occur at the cell surface of fungi; such as cell adhesion, secretion, cytoskeletal interactions, membrane dynamics and cell wall deposition. Investigating such phenomena is easily approached by selective visualization of surface regions of fungal and other eukaryotic cell systems using advanced microscopical techniques. The paper emphasizes the recent applications of various microscopic techniques to study cellular morphology and its alteration under stress conditions and biofilm monitoring in fungi.

Keywords: Confocal laser scanning microscopy; electron microscopy; atomic force microscopy; biofilms; cell surface.

1. Introduction

Fungi is one of the most diverse groups of heterotrophic organisms on earth, approximately 1.5 million species have been estimated, of which approximately 70.000 have been described [1]. Many fungal species contribute to ecosystem stability, traditional and modern forms of biotechnology, or are experimental model systems and sources for various industrial and therapeutic products. Others have threatened the humans in terms of food spoilage or infectious diseases. These substantial positive and negative impacts of fungi on human and ecosystem activities make it essential for us to better understand the fungal diversity, biology, and ecology. Fungi have been identified and characterized on the basis of macroscopic features such as colony morphology and cultural activities but there insight information regarding cellular structures and physiology needs microscopic examination. Cells are composed of genetic materials such as DNA and RNA, gene products like proteins and metabolic products viz. carbohydrates, lipids, metabolites, including inorganic components and other cytoplasmic contents. Most cells are less than 10 mm in at least one dimension, therefore microscopy is used to study their properties across the range from sub-cellular structures to multi-cellular assemblies [2]. Earlier in the science of microbiology, culture or community-based approaches where millions of organisms are studied collectively as a single entity were considered adequate for general physiological studies. However, microscopy of single cells and subpopulations has lately been realized as an important and sometimes indispensable tool when a community of apparently homogenous cells is investigated for phenotypic diversity. Today, new technologies provide the possibility to differentiate between subpopulations and to analyze single cells for altered gene expression using advanced microscopy, cell sorting and a growing number of fluorescent reporter tools and provides a more efficient and direct treatment of bacterial and fungal infections [3, 4]. Microscopy has played an important role in the analysis of microbial cells since Antonie van Leeuwenhoek described microorganisms using compound microscope in the late 1600s. Since then enormous improvements have been done in microscopy techniques such as phase contrast principle, invention of a variety of fluorescence imaging microscopes, the confocal microscope, as well as relatively new super-resolution microscopy methods like electron microscope, the atomic force microscope [5], and still the advancement is going on. Imaging the cellular and sub-cellular events had been an important technique required to answer many cell biological questions. Furthermore, a better insight into the regulation of differentiation in bacteria and fungi can lead to a wider application in biotechnology and medicine. Microscopic analysis allows finding characters that are not described by the macroscopic study of the structures and interactions: toxic effects on unltrastructures, changes of the isolates when they coexist in the same substratum, antagonistic mechanisms, new morphologic characters of the fungi such as biofilms, etc. In this chapter, we have

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

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described the utility of the art microscopy in studying cellular structures and interactions and biofilm development in fungi.

2. Light Microscopy

Light microscopy (LM) is regularly used to obtain rapid, inexpensive qualitative and quantitative information about fungal cells. LM techniques used in mycological research includes (i) bright-field, (ii) polarizing, and (iii) fluorescent microscopy. The basic instrument is a conventional compound (bright-field) microscope, to which polarizing and fluorescence accessories are easily attached. A wide variety of stains can be used in combination with bright-field LM. For example, Toluidine Blue O (TBO) is a metachromatic dye that produces different colors depending on the nature of the component to which it is bound. Polarized light is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials when compared to dark field and bright field illumination, differential interference contrast, phase contrast, Hoffman modulation contrast, and fluorescence [6]. Polarized light microscopes have a high degree of sensitivity and can be utilized for both quantitative and qualitative studies targeted at a wide range of anisotropic specimens. Qualitative polarizing microscopy is very popular in practice, with numerous volumes dedicated to the subject. In contrast, the quantitative aspects of polarized light microscopy, which is primarily employed in crystallography, represent a far more difficult subject that is usually restricted to geologists, mineralogists, and chemists. However, steady advances made over the past few years have enabled biologists to study the birefringent character of many anisotropic sub-cellular assemblies [6]. Fluorescence provides a sensitivity that is not available in other forms of LM, and allows detection of fluorescing compounds present in amounts as little as 10-18 mol. The fluorescence may be induced by a wide range of compounds such as fluorescent dyes, specific antibodies or lectins that are conjugated to fluorescent markers and substances that fluoresce only in specific chemical environments [7]. For example, calcofluor, a fluorescent brightener, can be used as a highly specific probe to localize the mixed linkage (1-3), (1-4)-β-D-glucan. Fluorescence techniques have become an effective tool in microscopy. With the help of sophisticated equipment and an increasing number of fluorochromes, it is now possible to observe and differentiate a range of materials, microorganisms, and even living and dead cells or tissues. Most of the fluorochromes also allow the unrestricted observation of living cells [8]. The precise identification and localization of various components permitted by such compounds, coupled with the sensitivity of the fluorescence technique, has made fluorescence microscopy a valuable tool in fungal biology.

3. Confocal Microscopy

Confocal microscopy has also been developed as a technique with some advantages over light microscopy. The confocal microscopy differs with the conventional light microscope in the way the image is captured and conveyed to a computer screen [9]. The major difference between a confocal and a conventional microscope is the placement of a pinhole at the focal plane of the image in the case of the confocal instrument. A conventional light microscope captures light from below that passes through the specimen and on up through the objective and eyepieces to the eye. With confocal microscopy, a laser light beam enters the head of the microscope, passes down and out of the objective onto the microscope slide, and excites a fluorescent dye in a stained specimen. The fluorescent dye in the specimen is excited by the laser light and emits light of longer wavelengths that scatters in all directions. The fluorescent light is then captured by the objective. A module that sits on top of the head of the microscope receives the fluorescent image of the specimen and transfers it to a computer screen, only light from the plane of focus is captured by the computer. This removes out-of-focus light and generates a clearer image and allow optical sectioning of the specimen. The image is considerably sharper than that observed by conventional fluorescence microscopy which collects all the fluorescence emitted. The way this fluorescent light is manipulated makes confocal microscopy distinctive. There are two basic types of confocal microscopy: (i) confocal scanning tandem microscopy (CSTM) and (ii) confocal scanning laser microscopy (CSLM). CSTM uses mercury, tungsten, or xenon illuminators and has the advantage of allowing real-time observation of the specimen. Low light intensity can be a problem with this technique. CSLM uses laser illumination. A standard equipment includes an argon laser (488 and 514nm wavelengths) with or without a helium-neon laser (633nm wavelength). In many of the CSLM systems currently in use, real-time observation is not available because of potential damage to the eye by laser emissions. Images are produced, stored, and manipulated by image-handling software. Light intensity is not a problem with this method. CSLM can provide focused images to a depth of up to several hundred micrometers, depending on the nature of the sample, so that sequential sections may be obtained for three-dimensional reconstruction of the image [10]. The invention of CLSM known simply as a confocal microscope, have been the biggest breakthroughs in microscopy for decades. Confocal microscopy offers one big advantage: the ability to perform optical sectioning at the cellular level in a noninvasive way. CLSM can serially produce thin (0.5 to 1.5 micrometer) optical sections through fluorescent specimens that have a thickness ranging up to 50 micrometers or more [11]. It eliminates the need to embed material



and cut serial sections. The main advantage of this technique is to observe an object in three-dimensions such as the location of microorganisms in a wide range of natural and host environments; and to gather quantitative data of cellular structures in fungi such as thickness, area, and volumes.

4. Electron Microscopy

Electron microscopy (EM) is used where a higher resolution than that obtainable by LM techniques is required, mainly in studying the ultrastructures of biological and non-biological samples. Since its invention, electron microscope has been a valuable tool in the development of scientific theory and had contributed greatly to biology, medicine and material sciences. Electron Microscopes were developed due to the limitations of LM for a maximum magnification of 500 X or 1000 X and a resolution of 0.2 micrometers. Electron Microscopes are scientific instruments that use a beam of highly energetic electrons for observation and characterization of materials on a nanometer (nm) to micrometer (μm) scale. Since the earliest research in transmission electron microscopy in the 1950s, mycologists have kept pace with the developments in all areas of electron microscopy and have used them to great advantage in generating fine structural information on fungi. These recent developments include the use of scanning electron microscopy in the 1960s, X-ray microanalysis, cryopreservation and immunoelectron microscopy in the 1970s and 1980s [12]. All of these techniques will continue to provide mycologists with the means to gain morphological and analytical data at the ultrastructural level. This examination can yield information about the topography i.e. surface features of an object, crystallography, morphology, and composition. This technique is exploited in two modes (i) Scanning electron microscopy (SEM) in conjunction with X-ray microanalysis, and (ii) transmission electron microscopy (TEM) in the electron energy loss spectrometry (EELS) mode. TEM was developed before SEM, and is used to probe the structures of fixed, sectioned or freeze-etched fungal samples, producing two-dimensional (flat) images. These images can be combined, at first laboriously [13], and now computationally, [14] to provide detailed information about cytoplasmic organization. When used in conjunction with immunolocalization, TEM can be an effective method to study cell wall composition [15]. Chemical fixation of certain components such as proteins (using aldehydes such as glutaraldehyde, formaldehyde, and osmium tetraoxide,OsO4), unsaturated fats (using OsO4 in the presence of imidazole) and some polysaccharides (using RuO4) followed by embedding in a resin and staining of thin sections with heavy metals such as uranyl acetate and/or lead citrate, makes it possible to distinguish these components in fungal cell [16]. When first developed, SEM was used to image fixed/dehydrated/gold coated specimens, in three-dimensional surfaces. But more recent SEM developments have been made as they apply to fungal cells. CryoSEM can be used to image cells in a frozen hydrated state, and thus is a significant development for high-resolution imaging of fungi and plants [16]. Fungal cells are relatively easy to be freezed compared to preservation by chemical fixation [17]. Most freezing artifacts affect the cytoplasm and therefore mainly TEM is affected, whereas SEM is less. In CryoSEM specimens are studied in frozen [17] or slowly frozen under partial vacuum [18] conditions and visualized by SEM at low temperature. SEM of frozen hydrated samples and TEM of replicas obtained from samples fixed by rapid freezing provide images of cellular structures unaffected by chemical fixation. Environmental SEM is also called variable pressure SEM (VP-SEM). Typically, SEM imaging requires a high vacuum, ≤10-8 Torr [19]. Few biological specimens tolerate these conditions without rapid collapse and fewer still survive [16]. The environmental SEM uses a series of pressure limiting apertures [20] to allow the electron gun to operate at 10-7 Torr while preventing gas leakage from the specimen chamber, which can be maintained at 1-20 Torr. Dual beam SEM is a relatively recent development that adds manipulation (dissection, metal deposition, physical transfer) capabilities to SEM and TEM imaging, along with element detection using EDS. Thus, it becomes possible to select, prepare, image, and analyze specimens with high spatial precision [16]. Its improved resolution over light microscopy combined with the large depth of field makes it ideal for viewing a range of sample types, but the challenge for imaging biological specimens is preparing the samples for imaging without changing their morphology.

5. Atomic Force Microscopy

Significant structural changes are induced in fungal cells when processing for EM. To overcome this there is a need for development of novel methods, such as maintaining environmental stages of sample and use of metal-free processing, to achieve proper results. Uses of EM techniques are limited when used to image complex biopolymers such as the irregular proteins and polysaccharides that are abundant in biofilms mode of growth. Since samples for EM need to be coated with metals, the size of the metal grains restricts the structural details being observed. Knowledge of the structure and properties of microbial surfaces at the nanometer level is of major importance to understand their functions in the natural environment and to efficiently exploit their potential in applied research. Although the ultrastructures of microbial surfaces have been studied for more than 3 decades using electron microscopy direct information on native surfaces could not be obtained. Exciting new opportunities are now offered by atomic force



microscopy (AFM). The technique was invented in 1986 [21] to probe the ultrastructure and local properties of surfaces. In recent year, AFM has provided a range of new opportunities for viewing, manipulating and analyzing biomolecules in the environments. AFM yields three-dimensional images of bio-systems (single molecules adsorbed on surfaces, lipid membranes, 2D protein crystals, living cells) in aqueous solutions and with (sub) nanometer resolution. In addition, it can also measure forces with remarkable sensitivity and positional precision [22]. AFM creates an image by scanning a sharp stylus, which is attached to a flexible cantilever, across the sample surface. While scanning, the force between the tip/probe (nm-scale) and the sample is measured by monitoring the deflection of the cantilever (with picoNewton (pN) sensitivity) detected with an optical lever, in which a red laser reflects from the cantilever surface into a four-quadrant photodiode. A topographic image of the sample is obtained by plotting the deflection of the cantilever versus its position on the sample [23]. Alternatively, it is possible to plot the height position of the translation stage. This height is controlled by a feedback loop, which maintains a constant force between tip and sample. This simple and highly sensitive technique can measure deflections caused even by individual molecules and atoms. The two most commonly used modes for imaging are (i) contact mode and (ii) intermittent/tapping mode, or dynamic mode. In contact mode, the tip is dragged across the surface and irregularities on the surface cause the cantilever, which is carrying the tip, to bend up and down. A laser is recording the bend, which can be directly correlated to the topology of the surface. In tapping mode an oscillating frequency is applied to the cantilever that makes the tip move up and down towards the surface. The tip is subjected to a combination of attractive and repulsive forces, which influence the amplitude of the oscillation [5]. Further information on the principles of AFM and on the different modes of operation can be found in the literature [5, 23, 24]. The AFM has atomic resolution on crystalline surfaces and nanometer resolution on other surfaces. Understanding the functions of microbial cell surfaces requires knowledge of their structural and physical properties. Electron microscopy has long been recognized as a key technique in microbiology to elucidate cell surface ultrastructure. An exciting achievement has been the development of cryotechniques which allow high-resolution imaging of cell structures in conditions close to the native state. Yet direct observation in aqueous solution remained impossible [25]. As opposed to most surface analysis and electron microscopy techniques, AFM does not rely on an incident beam, as in electron or light microscopy, the specimen can be directly observed at high resolution in aqueous solution. This makes AFM to investigate biological samples under physiological conditions under living conditions [23]. This enables real-time biochemical and physiological processes to be monitored at a resolution similar to that obtained for the electron microscope. Applications of atomic force microscopy in biological research began in the early 1990s. As the AFM became more integrated in life science research, this new tool provided a new approach for the examination of biomolecules including proteins, DNA, and highly topographic samples, such as bacterial and yeast cells at nanoscale resolution [5]. AFM can also measure minute forces within or between biological molecules a method known as Force spectroscopy (FS). This can be used to probe elasticity, surface forces, surface charge and hydrophobicity and to measure inter- and intramolecular interactions, providing new insights into the molecular bases of processes such as protein folding and receptor-ligand interactions. In addition, mechanical stability of supramolecular assemblies, unfolding pathways of membrane proteins, molecular forces determining cell adhesion and cell aggregation can also be analyzed. These measurements provide new insight into the structure-function relationships of microbial surfaces [25]. In spite of providing images with extreme resolution, AFM is not suitable for analysis of intercellular processes because it facilitate analysis of surfaces from above only.

6. Applications of light, confocal and electron microscopy in mycological research

Microscopy is a key method in microbiological studies for visualization of shape, size, surface and internal ultrastructures of microorganisms belonging to prokaryotic and eukaryotic group. A number of investigators have used these microscopic techniques in diagnosis of infections, to study the structural variations produced in fungi due to the toxic agents or interactions with other organisms. These techniques have enabled researchers to study food borne, human and plant pathogens and pest for their inhibition with antimicrobial agents and microbial interaction. The conventional and modern application of various forms of microscopy in mycology is described here briefly.

6.1. Studying the morphology and diagnosis of fungal infections

The ultimate goal of diagnostic microbiology is the rapid and accurate identification of bacteria and fungi in their natural environments. Culture-based methods are time consuming. A large number of fungal infections are identified by the presence of characteristic hyphal septation, reproductive structures like conidia and spores in diseased sample. Infections caused by dermatophytes including Trichophyton spp and Candida spp are identified by microscopic visualization after specific staining viz. potassium hydroxide wet mount, gram stain, geimsa stain, Kinyoun’s acid fast-stain [26, 27]. The direct microscopic examination of clinical specimens using histopathologic studies or direct fluorescent stains (eg, calcofluor white) allows for rapid detection [28, 29]. The presence of hyphae within mucosa,



submucosa, vessels and bone at histopathological examinations identifies invasive forms of paranasal fungal sinusitis [30]. Molecular techniques like PCR and subsequent hybridization or sequencing have revolutionized all fields of microbiology, and assisted in sensitive detection and exact identification of fungi. A very important tool for diagnosis is fluorescent in situ hybridization (FISH) that combines the precision of molecular genetics with the visual information from microscopy, to permit visualization and identification of individual microbial cells within their natural microhabitat or diseased tissue. As a technique it allows simultaneous visualization, identification, enumeration and localization of individual microbial cells. Fluorochromes with different excitation and emission maxima allow simultaneous detection of two or more microorganisms [31].

6.2. Studying the morphological alterations in fungi under stress condition

A large number of investigators have reported antifungal activity of natural or synthetic compounds against human and plant pathogenic fungi. Alteration in the morphology of fungal cells or hyphae in the presence of heavy metals has been shown by many workers. In a study, Chiou et al. [32] exploited light and electron microscopic techniques to show the combinational effects of Nikkomycin Z and the Echinocandin FK463 against Aspergillus fumigatus. The fungal cells treated individually resulted in no hyphal destruction or subtle changes in morphology were observed. When drugs were combined to evaluate efficacy of drugs, there were striking changes in cell wall, cell membrane, and cytoplasm under the influence of the combination of NZ and FK. Taken together these observations and other in vitro assays they concluded that these two cell wall-targeted antifungal agents, FK and NZ, are in synergistic mode of activity against A. fumigatus. The methods used in this study may be applicable to elucidating the activity and interaction of other cell wall-active agents. Some workers have used microscopic techniques to unfold the mechanism of action of natural products and given more insight of ultrastructural alterations occurring in the hyphae or fungal cell in the presence of such kinds of antimicrobial agents. Gahfarokhi et al. [33] elucidated the toxic effects of onion extracts on to the hyphal morphology of Trichophyton rubrum and T. mentagrophytes. The observation under light and transmission electron microscopy revealed cell membrane and cell wall damaging effects such as loss of cytoplasm in fungal hyphae, and budding of hyphal tip, thinner hyphal wall, flattened and empty hyphal tips. Similar observations were reported by Sharma and Tripathi [34] for oils of Citrus sinensis against A. fumigatus. Barodka et al. [35] used microscopic techniques to study the viability of Candida cells in the presence of liquid anesthetic isoflurane. These researchers highlighted the toxic effects of isoflurane over amphotericin B in terms of Candida germ tube production and hyphal deformation as observed under microscopy. In vitro effects of methylene bisthiocyanate (MBT) on hyphal morphology and ultra-structure of Ophiostoma floccosum were examined by Singh et al. [36] using differential interference contrast, epifluorescence and transmission electron microscopy (TEM). Differential interference contrast microscopy indicated that MBT caused rapid changes in O. floccosum hyphae resulting in extensive vaculoation and accumulation of granular materials within the cytoplasm. Epifluorescence microscopy provided evidence that MBT treatment causes a loss in the permeability properties of the plasma membrane. TEM showed retraction of the plasma membrane from the cell wall, aggregation of cytoplasmic contents, vesiculation of membranous components, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles. To understand the mode of action of MBT, in addition, they undertook to measure potassium ion (K+) leakage from cells, oxygen consumption, glucose and ATP levels. Taken together, there observations suggested that the target site of MBT in O. floccosum alters membrane properties and uncouplees oxidative phosphorylation from the respiratory chain. Wang et al. [37] using light and scanning electron microscopy and other assays suggested that the antifungal activity of eugenol against Botrytis cinerea is due to membrane binding and permeability alteration, leading to destabilization and disruption of the plasma membrane. Oils of Origanum syriacum, Thymbra spicata, Lavandula stoechas, Rosmarinus officinalis, Foeniculum vulgare, and Laurus nobilis were assessed for their antifungal activity against phytopathogens namely Phytophthora infestans and Botrytis cinerea by a group of Soylu et al. [38, 39]. They exploited light and scanning electron microscopic (SEM) observation on pathogen hyphae, exposed to both volatile and contact phase of oil. Their study revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage. Unusual pattern of hyphal growth, as well as alterations in cell shape and size were also demonstrated by SEM. This study has demonstrated that the essential oils are potential and promising antifungal agents which could be used as biofungicide. Some other investigators using light and electron microscopic techniques reported antifungal efficacy of Cinnamomum cassia Blume against phytopathogens Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera [40] and Chinese propolis ethyl acetate extract (PEAE) to control Penicillium digitatum and Penicillium italicum causing green and blue molds, respectively, which are economically important post harvest diseases of citrus fruits [41]. Recently, our group [42] has reported the antifungal activity of oils of Cinnamomum verum and Syzygium aromaticum and their major active compounds cinnamaldehyde and eugenol. This study has highlighted the time and concentration dependent toxic effects of these oils on to the growth and morphology of drug resistant strains T. rubrum



and Aspergillus fumigatus. Light microscopic observation revealed the hyphal necrosis, blistering and autolysis of cytoplasm (figure 1). The structural changes were confirmed by scanning and transmission electron microscopy of treated fungi and suggested that plasma membrane, nuclear membrane and other endo- membranous systems are targeted by these oils (figures 2 and 3)

(1.a) (1.b)

Figure 1. Concentration and time dependent toxicity of essential oils on hyphal morphology of T. rubrum IOA-9 (Light microscopy, 40 X) (a) control, 8h (b) C. verum oil 0.02% v/v, 20h ((1) Autolysis of cytoplasm (2) Chlamydoconidia formation)

(2.a) (2.b)

Figure 2. Scanning electron microscopy of hyphal morphology of A. fumigatus MTCC2550 treated with the 0.04% v/v of cinnamaldehyde (a) control, healthy hyphae (b) hyphae without cytoplasm (3).

(3.a) (3.b)

Figure 3. Transmission electron microscopy of A. fumigatus MTCC2550 treated with the 0.04% v/v of cinnamaldehyde (a) Control, intact cell wall, membrane and other organelles; (d) autolysis and degradation of cytoplasm content (4), abnormal distribution of polysaccharides (5).

These microscopic techniques have played important role in studying fungi-bacteria, fungi-fungi and fungi-metal interactions in environment and host. A very common symbiotic association of fungi and algae has been studied frequently using light and electron microscopy. Mycorhizal fungi forming speculated structures with plant roots both

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ectophytic and endophytic are mostly characterized by these techniques. Mycorhizal fungi especially VAM are identified on the basis of spore structures and surface of the spores and sizes. Li et al. [43] studied the mycoparasitism of Gliocladium roseum on Botrytis cinerea using light microscopy, scanning and transmission electron microscopy. Scanning electron microscopic studies revealed that infection of conidia and germ tubes occurred through direct penetration by hyphal tips of G. roseum without the formation of appressoria. Transmission electron microscopic studies indicated indentation and rupture of the host cell walls at penetration sites. The parasitized conidia and germ tubes of B. cinerea showed signs of cytoplasmic disintegration and the presence of hyphae of G. roseum. These studies indicated that G. roseum can be used as promising biocontrol agent against phytopathogen B. cinerea. Information on such kind of mycoparasitism was added by a study conducted by Zivkovic et al. [44]. These workers identified Trichoderma harzianum, Gliocladium roseum, Bacillus subtilis, Streptomyces noursei and Streptomyces natalensis, as promising biological control agents against Colletotrichum acutatum and Colletotrichum gloeosporioides, the causal agents of anthracnose disease in fruit crops. Mycoparasitism features as observed under microscopy were coiling, penetration, direct contact and parallel growth alongside the host hyphae. T. harzianum hyphae grew initially alongside and coiled compactly around the hyphae of the isolates of C. acutatum and C. gloeosporioides. Recently a report from Lima et al. [45] highlighted the biocontrol potential of Trichoderma harzianum in removing cadmium heavy metals from contaminated soil on the basis of optical and scanning electron microscopy observations made to hyphae in the presence of cadmium. The mycorrhizal association is a well known mutual relationship between two organisms, a fungus and the root of a vascular plant. A number of mycorrhizal types such as arbusclar mycorhizla (AM) and Vasicular arbuscular mycorhiza (VAM) have been recognized based on the fungal group involved and by the morphological features, resulting in the complex interaction between the symbionts. Various microscopic methods have been used to characterize morphological, anatomical and molecular features of orchid mycorrhizae. Fluorescence microscopy allows optical sectioning of images obtained from different reconstructions which are useful for studying complex fungal structures within the host [46]. Recently, CLSM has stimulated research in the development of new fluorochromes and dyes used routinely for light microscopy. Staining and microscopic methods not only provide reliable data on the degree of root colonization but also permit to visualize the presence of key features such as arbuscules, which are the morphological criteria that define AM associations [47]. Walker et al. [48] studied roots of Ranunculus from different latitudes for colonization by arbuscular mycorrhizae, fine endophyte, and septate endophyte fungi using lactofuchsin-stained material imaged with epifluorescence microscopy. Endorhizal quantitation was assessed for each endorhizal morphotype. There studies suggested that fine endophytes are important contributors to soil microbial diversity as related to plant survival and competitiveness in the high latitudes.

6.3. Biofilm examination and measurement

Biofilm has three-dimensional (3D) structured, heterogeneous community of microbial cells enclosed in an exopolysaccharide matrix (also called glycocalyx) that are irreversibly attached to an inert or living surface. The EPS plays various roles in structure and function of different biofilm communities. Adhesion to the surface provides considerable advantages such as protection against antimicrobial agents, acquisition of new genetic traits, and the nutrient availability and metabolic co-operability [49]. For most of the history of microbiology, microorganisms have primarily been characterized as planktonic, freely suspended cells and described on the basis of their growth characteristics in nutritionally rich culture media. Although the most common mode of growth for microorganisms on earth is in surface associated communities [50], the first reported findings of microorganisms “attached in layers” were not made until the 1940s. Anthony Van Leeuwenhoek, who discovered microbial attachment to his own tooth surface, is credited with the discovery of biofilms [49]. Our understanding of biofilms has developed as the methods for biofilm examination and characterization have evolved. Much of the early investigative work on bacterial and yeast biofilms relied heavily on SEM and CLSM. The EM technique utilizes graded solvents (alcohol, acetone, and xylene) to gradually dehydrate the specimen prior to examination, since water of hydration is not compatible with the vacuum used with the electron beam. This dehydration process results in significant sample distortion and artifacts since extracellular polymeric substances consist of ~95% water. But the use of transmission electron microscopy and specific polysaccharide stains like ruthenium red allowed researchers both to identify the nature of these extracellular fibers in biofilms and to better elucidate their association with the cells. Because of its excellent resolution properties, the electron microscopy, in spite of its limitations, continued to be an important tool for the biofilm scientists. Electron microscopy has been used for the examination and characterization of biofilms on medical devices and in human infections [51]. CLSM allows nondestructive optical sectioning of samples and provides images free of focus blur [52]. CLSM has provided researchers with the ability to examine biofilms in situ without the limitations encountered with the scanning electron microscope, albeit at lower magnifications. This has enabled researchers to examine the biofilm matrix unaltered and intact. The use of both CLSM and epifluorescence microscopy requires staining of biofilm organisms with fluorescent stains. These stains are designed to emit light at specific wavelengths and can be used to probe specific cellular functions. For example, nucleic acid stains such as DAPI (4’,6-diamidino-2-phenylindole), acridine orange, and Syto 9 will stain the DNA and RNA of all cells regardless of their viability. Other stains have been developed for



probing cell viability. Propidium iodide is taken up only by cells with damaged cytoplasmic membranes, and 5-cyano-2,3-ditolyl tetrazolium chloride is taken up and reduced to 5-cyano-2,3-ditolyl tetrazolium chloride-formazan only by cells that have a functioning cytochrome system. Using a suite of such stains allows the biofilm researchers to quantify all the cells and determine which ones are viable. Fluorescent antisera and fluorescent in situ hybridization probes have enabled workers to identify specific organisms within a mixed biofilm community. Green fluorescent protein, a constitutively produced, plasmid-mediated molecule, can allow biofilms to be examined noninvasively, without fixation or staining [53]. This chapter summarizes some the studies reporting use of these techniques in their research in studying the biofilms of yeasts and filamentous fungi. Li et al. [54] studied the quantitative variation of Candida albicans biofilms by staining biofilms by FUN-1 and Con A dyes for actively metabolizing cells and polysaccharides of cell wall and biofilms, respectively. The resulted red, green and yellow fluorescence was captured by CLSM and quantified in terms of stained cells. Martinez and Casadevall [55] studied the biofilm structure and their formation in Cryptococcus neoformans at different stages using light, scanning and confocal microscopy. Observations made by them under light microscopy revealed initial stage of adhesion of yeast cells followed by formation of microcolonies and a dense network of yeast cells enmeshed in polysaccharide matrix as revealed by SEM. CLSM images of a mature C. neoformans biofilm grown on polystyrene plates reveal a highly organized architecture. Intense green fluorescence resulting from ConA binding to polysaccharides outlined the cell walls of the yeast, while the red color due to FUN-1 staining localized in dense aggregates in the cytoplasm of metabolically active cells. Villena et al. [56] performed structural analysis of biofilms and pellets of Aspergillus niger by confocal laser scanning microscopy and cryo scanning electron microscopy. Biomass organization of Aspergillus niger biofilms and pellets stained with fluorescein isothiocyanate were analyzed by means of confocal laser scanning microscopy and detectable differences between both types of growth were found. While biofilms showed a spatially ordered mycelium and well structured hyphal channels, pellets were characterized by an entangled and notoriously compacted mycelium. These findings revealed common structural characteristics between A. niger biofilms and those found in other microbial biofilms. They suggested that biofilm microstructure may represent a key determinant of biofilm growth and physiology of filamentous fungi. The proper demonstration of mixed biofilms is challenging because staining both the mixture of yeasts and bacteria and the glycocalyx matrix is difficult. So far, mixed communities of yeasts and bacteria organized in a structured biofilm on the surface of TVPs have usually been visualized by SEM. Kania et al. [57] demonstrated the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with CLSM. Biofilms of 12 h removed TVPs were visualized by scanning electron microscopy, then stained with ConA FITC and propidium iodide for CLSM. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network on the surface of TVPs. A study from Bandara et al. [58] has reported the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species mixed biofilm development. Using microscopic techniques they were able to suggest this kind of interaction where P. aeruginosa inhibits biofilm formation by Candida spp. In their study they found that most of the bacteria were attached to the blastospores. Bacterial density varied in the presence of different Candida species at different time intervals. In general, P. aeruginosa distribution was scanty and nondescript in the dual species environment. As establish, biofilm formation has a serious implications in public health and medicine. In the case of human health, a number of microbial infections are associated with surface colonization not only on live surfaces (sinusitis, pulmonary infection in cystic fibrosis patients, periodontitis, etc. but also on medical implants (contact lenses, dental implants, intravascular catheters, urinary stents) etc. [50, 51]. This combined with increasing antimicrobial drug resistance is making many infectious diseases incurable. The detection of such biofilms and their inhibition studies regarding structural and phenotypic variations had been made possible using microscopic techniques. Ferreira et al. [59] exploited light microscopy, filipin fluorescent and scanning electron microscopy to show the effect of Gup1 in virulence of Candida albicans by observing morphemic alterations and biofilm forming ability of wild type and Cagup1Δ null mutant strains. They showed that Candida albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. The mutant colonies exhibited an aberrant morphology/differentiation pattern as observed under microscopy. Cagup1Δ null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane as observed under filipin fluorescent microscopy. Martins et al. [60] analyzed the effect of DNase treatment on the biofilm formation by Candida albicans. DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. They visually confirmed these results by light microscopy of treated and untreated biofilms. A study from Martinez et al. [61] has opened a new dimension to prevent fungal biofilms on indwelling devices. They coated indwelling medical devices such as pace maker and cathater with chitosan and analyzed the inhibition of



bioofilm formation by Cryptococcus neoformans using scanning electron and confocal laser scanning microscopy. Recently our group Khan and Ahmad [62] had reported antibiofilm activity from oils of Cymbopogon citratus and Syzygium aromaticum against drug resistant strains of Candida albicans. Light and scanning electron microscopic studies revealed the deformity of three dimensional structures of biofilms formed in the presence of sub-MICs of oils. The cell membranes appeared to be the target site of compounds in biofilm cells as displayed by SEM observations in our study (figure 4).

(a) (b)

Figure 4. Scanning electron micrograph of the 48 h biofilm formed in C. albicans 04 on catheter discs in the absence and presence of C. citratus. (a) biofilm formed without oils, smooth cell membrane of normal cell (b) biofilm formed in the presence of C. citratus at 45 µg/mL, cell membrane shrinkage in sessile cells, bursting of cells leading to vesicle formation due to lytic material. In the case of metallic materials, undesirable changes in material properties due to a biofilms are referred to as biocorrosion or microbially influenced corrosion. Biofouling and biocorrosion occur in terrestrial and aquatic habitats varying in nutrient content, temperature, pressure and pH [63]. Formation of biofilm is responsible for deterioration of water quality and the contamination of food. Understanding of microbial corrosion requires interdisciplinary approach including well defined microbiological and electrochemical methodologies. Such as microscopical techniques viz. environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy and, new spectroscopical techniques used for the study of corrosion products and biofilms and electrochemistry like electrochemical impedance spectroscopy and electrochemical noise analysis. A brief survey of studies performed using such techniques have been summarized below. Biofilms of a municipal water distribution system were characterized by Doggett et al. [64] to assess the occurrence of fungi within surface matrixes. Observations by SEM suggested that spores, not hyphae or vegetative cells, comprised the primary source of viable propagules. Fungal spores were observed in each of the four samples and were usually loosely embedded on biofilm-water interface or within crevices and encrustations. Soncini-Junior et al. [65] evaluated biodeterioration and biodegradation potential of Curvularia geniculata, based on the presence of a strongly adhered biofilm investigated by SEM. They undertook microscopical study to analyze the eventual fungi adhesion over a polyester imide surface in enameled copper wires. SEM allowed observing these adhered biofilms for high amount of pigments, hyphae and its enzymatic arsenal probably acting in the surface of the polymer.

7. Application of atomic force microscopy

In recent years the atomic force microscope (AFM) has rapidly developed as a useful imaging tool in biological research. It is utilized for structural biology, with emphasis on high-resolution imaging of cell membranes, DNA and soluble and membrane proteins, protein fibrils, polysaccharides, and interfaces. AFM can be used to recognize individual surface components on fungi [66] whereas, two surface molecules can also be studied simultaneously [67]. AFM has found potential application in extraction of single mRNA molecules [68] from living cells of fungi for studying the relationship between mRNA localization and cell polarity, and transfection using plasmids [69]. Colloid probe AFM has been used to study the molecular scale characteristics responsible for adhesion to surfaces [70], which has direct applications for fungal biofilms. This chapter provides a survey of the various applications offered by AFM for probing microbial cell surfaces at high spatial resolution: (i) visualization of surface ultrastructure, (ii) measurement of surface forces, and (iii) characterization of mechanical properties.

7.1. Surface ultrastructure at nano scale imaging

Because of the small size of microorganisms, the physical properties of their surfaces have been difficult to study. Although the chemical composition of many fungal cell walls is known, the spatial organization and interactions of the individual macromolecules is not well studied. Two classes of single-molecule techniques can be distinguished (i) fluorescence techniques, which use fluorescence to track the real-time position and dynamics of single molecules, and (ii) force measuring techniques, which exert force on the probed molecules and include flow chamber experiments,

i

ii

iii



microneedles, the biomembrane force probe (BFP), optical and magnetic tweezers and AFM techniques. Currently, AFM is the only force-measuring technique that can simultaneously localize and force probe single molecules on live cells. The ability of AFM to image single live cells in real time provides novel insight into the processes of cell wall remodeling [23, 24, 25]. Yeast cell surfaces mapped using AFM probes decorated with antibodies [71] indicated a non-uniform distribution of mannan [72], which relates to wall maturation and to changes associated with bud scars. Ahimou et al. [73] described AFM imaging of S. cerevisiae surface topography with 2 nm lateral resolutions to monitor the effects of protease digestion on the cell wall, showing that the surface became progressively rougher, consistent with the erosion of mannoprotein. AFM probes covalently linked with antibodies have been used to assess adhesion between AFM tips and fungal cell surfaces [71]. Thus, it is possible to understand at least some of the chemical changes that accompany yeast wall maturation using AFM based methods. Recent breakthroughs include the visualization of the structural dynamics of single cells as they grow or interact with drugs, the quantification and nanoscale imaging of cell surface hydrophobicity and the measurement of the molecular elasticity of cell wall polysaccharides and proteins [24]. Dufrene et al. [74] characterized the surface ultrastructure and molecular characteristics of Phanerochaete chrysosporium spores using AFM probes functionalized with hydroxy- or methyl-groups to map their topography and relative hydrophobicity. Dufrene et al. ]23] used FS to probe fungal spores under various physiological and environmental conditions, showing a change from dormancy to germination. Ma et al. [75] showed that filamentous fungi are capable of remodeling hyphal as well as spore cell walls. The surface of many fungal structures (spores, hyphae) is covered by a thin layer of regularly arranged proteinaceous rodlets, which are 5-10 nm thick and up to 250 nm in length [76]. AFM was used by de Vocht et al. [77] to examine the patterns of rodlets of 9-15 nm diameter after drying a solution of amphiphilic hydrophobin SC3 isolated from Schizophyllum commune on a mica surface. Dufrene et al. [74] studied the surface ultrastructure of living spores of the filamentous fungus Phanerochaete chrysosporium immobilized into a porous membrane. High-resolution images of the surface of dormant spores showed patterns of rodlets having a periodicity of 10 nm, consistent with freeze-etching measurements. An elegant example is the use of AFM to probe the high-resolution structural dynamics of single Aspergillus spores during germination. Zhao et al. [78] exploited AFM as a nanoindentor to measure cell wall mechanical properties of the model fungus Aspergillus nidulans in both hyphal and spore forms. They described both surface morphology and adhesive properties of fungal spores. AFM images showed characteristics “rodlet” protein structures covering spore surfaces. This group further studied both surface topography and micromechanical properties of Aspergillus nidulans spores using AFM. These results implied that the rodlet layer is significantly softer than the underlying portion of the cell wall. Dague et al. [79] used real-time atomic force microscopy with a temperature-controlled stage (37 °C) to probe the structural and physicochemical dynamics of single Aspergillus fumigatus conidia during germination. Nanoscale topographic images of dormant spores revealed the presence of a layer of rodlets made of hydrophobins, in agreement with earlier electron microscopy observations. The rodlet surface was uniformly hydrophobic due to the presence of hydrophobins, whereas the cell wall material appearing upon germination was purely hydrophilic. These studies illustrated the potential of real-time atomic force microscopy imaging and force spectroscopy for tracking cell-surface dynamics.

7.2. Surface and intermolecular forces

A major advantage of the AFM over other microscopy techniques is that it can simultaneously provide information on local surface properties and interaction forces. Several investigators have exploited the AFM to measure surface forces in aqueous environments such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces associated with polymer covered surfaces [23]. Hydrophobic interactions mediate various crucial biological events, including protein folding, membrane fusion and cell adhesion. These forces are of particular importance in microbiology, since they are a driving force for the adhesion of microbes, including pathogens. [24]. With respect to biological applications, many studies have focused on the forces associated with individual biomolecules (23, 24, 80, 81]. To gain insight into the forces involved in microbial adhesion, several experimental approaches using AFM have been developed. A few investigators immobilized microbial cells on AFM probes and measured the forces between the modified probes and solid substrata. Interaction forces were measured between probes coated with single S. cerevisiae cells and mica [82] or polymeric membranes [83]. Using this procedure, it was demonstrated that changes in surface ultrastructure of P. chrysosporium spores occurring during germination are correlated with profound modifications of molecular interactions. Probe functionalization was employed to map the surface hydrophobicity of dormant P. chrysosporium spores [74]. Dague et al. [84] demonstrated the ability of chemical force microscopy (CFM) to probe the hydrophobic character of live A. fumigatus cells, on a scale of only ~25 functional groups. In CFM, AFM tips are modified with specific functional groups in order to map the spatial arrangement of chemical groups and to measure their interactions [85]. AFM force curves recorded across the surface of native spores in aqueous solution with a hydrophobic tip showed large adhesion forces, revealing that the surface was markedly hydrophobic. This study provided direct evidence that, in vivo, cell surface hydrophobins have a strong hydrophobic character, thus explaining their roles as dispersion and adherence structures.



7.3. Probing polysaccharide conformation and biofilm with AFM

Polysaccharides represent a major class of biological macromolecules that occur throughout nature, including in plant, algal, bacterial, fungal and animal cells. In the microbial realm, polysaccharides encapsulate bacteria and fungi and are implicated in cell-to-cell communication, biofilm formation, dental plaque formation, microbial-induced metal corrosion, biofouling and the propagation of infection in certain pathogenic micro-organisms and resistance to heavy metal toxicity [86]. Polysaccharides form eukaryotic cell surface receptors and are involved in cell–cell recognition and adhesion [87]. The height and thickness of polysaccharide chains can be determined from trace analyses,as has been done for many types of polysaccharides. On the other hand, properties of the polysaccharide chains can be measured under different conditions and used to reconstruct a conceptual picture of the polysaccharide’s conformation. Contour lengths, persistence lengths and end-to-end distances can be obtained from imaging and image analysis [86]. Aggregation of microbial cells mediated by specific interactions plays a pivotal role in the natural environment, in medicine and in biotechnological processes. Touhami et al. [66] used AFM to measure individual lectin–carbohydrate interactions involved in the flocculation of yeast cells, an aggregation event of crucial importance in fermentation technology. By contrast, specific interaction forces were not observed in non-flocculating conditions, i.e. in the presence of mannose or when using non-flocculating cells, pointing to their involvement in yeast flocculation. The single molecule force spectroscopy measurements presented here provided a means to study a variety of cellular interactions at the molecular level, such as the adhesion of micororganisms to animal and plant tissues. AFM has proved useful in imaging the morphology of fungal biofilms on solid surfaces, both in the dried and hydrated states. While nanometer scale information could generally not be obtained due to the high sample softness, AFM allowed direct visualization of hydrated extracellular polymeric substances to a resolution that was not previously accessible by any other microscopy technique. Many investigators have studied the biofilm structure of yeast and filamentous fungi using AFM to reveal efficacy of antibiofilm agents and morphogenetic variations compared to planktonic growth of cells. Recently, Tyagi and Malik [88] made microscopic observations on Candida biofilm cells treated with the oil of Cymbopogon citratus. Observation made under SEM and AFM led the workers to differentiate the effect of liquid and vapour phase of oils on to biofilm structure compared to untreated cells. The AFM picture of untreated cells showed cluster of C. albicans cells in intact shape. In the lemon grass essential oil treated cells, cells loose their original shapes, appear shrunken and partially deformed. Although length and width seems to be less affected, cells appear relatively flattened. The height of untreated, lemon grass essential oil treated and lemon grass essential oil vapour treated cells was found to be 350 nm, 150 nm and 37.5 nm, respectively. Atomic force microscopes simultaneously measure surfaces in the x, y, and z dimensions and provide a true three-dimensional map of the surface of cell samples on a submicron scale. The three dimensional structure of the C. albicans cells also shows significant differences in the Z axis value which was 700 nm/div, 500 nm/div and 100 nm/div in untreated, lemon grass essential oil treated and lemon grass essential oil vapour treated samples, respectively.. It was clear from there results that the vapour treatment not only perceptibly alters the cell dimensions and the overall morphology, but a great impact on the cell surface properties was also noticed (figure 5). The most commonly studied relation involves biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels. A study from Lugauskas et al. [63] presented corrosion of steel and aluminium by Chrysosporium merdarium, Penicillium cyclopium, Arthrinium phaeospermum, Cladosporium herbarum, Aspergillus niger, obtained from metals exposed to natural environment conditions for a long time (0.5 to 2 years). The investigation was carried out for fungal interaction with metal (steel, Al) surface; products of interaction of metal with fungi; metal surface morphology after the impact of fungi: nanometric evaluation of changes in the surface of metals. The influence of fungi on the corrosion rate was evaluated using polarization resistance as a criterion using AFM.

Figure 5. Atomic force micrographs showing variation in the height of untreated and treated (24 h) C. albicans cells from glass surface: (a) Untreated (h 350 nm), (b) Cymbopogon citratus essential oil treated (h 150 nm).



Conclusions

Fungal ultrastructure has been, and continues to be, a key research element in the study of spore development and germination, host-pathogen interactions, and studies of sub-cellular organelles structure and function. Development of biofilms by microorganism have played crucial role affecting clinical and environmental set up and waste water management. Use of modern microscopic techniques including fluorescent and electron microscopy is a prerequisite in advancement of the above area of research. Further, evaluation of toxic and anti-biofilm effects of natural or synthetic compounds have contributed largely in clinical microbiology in drug discovery. These microscopic techniques have speeded up the search for potential alternatives for synthetic fungicides with modification as their structures could lead to the development of new classes of antifungal compounds against pathogenic fungi. The optical and electron microscopy of cellular morphology and ultrastructures has been visualized in the pathology, ontogeny, and taxonomy of many organisms. The newly developed atomic force microscope is a valuable tool for studying physical and biological structures and provides a unique window to the microbial world, sub-cellular structures, and biomolecules. The AFM has been used in viewing and analyzing the ultra structure of microbial cell surface studies and analyzing structure of native membrane proteins at sub-nanometre resolution, function-related conformational changes in single proteins, and surface ultra structure of living cells, cell surface dynamics, and organization of biofilm communities especially in fungi.

References

[1] Hawksworth DL. The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycological research. 1991;95:641-655.

[2] Kaminskyj SGW, Dahms TES. High spatial resolution surface imaging and analysis of fungal cells using SEM and AFM. Micron. 2008;39:349-361.

[3] Smits WK, Kuipers OP, Veening J-W. Phenotypic variation in bacteria: the role of feed back regulation. Nature Reviews Microbiology. 2006;4:259-271. [4] Veening J-W, Smits WK, Hamoen LW. Single cell analysis of gene expression patterns of competence development and

initiation of sporulation in Bacillus subtilis grown on chemically defined media. Journal of Applied Microbiology. 2006;101:531-541.

[5] Haagensen JAJ, Regenberg B, Sternberg C. Advanced microscopy of microbial cells. Advances in Biochemical Engineering/Biotechnology. 2011;124:21-54.

[6] Colomb T, Durr F, Cuche E, Marquet P, Limberger HG, Salathe RP, Depeursinge C. Polarization microscopy by use of digital holography: application to optical-fiber birefringence measurements. Applied Optics. 2005;44:4461-4469.

[7] Amos WB. Instruments for fluorescence imaging, in V. J. Allan (ed.), Protein Localization by Fluorescence Microscopy: A Practical Approach, New York: Oxford University Press, 67-108, 2000.

[8] Conn PM. Green Fluorescent Protein, Methods inEnzymology, Volume 302, New York, Academic Press, 1999. [9] Schmolze DB, Standley C, Fogarty KE, Fischer AH. Advances in microscopy techniques. Archives of Pathology & Laboratory

Medicine. 2011;135:255-263. [10] Paddock SW. Confocal Microscopy: Methods and Protocols, Totowa, New Jersey: Humana Press, 1999. [11] Sandison D, Webb W. Background rejection and signal-to- noise optimization in the confocal and alternative fluorescence

microscopes. Applied Optics. 1994;33:603-610. [12] Klomparens KL. The development and application of ultrastructural research in mycology. Mycopathologia. 1990;109:139-148. [13] Heath IB, Kaminskyj S. The organization of tip-growth-related organelles and microtubules revealed by quantitative analysis of

freeze substituted oomycete hyphae. Journal of Cell Science. 1989;93:41-52. [14] Hohmann-Marriott MF, Uchida M, van de Meene AML, Garret M, Hjelm BE, Kokoori S, Roberson RW. Application of

electron tomography to fungal ultrastructure studies. New Phytologist. 2006;172:208-220. [15] Wolski E, Maldonado S, Daleo G, Andreu A. Cell wall α-1,3-glucans from a biocontrol isolate of Rhizoctonia:

immunocytolocalization and relationship with a-glucanase activity from potato sprouts. Mycological Research. 2007;111:976-984.

[16] Kaminskyj SGW, Dahms TES. High spatial resolution surface imaging and analysis of fungal cells using SEM and AFM. Micron. 2008;39:349-361.

[17] Hess MW. Cryopreparation methodology for plant cell biology. In: Cellular Electron Microscopy, Academic Press, New York.2007.

[18] Craig S, Beaton CD. A simple cryo-SEM method for delicate plant tissues. Journal of Microscopy. 1996;182:102-105. [19] Stewart ADG. The origins and development of scanning electron microscopy. Journal of Microscopy. 1985;139:121-127. [20] Muscariello L, Rosso F, Marino G, Giordano A, Barbarisi M, Cafiero G, Barbarisi A. A critical overview of ESEM applications

in the biological field. Journal of Cellular Physiology. 2005;205:328-334. [21] Binnig G, Quate CF, Gerber C. Atomic force microscope. Physical Review Letters. 1986;56:930-933. [22] El Kirat K, Burton I, Dupres V, Dufrene YF. Sample preparation procedures for biological atomic force microscopy. Journal of

Microscopy. 2005;218:199-207. [23] Dufrene YF. Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

Micron. 2001;32:153-165. [24] Dufrene YF. Atomic force microscopy of fungal cell walls: an update. Yeast 2010; 27: 465-471. [25] Dufrene YF. Atomic force microscopy, a powerful tool in microbiology. Journal of Bacteriology. 2002;184:5205-5213.



[26] Elewski BE. Onychomycosis: Pathogenesis, diagnosis, and management. Clinical Microbiology Review. 1998;11:415-429. [27] Bharathi MJ, Ramakrishnan R, Meenakshi R, Mittal S, Shivakumar C, Srinivasan M. Microbiological diagnosis of infective

keratitis: comparative evaluation of direct microscopy and culture results. British Journal of Ophthalmology. 2006;90:1271-1276.

[28] Souza PR, Vettorato G, Pinto GM, Duquia RP, Amaro TG, Almeira Junior HL, Breunig Jde A. Concordance between direct microscopy and fungical culture for the diagnostic of feet's onychomycosis. Anais brasileiros de dermatologia. 2012;87:157-159.

[29] Wengenack NL, Binnicker MJ. Fungal molecular diagnostics. Clinics in Chest Medicine. 2009;30:391-408. [30] Pagella F, Matti E, De Bernardi F, Semino L, Cavanna C, Marone P, Farina C, Castelnuovo P. Paranasal sinus fungus ball:

diagnosis and management. Mycoses. 2007; 50:451-456. [31] Moter A, Gobel UB. Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. Journal of

Microbiological Methods. 2000;41:85-112. [32] Chiou CC, Mavrogiorgos N, Tillem E, Hector R, Walsh TJ. Synergy, pharmacodynamics, and time-sequenced ultrastructural

changes of the interaction between nikkomycin z and the echinocandin fk463 against aspergillus fumigatus. Antimicrobial Agents and Chemotherapy. 2001;45:3310-3321.

[33] Ghahfarokhi MS, Goodarzi M, Abyaneh MR, Al-Tiraihi T, Seyedipour G. Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and Trichophyton mentagrophytes. Fitoterapia 2004;75:645-655.

[34] Sharma N, Tripathi A. Effects of Citrus sinensis (L.) Osbeck epicarp essential oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem. Microbiological Research. 2008;163:337-344.

[35] Barodka VM, Acheampong E, Powell G, Lobach L, Logan DA, Parveen Z, Armstead V, Mukhtar M. Antimicrobial effects of liquid anesthetic isoflurane on Candida albicans. Journal of Translational Medicine. 2006, 4:46.

[36] Singh T, Kreber B, Singh A, Stewart A, Jaspers M. Microscopic, biochemical and physiological assessment of the effect of methylene bisthiocyanate on the sapstain fungus Ophiostoma floccosum. European Journal of Plant Pathology. 2006;114:317-328.

[37] Wang C, Zhang J, Chen H, Fan Y, Shi Z. Antifungal activity of eugenol against Botrytis cinerea. Tropical Plant Pathology. 2010;35:137-143.

[38] Soylu EM, Soylu S, Kurt S. Antimicrobial activities of the essential oils of various plants against tomato late blight disease agent Phytophthora infestans. Mycopathologia. 2006;161:119-128.

[39] Soylu EM, Kurt S, Soylu S. In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea. International Journal of Food Microbiology. 2010;243:83-189.

[40] Nguyen VN, Nguyen DMC, Seo DJ, Park RD, Jung WJ. Antimycotic activities of Cinnamon-derived compounds against Rhizoctonia solani in vitro. BioControl. 2009; 54:697-707.

[41] Yang S, Peng L, Cheng Y, Chen F, Pan S. Control of citrus green and blue molds by chinese propolis. Food Science and Biotechnology. 2010;19:1303-1308.

[42] Khan MSA, Ahmad I. In vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of Cinnamomum-, Syzygium- and Cymbopogon-species against Aspergillus fumigatus and Trichophyton rubrum. Phytomedicine. 2011;19:48-55.

[43] Li GQ, Huang HC, Kokko EG, Acharya SN. Ultrastructural study of mycoparasitism of Gliocladium roseum on Botrytis cinerea. Botanical Bulletin of Academia Sinica. 2002;43:211-218.

[44] Zivkovic S, Stojanovic S, Ivanovic Z, Gavrilovic V, Popovic T, Balaz J. Screening of antagonistic activity of microorganisms against Colletotrichum acutatum and colletotrichum gloeosporioides. Archives of Biological Sciences. 2010;62:611-623.

[45] Lima AF, Moura GF, de Lima MAB, de Souza PM, da Silva CAA, Takaki GMC, do Nascimento AE. Role of the morphology and polyphosphate in trichoderma harzianum related to cadmium removal. Molecules. 2011;16:2486-2500.

[46] Schelkle M, Ursic M, Farquhar M, Peterson RL. Mycorrhiza. 1996:6:431-440. [47] Brundrett MC. Diversity and classification of mycorrhizal associations. Biological Reviews. 2004;78:473-495. [48] Walker XJ, Basinger JF, Kaminsky SGW. Endorhizal fungi in ranunculus from western and arctic Canada: predominance of

fine endophytes at high latitudes. The Open Mycology Journal. 2010;4:1-9. [49] Kokare CP, Chakraborty S, Khopade AN, Mahadik KR. Biofilm: Importance and applications. Indian Journal of Biotechnology.

2009;8:159-168. [50] Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated communities. Annual Review of

Microbiology. 2002;9:187-209. [51] Donlan RM, Costerton JW. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev.

2002;15:167-193. [52] Conchello JA, Lichtman J. Optical sectioning microscopy. Nature Methods. 2005;2:20-931. [53] Bloemberg GV, O’Toole GA, Lugtenberg BJJ, Kolter R. Green fluorescent protein as a marker for Pseudomonas spp. Applied

and Environmental Microbiology. 1997;63:4543-4551. [54] Li X, Yan Z, Xu J. Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology.

2003;149:353-362. [55] Martinez LR, Casadevall A. Specific antibody can prevent fungal biofilm formation and this effect correlates with protective

efficacy. Infection and Immunity. 2005;73:6350-6362. [56] Villena GK, Fujikawa T, Tsuyumu S, Gutiérrez-Correa M. Structural analysis of biofilms and pellets of Aspergillus niger by

confocal laser scanning microscopy and cryo scanning electron microscopy. Bioresource Technology. 2010;101:1920-1926. [57] Kania RE, Lamers GEM, van de Laar N, Dijkhuizen M, Lagendijk E, Huy PTB, Herman P, Hiemstra P, Grote JJ, Frijns J,

Bloemberg GV. Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms. Biofouling. 2010;26:519-526.

[58] Bandara HMHN, Yau JYY, Watt RM, Jin LJ, Samaranayake LP. Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development. BMC Microbiology 2010;10:125.



[59] Ferreira C, Silva S, Faria-Oliveira F, Pinho E, Henriques M, Lucas C. Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p. BMC Microbiology. 2010;10:238.

[60] Martins M, Uppuluri P, Thomas DP, Cleary IA, Henriques M, Lopez-Ribot JL, Oliveira R. Presence of extracellular dna in the Candida albicans biofilm matrix and its contribution to biofilms. Mycopathologia. 2010;169:323-331.

[61] Martinez LR, Mihu MR, Han G, Frases S, Cordero RJB, Casadevall A, Friedman AJ, Friedman JM, Nosanchuk JD. The use of chitosan to damage Cryptococcus neoformans biofilms. Biomaterials. 2010;31:669-679.

[62] Khan MSA, Ahmad I. Biofilm inhibition by Cymbopogon citratus and Syzygium aromaticum essential oils in the strains of Candida albicans. Journal of Ethnopharmacology. 2012;140:416-423.

[63] Lugauskas A, Prosyčevas I, Ramanauskas R, Grigucevičienė A, Selskienė A, Pakštas V. The influence of micromycetes on the corrosion behaviour of metals (steel, al) under conditions of the environment polluted with organic substances. Materials Science. 2009;15:224-235.

[64] Doggett MS. Characterization of fungal biofilms within a municipal water distribution system. Applied and Environmental Microbiology. 2000;66:1249-1251.

[65] Soncini-Júnior G, Franchetti SMM, Marconato JC. Architecture and relevance of several strongly adhered biofilms over a polyester imide (pei) surface. Brazilian Journal of Microbiology. 2003;34:105-10.

[66] Touhami A, Hoffman B, Vasella A, Denis FA, Dufrene YF. Probing specific lectin–carbohydrate interactions using atomic force microscopy imaging and force measurements. Langmuir. 2003;19:9937-9941.

[67] Wang YL, Zhao XZ, Zhou FQ. Improved parallel scan method for nanofriction force measurement with atomic force microscopy. The Review of Scientific Instruments. 2007;78.

[68] Uehara H, Osada T, Ikai A. Quantitative measurement of mRNA at different loci within an individual living cell. Ultramicroscopy. 2004;100:197-201.

[69] Cuerrier CM, Lebel R, Grandbois M. Single cell transfection using plasmid decorated AFM probes. Biochemical and Biophysical Research Communications. 2007;355:632-636.

[70] Salerno MB, Li X, Logan BE. Adhesion characteristics of two Burkholderia cepacia strains examined using colloid probe microscopy and gradient force analysis. Colloids Surf. B: Biointerfaces. 2007;59:46-51.

[71] Hinterdorfer P, Dufrene YF. Detection and localization of single molecular recognition events using atomic force microscopy. Nat. Methods. 2006;3:347-355.

[72] Gad M, Itoh A, Ikai A. Mapping cell wall polysaccharides of living microbial cells using atomic force microscopy. Cell Biology International reports. 1997;21:697-706.

[73] Ahimou F, Touhami A, Dufrene Y F. Real-time imaging of the surface topography of living yeast cells by atomic force microscopy. Yeast. 2003;20:25-30.

[74] Dufrene Y F, Boonaert CJP, Gerin PA, Asther M, Rouxhet PG. Direct probing of the surface ultrastructure and molecular interactions of dormant and germinating spores of Phanerochaete chrysosporium. Journal of Bacteriol. 1999;181:5350-5354.

[75] Ma H, Snook LA, Kaminskyj SGW, Dahms TES. Surface ultrastructure and elasticity in growing tips and mature regions of Aspergillus hyphae describe wall maturation. Microbiology. 2005;151:3679-3688.

[76] Wessels JGH. Hydrophobins: proteins that change the nature of the fungal surface. Advances in Microbial Physiology. 1997;38:1-45.

[77] de Vocht ML, Scholtmeijer K, van der Vegte EW, de Vries OMH, Sonveaux N, Wosten HAB, Ruysschaert JM, Hadziioannou G, Wessels JGH, Robillard GT. Structural characterization of the hydrophobin SC3, as a monomer and after self-assembly at hydrophobic/hydrophilic interfaces. Biophysical Journal. 1988;74:2059-2068.

[78] Zhao L, Schaefer D, Haixin X, Modi SJ, LaCourse WR, Martin R. Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy. Biotechnol Progress. 2005;21:292-299.

[79] Dague E, Alsteens D, Latge JP, Dufrene YF. High resolution cell surface dynamics of germinating Aspergillus fumigatus conidia. Biophysical Journal. 2008;94: 656-660.

[80] Oberhauser AF, Marszalek PE, Erickson HP, Fernandez JM. The molecular elasticity of the extracellular matrix protein tenascin. Nature. 1998;393:181-185.

[81] Rief M, Oesterhelt F, Heymann B, Gaub HE. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science. 1997;275:1900-1905.

[82] Bowen WR, Hilal N, Lovitt RW, Wright CJ. Direct measurement of the force of adhesion of a single biological cell using an atomic force microscope. Colloids Surfaces A. 1998; 136:231-234.

[83] Bowen WR, Hilal N, Lovitt RW, Wright CJ. Characterization of membrane surfaces: direct measurement of biological adhesion using an atomic force microscope. Journal of Membrane Science. 1999;154:205-212.

[84] Dague E, Alsteens D, Latge JP. Chemical force microscopy of single live cells. Nano Letters. 2007;7:3026-3030. [85] Dufrene YF. Atomic force microscopy and chemical force microscopy of microbial cells. Nature Protocols. 2008;3:132-1138. [86] Abu-Lail NI, Camesano TA. Polysaccharide properties probed with atomic force microscopy. Journal of Microscopy.

2003;212:217-238. [87] McEver RP, Moore KL, Cummings RD. Leukocyte trafficking mediated by selectin–carbohydrate interactions. Journal of

Biological Chemistry. 1995;270:11025-11028. [88] Tyagi AK, Malik A. Liquid and vapour-phase antifungal activities of selected essential oils against Candida albicans:

microscopic observations and chemical characterization of Cymbopogon citratus. BMC Complementary and Alternative Medicine. 2010;10:65.



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Microscopy in mycological research with especial reference ...formatex.info/microscopy5/book/646-659.pdf · Microscopy in mycological research with especial reference to ultrastructures